The photosynthesis
of carbon compounds

The

Photosynthesis of
Carbon Compounds

Melvin Calvin
J. A. Bassham

University of California
Berkeley, California

W. A. Benjamin, Inc.

New York

1962

THE PHOTOSYNTHESIS OF CARBON COMPOUNDS

Copyright © 1962 by W. A. Benjamin, Inc.
All rights reserved

Library of Congress Catalog Card Number: 62-10567
Manufactured in the United States of America

The manuscript was received November 15, 1961, and published
February 27, 1962.

W. A. BENJAMIN, INC.

2465 Broadway

New York 25, New York

1»

Prolog

ue

Nearly sixty years ago, Emil Fischer described his experi-
ments which led to the discovery of the structure of glucose
and related sugars. In the past fifteen years Melvin Calvin
and his associates have performed experiments leading to an
understanding of the reactions used by photosynthetic organ-
isms to make these sugars and many other compounds from
carbon dioxide, water, and minerals, using the energy of light.

It was not long after the basic reaction of photosynthesis
was recognized that speculation regarding its mechanism com-
menced. These discussions were carried forward first by Justus
von Liebig and then by Adolf von Baeyer and, finally, by
Richard Wilstatter and Arthur Stoll, into this century. How-
ever, it was the mechanism of the reverse pathway, that is,
the combustion of carbohydrate to carbon dioxide and water
with the utilization of the energy, which was first successfully
mapped. This pathway was elucidated primarily by Otto
Meyerhof and Hans Krebs.

Professor Calvin's interest in the basic process of solar
energy conversion by green plants began about 1935, when he
was studying with Professor Michael Polyani at Manchester,
There he became interested in the remarkable properties of

coordinated metal compounds, particularly metalloporphy-
rins, as represented by heme and chlorophyll. He began a
study on the electronic behavior of such compounds at that
time. When Professor Calvin joined the Chemistry Depart-
ment at Berkeley, these studies were encouraged by Professor
Gilbert N, Lewis, and they have been continued to the present
time. In time they will contribute to our understanding of the
precise way in which chlorophyll and its relatives accomplish
the primary quantum conversion into chemical potential,
which is then used to drive the carbohydrate synthesis.

It has long been known that the reduction of carbon di-
oxide to carbohydrate is probably a dark reaction, separate
from the primary quantum conversion act. This knowledge
stemmed from the early work of F. F. Blackman on the dark
reactions of photosynthesis and its interpretation by Otto
Warburg, and particularly from the comparative biochemical
studies of Cornelius van Niel. Finally, Robert Hill separated
the photo-induced production of molecular oxygen chemi-
cally and physically from the reduction of carbon dioxide
when he demonstrated oxygen evolution by illuminated
chloroplasts, using ferric ion as an oxidant in place of carbon
dioxide.

We can summarize the over-all conversion of light energy
into chemical energy in the form of carbohydrate and
oxygen by several steps. First, the light energy absorbed by
chlorophyll and related pigments is converted into the high
chemical potential energy of some compounds. Second, these
compounds react with water and produce oxygen and good
reducing agents as well as other cofactors containing high
chemical f)otential energy. Finally, these reducing and ener-
getic cofactors react with carbon dioxide and other inorganic
compounds to produce organic compounds.

One of the principal difficulties in studying the synthetic
pathway is that the machinery which converts carbon dioxide
and minerals to organic compounds is itself composed of
organic compounds made up of the same elements. Ordinary
analytical methods do not allow us to distinguish easily be-

VI

tween the machinery and its substrate. Fortunately, the dis-
covery of the long-lived isotopic carbons (carbon- 14) by
Samual Rubin and Martin Kamen in 1940 provided the
ideal tool for tracing these synthetic routes.

In 1945, carbon- 14, radiocarbon, became available in
large amounts as a product from nuclear reactors. With the
encouragement and support of Professor Ernest O. Lawrence,
the Director of the Radiation Laboratory in Berkeley, Pro-
fessor Calvin began to study the pathway of carbon reduction
during photosynthesis, using carbon- 14 as his principal tool.

Among a number of people who were to be associated
with him during the next few years of this work and who
would all contribute to the success of the research. Dr. Andrew
A. Benson was particularly instrumental, especially in the
identification of the early products of photosynthetic carbon
reduction. Key contributions to the development of the
carbon reduction cycle were made by Dr. Peter Massini and
Dr. Alex Wilson. Beginning as a graduate student with Pro-
fessor Calvin in 1947, I have had the pleasure of being asso-
ciated with him in this work to the present time.

The first big success came with Professor Calvin's identi-
fication of phosphoglyceric acid as the first stable product of
carbon reduction during photosynthesis. Soon thereafter the
application of two-dimensional paper chromatography com-
bined with radioautography became an invaluable analytical
tool for separating the minute amounts of radioactive ingre-
dients formed in the plant. Identification of the remaining
intermediates in the carbon reduction cycle soon followed,
and these turned out to be all sugar phosphates.

A combination of kinetic studies on the appearance of
carbon- 14 in these intermediates, with degradation of the
compounds that revealed the location of the radiocarbon in
individual atoms, soon led to a linking together of a reaction
sequence leading from phosphoglyceric acid through the
several sugar phosphates. The experiments of Massini and
Wilson helped to establish the carboxylation and reduction
reactions of photosynthesis, and the cycle was complete.

vn

In succeeding years much work has been done to check
the validity of the cycle, to investigate details of its mecha-
nism, and to establish its quantitative importance.

From almost the beginning of these studies we have been
interested in reactions leading from the cycle to various other
synthetic intermediates and end products, such as amino acids,
sucrose and polysaccharides, and carboxylic acids. As a result
of this work we have found that the photosynthetic machine,
the chloroplast, is an even more complex and diversified ap-
paratus than had been suspected. Not only does it manufac-
ture sugars and other carbohydrates, but apparently nearly all
other organic materials necessary for its continued growth as
well.

In this book we review the evidence leading to the formu-
lation of the carbon reduction cycle and discuss its quantita-
tive importance. We describe as far as possible the biosynthetic
pathways which we believe exist in the chloroplast. We show
how newly reduced carbon from the carbon reduction cycle
provides the starting material for these pathways. Our ob-
jective is to map complete synthetic sequences from carbon
dioxide to final products. Three papers, of fundamental im-
portance in the development of the theory regarding the path
of carbon in photosynthesis, are included as reprints.

We are now just at the threshold of discovery of many of
the biosynthetic pathways. There is good experimental evi-
dence for some and a few clues for others, but for many we
must speculate, relying on known, but nonphotosynthetic,
pathways. We have called on our experience of some fifteen
years' study of carbon fixation patterns during photosynthesis
to provide us with clues. The clues help us to predict which
reactions, which pathways, and which intermediates may be
considered to be likely participants in the photosynthesis of
carbon compounds.

This year Professor Calvin was awarded the Nobel Prize
for his work on the assimilation of carbon dioxide during
photosynthesis. Those who have worked with him and have
experienced the stimulation provided by his enthusiasm and

vni

insight are especially delighted by this most well-deserved
recognition of one of his many scientific achievements. Those
of us who, under his leadership, have contributed something
to the development of the carbon reduction cycle are particu-
larly pleased to have been a part^iDf this exciting work.

James A. Bassham

Berkeley, California
December 1961

IX

Acknowledgments

The publisher and the authors wish to acknowledge the
assistance of the following organizations in the preparation
of this volume:

The United States Atomic Energy Commission, which
sponsored the preparation of this volume.

Verlag-Birkhauser A.-G., Basel, for permission to reprint
the article from Experientia.

The American Chemical Society, Washington, D.C., for
permission to reprint the article from the Journal of the
American Chemical Society.

Elsevier Publishing Co., Inc., Amsterdam, for permission
to reprint the article from Biochimica et Biophysica Acta.

/uj/

1»

^^^ Contents

Preface v

Acknowledgments x

Introduction 3

Carbon reduction cycle of photosynthesis 8

Evidence for the carbon reduction cycle 12

The carboxylation reactions 21

Balance among synthetic pathways 25

Photosynthesis vs. other forms of biosynthesis 27

Amino acid synthesis 29

Carboxylic acids 37

Carbohydrates 49

Fats * 56

Pigments 60

Aromatic nuclei 65

Other biosynthetic products 67

References 69

XI

8094

i

Reprints

The Path of Carbon in Photosynthesis: XX. The
Steady State, by M. Calvin and P. Massini, Experi-
entia, VIII/12, 445-457 (1952) 79

The Path of Carbon in Photosynthesis: XXI. The
Cyclic Regeneration of Carbon Dioxide Acceptor,
by J. A. Bassham, A. A. Benson, Lorel D. Kay,
Anne Z. Harris, A. T. Wilson, and M. Calvin,
;. Am. Chem. Soc, 76, 1760-1770 (1954) 92

Dynamics of the Photosynthesis of Carbon Com-
pounds: I. Carboxylation Reactions, by J. A. Bass-
ham and Martha Kirk, Biochim. et Biophys. Acta,
43,447-464 (1960) 103

Index 121

xn

The photosynthesis
of carbon compounds

'^^ Introduction

Biosynthesis begins with photosynthesis. Green plants
and other photosynthetic organisms use the energy of ab-
sorbed visible light to make organic compounds from in-
organic compounds. These organic compounds are the
starting point for all other biosynthetic pathways.

The products of photosynthesis provide not only the
substrate material but also chemical energy for all subsequent
biosynthesis. For example, nonphotosynthetic organisms
making fats from sugars would first break down the sugars
to smaller organic molecules. Some of the smaller molecules
might be oxidized with O2 to CO2 and water. These reac-
tions are accompanied by a release of chemical energy, be-
cause O2 and sugar have a high chemical potential energy
toward conversion to CO2 and H2O. In a biochemical system
only part of this energy would be released as heat. The rest
would be used to bring about the conversion of certain
enzymic cofactors to their more energetic forms. These co-
factors would then enter into specific enzymic reactions in
such a way as to supply energy to drive reactions in the
direction of fat synthesis. Fats would be formed from the
small organic molecules resulting from the breakdown of

sugars. Thus sugar, a photosynthetic product, can supply
both the energy and the material for the biosynthesis of fats.

Photosynthetic organisms achieve energy storage through
their ability to convert electromagnetic energy to chemical
potential energy. The conversion begins when pigments
absorb light energy. The absorbed energy changes the elec-
tronic configuration of the pigment molecule (chlorophyll)
from its ground energy state to an excited state. The return
of the pigment molecule to its ground-state energy level is
accompanied by a (chemical) reaction that would not proceed
without energy input; i.e., the products of this reaction have
a smaller negative free energy of formation from their ele-
ments than do the reactants (in the same reaction). Thus some
of the light energy is converted to chemical potential.

The detailed mechanism of all these energy-conversion
steps is not known. However, the net result is often formu-
lated by two chemical equations. One of these is an oxida-
tion-reduction reaction resulting in the transfer of hydrogen
from water to triphosphopyridine nucleotide (TPN):

(1) HOH + TPN+ -^ iOa + TPNH + H+

AF' = +52.6 kcal*

The other reaction is the formation of an anhydride, adeno-
sine triphosphate (ATP), from the ions of two phosphoric
acids, adenosine diphosphate and orthophosphate:

(2) ADP3- + HP04= ^^ HOH + ATP^" + H +

AF' = +11 kcal*

In each of these reactions some of the light energy is stored
as chemical potential, as indicated by the positive quantities
for free energy change.

The structural formulas of these two cofactors are shown
in Figure 1. TPNH and its close relative DPNH (reduced
diphosphopyridine nucleotide) serve a double function in
photosynthesis and in all biosynthesis. Both TPNH and

* Assuming these concentrations: (TPNH) = (TPN + ),
(ATP*-) = (ADP-^-), (H + ) = 10-' M, (HP04=) = 10-=^ M.

H

HC
H

I

NHj

NHe

r

I

HC

I
HC-OH

I
HC-OH

I
HC

I

N

I

HCa,

HC — 0 — P-
H II

0

0"

I

-P-

II CH

^C-N^

I
HC

I
HC-OPOsH-

I
HC-OH

I
HC

I

NHe

I

N C — N,

I II

N I

HC-
I

CH

HC-OH

I
HC-OH

I
HC

-CH
H

I

0"

I

0'

I

Triphosphopyridine nucleotide (oxidized form)
(TPN+)

"-C-"

HC — O — P-0-P-O-P-OH
H II II II

0 0 0

Adenosine triphoiphote (ATP)

In Adenosine diphosphote (AOP),
terminal phosphate is replaced by -OH.

HC
II
HC>

II
,CH

N
I
R

'NHg

NIcotinomide portion of
TPNH (reduced TPN+)

Figure 1. Formulas of TPN and ATP.

DPNH are reducing agents and carriers of chemical poten-
tial, in other words, strong reducing agents. Thus, one of
their roles in biochemistry is analogous to that of H2 in syn-
thetic organic chemistry.

The function of ATP is to carry chemical potential and
to act as a powerful phosphorylating agent. In the reduction
of an acid to an aldehyde, important in photosynthesis, its
role may be compared to that of a mineral acid anhydride
in organic synthesis:

Organic synthesis:

Car- Acid

boxylic anhy-

acid dride

O

/
(3) R— C + iPCI,

OH

Acyl
deriv-
ative

Reduc-
ing
agent

0

H2

\

CI

catalyst

— > 4H3P03

Aldehyde

//

O

Acid

R— C -f HCl

H

Biosynthesis:

Carboxylic Acid Reducing

acid anhydride Acyl derivative agent

o o

/ /■ TPNH

(4) R— C + ADP— O— PO3H I > R— C >

\ \ enzyme

O- OPO3H-

> ADP

Aldehyde Acid

O

R— C + HOPO3H-

\

o-

Among the many other reactions of ATP in biosynthesis, one,
which is of considerable importance in photosynthesis, is the
formation of sugar phosphates from sugars:

(5) H+ + ROH + ADP— O— POgH" ->

R— OPO3H- + ADP + H2O

The only known reactions of the carbon reduction cycle in
photosynthesis which would require the use of TPNH and
ATP are of the type shown in Eqs. (4) and (5). These re-
actions are the means by which chemical potential, derived
from the absorbed light, is used to bring about the reduction
and transformation of carbon from CO2 to organic com-
pounds.

These two cofactors, ATP and TPNH, are at present
the only ones that are known to be generated by the light
reactions of photosynthesis and at the same time seem to be
required for steps in the carbon reduction cycle. The possi-
bility remains, however, that there are other energetic or
reduced cofactors acting as carriers of hydrogen and energy
from the light reactions to the carbon reduction cycle. Such
unknown cofactors might substitute for or replace TPNH
or ATP. They could, in fact, be more effective than the
known cofactors, particularly in vivo, where they might well
be built into the highly organized structure of the chloro-
plast. If such unknown cofactors do exist, they would have

to perform essentially the same functions as TPNH and ATP
and would presumably be about as effective as carriers of
chemical potential. In all discussions of the role of TPNH
and ATP, the possibility of their replacement by as-yet-
unidentified cofactors should be kept in mind.

For the purpose of discussion, let us consider the photo-
synthesis of carbon compounds as an isolated set of reactions.
The principal substrates for this set of reactions are CO2,
hydrogen (as TPNH), phosphate (as ATP), and NH4 + . The
ammonium ion may be contained in the plant nutrient or
it may be derived from the reduction of nitrate. If nitrate
reduction is the source of NH4 + , the energy for the reduc-
tion must also come from the light, at least indirectly. Other
probable inorganic substrates for photosynthesis of organic
compounds include sulfate, magnesium ion, and a number
of trace elements. Many of these are required for growth in
plants but may or may not be incorporated in organic com-
pounds by photosynthesis.

Carbon
^^ reduction cycle
"^i^r of photosynthesis

We believe the principal pathways for the photosyn-
thesis of simple organic compounds from CO2 to be those
shown in Figure 2 (1,2). The points at which ATP and
TPNH act in these pathways are indicated. Kinetic studies
(3) show that these pathways account for nearly all the car-
bon dioxide reduced during photosynthesis, at least in the
unicellular algae Chlorella pyrenoidosa. From other inves-
tigations (4) it appears that the general metabolic sequence
is the same in most respects for all photosynthetic organisms.
(We shall discuss the recently proposed role of glycolic acid
in CO2 reduction in the section on Carboxylic Acids.)

The central feature of carbon-compound metabolism
in photosynthesis is the carbon reduction cycle. Most of the
carbon dioxide used is incorporated via this cycle. Pathways
lead from intermediates in the cycle to various other impor-
tant metabolites. A few of these pathways are shown in Fig-
ure 2.

The initial step for carbon dioxide incorporation in
the cycle is the carboxylation of ribulose-l,5-diphosphate at
the number 2 carbon atom of the sugar to give a highly

labile ^-keto acid. Evidence for the existence of this unstable
intermediate has been adduced from in vivo studies (5) . It
has not been isolated in the in vitro reaction with the enzyme
carboxydismutase. The product of the reaction in vitro is 2
molecules of 3-phosphoglyceric acid (PGA). The products
in intact photosynthesizing cells may be 2 molecules of PGA
or, as kinetic studies indicate (3), 1 molecule of PGA and 1
molecule of triose phosphate.

Once formed, the PGA is transformed in two ways.
Some molecules are converted to products outside the cycle
while the remainder are reduced to 3-phosphoglyceraldehyde
via a reaction of the type shown in Eq. (4). The enzymes
responsiule for the two successive steps in the reduction are
probably similar to phosphoglycerylkinase (6) and triose
phosphate dehydrogenase (7-10).

The next phase of the carbon reduction cycle is the
conversion of 5 molecules of triose phosphate to 3 molecules
of pentose phosphate by a series of reactions. These reactions
include condensations (aldolase), carbon-chain-length dismu-
tations (transketolase), removal of phosphate groups (phos-
phatase), and interconversions of different pentose phos-
phates (isomerase, epimerase). Enzyme systems that catalyze
reactions similar to these steps are listed in Table 2. The
sequence of steps may be seen in the cycle diagram (Figure 2).

The various pentose phosphates are converted to ribu-
lose-5-phosphate. The final step is the formation of ribulose
diphosphate (RuDP) from ribulose-5-phosphate. This step
requires 1 molecule of ATP [Eq. (5)].

For every reaction in the cycle to occur at least once
(a complete turn of the cycle), the carboxylation reaction
must occur three times. The net result of each complete
turn of the cycle is the incorporation of 3 molecules of CO2
and the production of 1 three-carbon (or V2 six-carbon) or-
ganic molecule. Each complete turn of the cycle would re-
quire 6 molecules of TPNH or equivalent reducing cof actor
(2 per CO2) and 9 molecules of ATP, if each Ce carboxylation
product is split to 2 molecules of PGA and if all the PGA

©

00^09
0-0-0-0-0

X

is reduced to triose phosphate. If the carboxylation product
is reductively split (dashed line in Figure 2) the requirement
for TPNH would probably be the same, that is, 6 molecules
per complete turn of the cycle. In this case, however, the
cycle might require either 9 molecules of ATP or only 6.

Figure 2. Carbon reduction pathways in photosynthesis. Com-
pounds: (1) 2-carboxy-3-keto-l,5-diphosphoribitol, (2) 3-phospho-
glyceric acid (3-PGA), (3) glyceraldehyde-3-phosphate, (4) dihy-
droxyacetone phosphate, (5) fructose- 1,6-diphosphate, (6) ery-
throse-4-phosphate, (7) sedoheptulose-l,7-diphosphate, (8) xylu-
lose-5-phosphate, (9) ribose-5-phosphate, (10) ribuIose-5-phosphate,
(11) ribulose-l,5-diphosphate, (12) 2-phosphoglyceric acid (2-
PGA), (13) phosphoenolpyruvic acid (PEPA), (14) oxalacetic acid.
_@: fructose diphosphate and sedoheptulose diphosphate lose
one phosphate group before transketolase reaction occurs.

11

Evidence
^<f for the carbon
'T^ reduction cycle

The carbon reduction cycle in essentially the form
shown in Figure 2 was mapped during the period between
1946 and 1953 (11-17). The experiments, results, and inter-
pretations leading to its formulation have been extensively
discussed elsewhere (2). They will be briefly reviewed here,
not necessarily in chronological order.

The carbon that enters the plants' metabolism has been
followed through the various intermediate compounds by
labeling the carbon dioxide with radiocarbon, C^*. The
analysis of the labeled compounds has been carried out by
paper chromatography and radioautography. The interpre-
tation of results leading to the cycle formulation has been
based on the kinetics of the appearance of C^* in various
identified compounds as a function of time of photosynthesis
with C^*02 and other variables.

The methods are best described by an illustration. Con-
sider a simple experiment with a suspension of the algae
Chlorella pyrenoidosa, very extensively used in these studies.
These green unicellular plants, suspended in water contain-
ing the necessary inorganic ions (nitrate, phosphate, etc.)
and aerated with a stream of C^-Oo (ordinary carbon dioxide),

12

photosynthesize at a rapid rate if illuminated from each side
in a thin transparent vessel. The CO2 is continually taken
up from the solution (where it is in equilibrium with bicar-
bonate ion) and converted by the photosynthetic plant
through a series of biochemical intermediates to various
organic products.

A solution of radioactive bicarbonate, HC^'^Oa-, is sud-
denly introduced into the algae suspension. The plant does
not distinguish in any important way between the C^^ ^nd
C^*, which are chemically almost identical. Immediately some
of the C^* is incorporated into the first of the biochemical
intermediate compounds. As time passes the C^* gets into
subsequent intermediates in the chain. After a few seconds
exposure to the C^^02, the suspension of algae is run into
methanol to a final concentration of 80 per cent methanol.
This treatment denatures all the enzyme instantly and freezes
the pattern of C" labeling by preventing further change.
Now the dead plant material is analyzed for radioactive com-
pounds to see which are the first stable products of carbon
reduction during photosynthesis.

The first step in this analysis is to prepare an extract
of the soluble compounds. The early products of carbon
reduction have been found to be simple soluble molecules.
This extract is then concentrated and analyzed by the method
of two-dimensional paper chromatography (12). The impor-
tance of the method for these studies stems from the fact that
it permits the analysis of a few micrograms or less of dozens
of different substances in a single simple operation.

Of these many compounds, those into which the plant
incorporates C^^ during its few seconds of photosynthesis
with HC^'^Os- are radioactive and omit the particles result-
ing from radioactive decay of the C^". In this case these are
13 particles, and these may be detected by the fact that they
expose x-ray film. Thus, if a sheet of x-ray film is placed in
contact with the two-dimensional paper chromatogram, sub-
sequent development of the film will show a black spot on
the film corresponding to the exact shape and location of

13

each radioactive compound on the paper. A quantitative
determination of the amount of radiocarbon in each com-
pound may then be made by placing a Geiger-Miiller tube
with a very thin window over the radioactive compound on
the paper and counting electronically the emitted ^ particles.

The next stage in the method of radiochromatographic
analysis is the identification of the radioactive compounds.
This identification is accomplished in a variety of ways.
When a familiar set of chromatographic solvents has been
used, the position of an unknown compound compared to
the positions of known substances provides a clue to its iden-
tity. The next step may be elution or washing of the com-
pound off the paper and the determination of such chemical
and physical properties (e.g., the distribution coefficient) of
the substance as can be measured with a solution of a few
micrograms or less of the material. These properties are then
compared with those of known compounds. The final check
on the identity of the compound is frequently made by plac-
ing on the same spot on filter paper the radioactive com-
pound and 10 to 100 /xg of the pure nonradioactive substance
with which the radioactive compound is thought to be iden-
tical. The new chromatogram is then developed. A radio-
autograph is prepared to locate the radioactive substance,
after which the paper is sprayed with a chemical spray (for
example, ninhydrin for amino acids), which produces a color
where the carrier compound is located on the paper. Super-
position of the paper chromatogram and the radioautograph
(x-ray film) will show an exact coincidence between chem-
ically developed color on the paper and the black spot on
the film, provided the two substances are identical.

Once the identity of the radioactive compounds formed
during a short period of photosynthesis had been established,
experiments were performed under a variety of conditions
and times of exposure of the algae to radiocarbon.

The radioautogram from the experiment with Chlorella
described above is shown in Figure 3. Even after only 10
seconds of exposure to C^^, a dozen or more compounds are

14

found. Some of these (the sugar phosphates) are not sepa-
rated from each other by the first chromatography and must
be subjected to further analysis. When the sugar mono-
phosphates are hydrolyzed to remove the phosphate groups
and rechromatographed, separate spots are found of triose
(dihydroxyacetone), tetrose, pentoses (ribulose, xylulose, and
ribose), hexoses (glucose and fructose), and heptose (sedo-
heptulose). The radioactive sugar diphosphates area gives
free ribulose, fructose, glucose, and sedoheptulose.

After periods of photosynthesis with C^* of less than 5
seconds, 3-phosphoglyceric acid (PGA) was found to be the
predominant radioactive product. Chemical degradation of
this compound showed that the radioactivity first appears in
the carboxyl carbon (14). Later kinetic studies showed that
the rate of incorporation of C^^ into PGA at very short
times was much greater than the rate of labeling of any
other compound (18,1). Therefore, it was concluded that
PGA is the first stable product of carbon dioxide fixation
during photosynthesis, and, furthermore, that carbon dioxide
first enters the carboxyl group of PGA, presumably via a
carboxylation reaction.

Further reactions in the photosynthetic sequence were

^^^ MALIC ACIO
ALANINE

ASPARTIC ACID

TRIOSE PHOSPHATE ^^V PGA

10 SEC PHOTOSYNTHESIS *ITH C'«0, ^^^ PHOSPHATES

CHLO/KLLA g^

SUGAR DIPHOSPHATES

Figure 3. Radioautograph of two-dimensional paper chromato-
gram. Alcoholic extract of Chlorella pyrenoidosa after 10 seconds
photosynthesis with Ci'*02.

15

suggested by the already known pathways of the glycolytic
breakdown of sugars, which lead to PGA as an intermediate.
Since the sugar phosphates are important early products of
carbon reduction in photosynthesis, it was proposed that
they are formed from PGA by a reversal of the glycolytic
pathway. Degradation of the radioactive hexoses from short
experiments showed that they were labeled in the two center
carbon atoms (numbers 3 and 4) just as one would expect if
2 molecules of carboxyl-labeled PGA were first reduced to
triose and then linked together by the two labeled carbon
atoms to give hexose (Figure 4).

The hexose and triose phosphates may be converted by
aldolase or transaldolase and transketolase enzymes to pen-
tose and heptose phosphates (Figure 2 and Table 2). Deg-
radation of these sugars and comparison of the labeling pat-
terns within the molecules showed that this conversion did
occur, and in such a way that 5 molecules of triose phos-

Heptose
and pentose
phosphates

Hexose phosphotes

Triose phosphate

I

-C-

I
-CH

I

Ao,

CHs

I
HC-NHg

I
*COOH

Alanine

TPNH
ATP

HgC-OPOsH-

HC-OH

I
*COOH

3- PGA

2- PGA

CHg
II

C-OPO3H-
1
-«COOH

PEPA

Light

*C02

c

I

c

I

*c

I

»c
c

I

c

•COOH

I

CHt

I
HC-NHa

I
♦COOH

t .

♦COOH

I

CHe

I

C = 0

I
*COOH

Asportic
acid

DPNH

»COOH

I

CHg

I
HC-OH

I
♦COOH

Malic ocid

14

Figure 4. Labeling of compounds with C
during early steps in carbon dioxide reduction
during photosynthesis with C^*02-

16

phate were ultimately converted to 3 molecules of pentose
phosphate.

Other known metabolic pathways leading from PGA
(Figure 4) give rise first to phosphoenolpyruvic acid (PEP A),
which then may undergo further transformations, including
the following: (1) it may be carboxylated and transaminated
to give aspartic acid, (2) it may be carboxylated and reduced
to give malic acid, or (3) it may be dephosphorylated and
transaminated to give alanine. All these compounds are
labeled after short exposures of the algae to HC"0.s~ in the
light.

The enzyme system of plants, which during respiration
brings about the oxidation of triose phosphate to PGA in
the glycolytic pathway, was known to produce ATP and
TPNH (or DPNH). If PGA is to be reduced to triose phos-
phate during photosynthesis, it follows that ATP and TPNH
must be supplied. We have already seen that these two co-
factors, and possibly others, are produced as a consequence
of the light reaction and the splitting of water. It might be
expected that, if the light were turned off from plants photo-
synthesizing in ordinary carbon dioxide at precisely the same
time that C^^02 is introduced, PGA would no longer be
reduced to sugar phosphates but would still be formed (if
no light-produced cofactors are required for the carboxyla-
tion reaction). Moreover, the PGA would still be used in
other reactions not requiring these cofactors. In Figure 5,
the radioautograph from just such an experiment, this pre-
diction proves to be correct. Labeled PGA is still formed
by the algae from C^^02 during 20 seconds in the dark, but
only a very little of the PGA is reduced to sugar phosphates.
At the same time, a large amount of alanine is formed from
PGA via PEPA in reactions that do not require ATP.
The trace of labeled sugar phosphates that does appear may
be due to the residual ATP, or some unknown cofactor,
which was formed while the light was on but which had
not yet been used up when the C^^02 was introduced. The
formation of malic acid and of alanine and aspartic acid

17

in the dark indicates the presence of some reducing cofactors,
either remaining from the light or derived from some other
metabolic reaction.

Before we discuss the evidence for the remainder of the
carbon reduction cycle, we must describe another type of
experiment with C^^Oa and photosynthesizing algae. In these
experiments, algae are first permitted to photosynthesize for
20 minutes or more in the presence of a constant supply
of C^*02. During this time environmental conditions are
maintained nearly constant (temperature, CO2 pressure,
light intensity, etc.). After about 10 minutes of exposure to
C^*02, so much radiocarbon has passed through the various
biochemical intermediate compounds on its way to end
products that each carbon atom of each intermediate com-
pound contains, on the average, the same percentage of C^^
atoms as the CO2 being absorbed. In other words, the specific
radioactivities of all the carbon atoms of all the early inter-
mediates are the same as the specific radioactivity of the
entering radiocarbon, which can be measured.

At this point samples of the algae are removed without
disturbing the rest of the algae, and these samples are killed
and subsequently analyzed by the methods described. The

MALIC ACID

ALANINE

SERINE ASfWlTIC AQD

PEPA

i

20 SEC DARK C'^Oj FIXATION ^0 ^''^

AFTER PREILLUMINATION
CHLOiELLA SUGAR PHOSPHATE^

Figure 5. Radioautograph of chromatogram of products of
20 seconds C^^Oa fixation by Chlorella pyrenoidosa in the dark
following a period of photosynthesis.

18

total radioactivity of each intermediate is measured, and,
when this is divided by the known specific radioactivity of
the entering CO2, the total number of carbon atoms of each
intermediate compound in the sample can be calculated.
Thus the number of moles per unit volume of algae of the
various intermediates of the actively photosynthesizing sys-
tem may be determined. This number of moles per unit
volume of plant material is an average concentration, since
the distribution of molecules in such a heterogeneous system
is not homogeneous.

This determination of the concentrations of intermedi-
ates in vivo is an extremely valuable tool which has many
uses, but let us proceed for the moment with one particular
application. Having taken a sample of algae for later de-
termination of the concentrations of compounds, the experi-
menter turns off the light and proceeds to take a series of
samples of the algae as rapidly as possible, which is about
every 3 seconds. When the concentrations of compounds in
these samples are determined, any changes resulting from
turning off the light will be revealed. The two most striking
changes are found to be in the concentration of PGA, which
increases rapidly, and in the concentration of one particular
compound, ribulose diphosphate, which drops rapidly to
zero (16,20).

The increase in PGA on turning off the light is expected.
The cofactors, derived from the light reaction, are necessary
for the reduction of PGA. The rapid drop in ribulose diphos-
phate, taken together with the fact that other sugar phos-
phates initially do not drop rapidly in concentration, indi-
cates that the formation of ribulose diphosphate from other
sugar phosphates requires a light-formed cofactor. This con-
clusion agrees with the fact that the known enzyme, which
converts ribulose-5-phosphate to ribulose- 1,5-diphosphate
(RuDP), requires ATP (Table 2). The drop in ribulose di-
phosphate, alone among the sugar phosphates, means that it
is being used up by a reaction that does not require light.

Ribulose diphosphate, then, is used up by some reaction

19

that proceeds in the dark, and PGA continues to be formed
in the dark. Could the carboxylation of ribulose diphosphate
to form PGA be the first step in carbon dioxide reduction?
To answer this question, an experiment similar to the one
just described was performed. This time, however, instead
of turning off the light, the light was left on, and carbon
dioxide was suddenly removed (19). The result of this ex-
periment was that the concentration of ribulose diphosphate
now rose rapidly while PGA dropped rapidly. Thus the car-
boxylation of RuDP to give PGA was confirmed.

20

The
^^ carboxylation
'^^ reactions

Thus far we have mentioned two carboxylation reactions
in photosynthesis: carboxylation of RuDP (the carbon re-
duction cycle) and carboxylation of PEPA. When algae
have been allowed to photosynthesize for less than a minute,
virtually all the radioactivity found on the chromatogram
prepared from the algae is located in compounds apparently
derived from these two reactions. There still remained the
possibility that other carboxylation reactions might occur
which would involve intermediate compounds too unstable
or too volatile to be seen on the chromatograms. These pos-
sibilities were tested by making a quantitative comparison
between the rate of uptake of C^^02 from the medium and
the rate of appearance of C^* in compounds on the chromato-
grams (3).

For these experiments, the algae were kept, as close as
possible, in steady-state growth in the experimental vessel.
Light, temperature, pH, and supply of inorganic nutrients
were kept constant. Gas was circulated through the algae
suspension in a closed system by means of a pump. Levels of
CO2, O2, and, when present, C^^Oo, were continuously
measured and recorded. From the known gas volumes of the

21

system and the recorded rates of changes in gas tensions,
we calculated the total change in these gases as a function
of time. Then we added 0^*02 to the system and took sam-
ples of algae every few seconds for the first few minutes and
then less frequently up to an hour. Each sample of algae was
killed immediately and a portion analyzed as described
earlier. A part of each sample was reserved and was dried on
a planchet to determine the rate of appearance of C^* in all
stable nonvolatile compounds. This rate proved to be the
same as the externally measured rate of uptake of CO2 and
C^^ between 20 and 60 seconds after the addition of C^^. If
unstable or volatile intermediates do precede these stable
compounds, they are equivalent in micromoles of carbon
to no more than 5 seconds photosynthetic fixation, according
to the shape of the fixation curve during the first 20 seconds.
We analyzed each sample by paper chromatography
and determined the radioactivity in each compound in each
sample. On the basis of the externally measured uptake rates,
at least 85 per cent of the carbon was found to be incorpo-
rated into individual compounds on the paper chromato-
grams during the first 40 seconds. At least 70 per cent of the
total carbon uptake rate could be accounted for by the ap-
pearance of C^** in compounds apparently derived from the
RuDP carboxylation reaction of the carbon reduction cycle
via the pathways shown in Figure 2. Another 5 per cent or
more was found to be incorporated via C1-C3 carboxylation.
About 5 per cent was found in unidentified compounds or
in glutamic acid, whose photosynthetic pathway is not defi-
nitely known. Of the 15 per cent not accounted for, some
may be in nonextractable polysaccharides, whose sugar phos-
phate precursors become labeled very quickly. More of the
unaccounted-for radiocarbon is undoubtedly in a large num-
ber of unmeasured compounds on the chromatograms. Each
of these compounds contains by itself too little C^"* to be
readily determined. In any event, it is clear that the known
fixation pathways are the only quantitatively important

22

ones unless there are unknown pathways utilizing the same
intermediate compounds.

A kinetic analysis of the appearance of C^'' in PGA
and RuDP in this experiment indicated that the carboxy-
lation reaction results in the formation of only one free mole-
cule of PGA per molecule of CO2 entering the cycle. The
kinetic analysis cannot say what the other three-carbon
fragment would be. It might be merely a molecule of PGA
bound in some way so that its labeling remains distinct from
that of the PGA from the other half of the six-carbon addi-
tion product. The only other compounds that seem to satisfy
the kinetic requirements and that could readily result from
the splitting of the six-carbon addition product are the triose
phosphates. The formation of a molecule of triose phosphate
in this way would require a reductive split of the addition
product, as indicated by the dashed line in Figure 2.

That such a pathway differing from the in vitro reaction
may exist seems entirely reasonable, since the enzymes of the
carbon reduction cycle appear to be closely associated with
the molecular structures in which the TPNH is formed in
the chloroplast (21). In the intact plant the carboxylation
enzyme, as well as the enzyme responsible for the splitting of
the product and the enzyme that brings about the reduction
of TPN+ to TPNH, might be part of a structurally organ-
ized system. In fact, if a reductive scission does occur, the
reducing agent could be a substance formed from the oxida-
tion of water and preceding TPNH in the electron transport
chain. This substance might never be available in sufficient
concentration to be a factor in in vitro systems in which
carboxydismutase is coupled with isolated or broken chloro-
plasts. Such an explanation of the experimentally observed
kinetic result is purely hypothetical. We mention it to focus
attention on the possibility that a given biosynthetic pathway
may follow a different course in an intact cell than that which
would be predicted on the basis of studies with fragmented
cells or enzymes alone.

23

In higher plants much of the product of photosynthesis
must be transported to a nonphotosynthetic part of the plant.
This requires that higher proportions of easily transported
molecules such as sucrose are formed (4). In all higher plants
that have been studied, however, there is appreciable direct
photosynthesis of amino acids and fats, not just carbohydrates.

24

^g^ Balance among
"^!^ synthetic pathways

We have seen that in each complete turn of the carbon-
reduction cycle 3 molecules of RuDP (15 carbon atoms) are
carboxylated by 3 molecules of CO2 to give 6 three-carbon
compounds (18 carbon atoms). Thus there is a net gain of 3
reduced carbon atoms. These atoms are withdrawn from the
cycle for further synthesis. They may be withdrawn from
the cycle as PGA or as any of the sugar phosphates in the
cycle. Before the photosynthetic reactions had been mapped,
it was commonly believed that photosynthesis leads first to
carbohydrates only and that these carbohydrates are then
converted via nonphotosynthetic reactions to other com-
pounds such as amino acids and fatty acids. We now know
that pathways leading from the carbon reduction cycle to
amino acids and fatty acids and other substances can be just
as important quantitatively as those leading to carbohydrates.
This is particularly true in a unicellular algae, as exempli-
fied by Chlorella pyrenoidosa, where under some conditions
less than half of the assimilated carbon is directly converted
into carbohydrate. This carbohydrate synthesis draws its
carbon from the cycle in the form of sugar phosphates. Con-
sequently, more than half of the carbon drained from the

25

carbon reduction cycle as PGA or sugar phosphates may be
used in fat and protein synthesis.

It is interesting to consider an extreme case in which
all the carbon assimilated by the carbon reduction cycle
would be withdrawn from the cycle as PGA, converted to
PEPA, and then carboxylated to give four-carbon compounds.
In this case, 75 per cent of the assimilated carbon would
enter the photosynthetic pathways via the carbon reduction
cycle, while the remaining 25 per cent would enter via the
carboxylation of PEPA.

With normal conditions of steady-state growth under
high light intensity, the ratios of various fixation pathways
must be determined to a large extent by the requirements
of the plant for the small molecules from which the protein,
carbohydrate, fat, and other substances of the plant are syn-
thesized.

26

Photosynthesis
^<p vs. other forms
'^^ of biosynthesis

Biosyntheic reactions in plants cannot be classified as
photosynthetic or nonphotosynthetic on the basis of direct
photochemical action because all reactions in the synthetic
pathways are probably "dark" reactions. However, we can
make a classification on the basis of the immediate source of
the required cofactors. The conversion of light energy results
in the formation of ATP and TPNH and perhaps other un-
known cofactors. When these cofactors are formed by the
light reaction and are used to bring about the synthesis of
carbon compounds, we may consider the reactions to be
photosynthetic. Also included in this category would be
preliminary and intermediate steps such as hydrations, con-
densations, and carboxylations.

It may well be that all photosynthetic reactions, as just
defined, occur in the chloroplasts while the light is on.
If this is true, reactions outside the chloroplast would derive
their energy from substrate carbon compounds which diffuse
from the chloroplast to the extrachloroplastic spaces of the
cell. Such an interpretation is suggested by the report by
Tolbert (22), who found that chloroplasts isolated from Swiss
chard, when allowed to photosynthesize with HC^^Oa", ex-

27

creted mainly glycolic acid into the medium. Phosphate
esters, of importance to the carbon reduction cycle, were
retained in the chloroplasts. Isolated chloroplasts have a
carbon metabolism that is much more limited than photo-
synthesis in intact cells. This is probably due to loss of
enzymic activity by chloroplasts during the isolation process.
In all probability the carbon compounds excreted by intact
chloroplasts in vivo include substances other than glycolate.
There is more than a semantic reason for making a dis-
tinction between photosynthetic and nonphotosynthetic
pathways. The environment of the photosynthetic metab-
olism is unique. There is an abundance of the reduced and
energetic form of the coenzymes. Hence synthetic pathways
do not require energy derived from degradative reactions
such as decarboxylations and oxidations. For example, a
well-known biosynthetic pathway leading to glutamic acid
from acetate includes oxidative and decarboxylation steps.
Such a pathway is to be expected in a nonphotosynthetic
system, where degradation of part of the substrate is the
only means of obtaining the energy and reducing power for
synthetic reactions. In a photosynthetic system one might
expect instead a pathway involving only condensations, re
ductions, and carboxylations. We cannot say that this differ-
ence in type of reaction will always be borne out by the
actual mechanisms when they are known. This proposed dif-
ference in reaction type may be a useful working hypothesis
to those who attempt to map photosynthetic pathways from
experimental data.

28

WP

Amino acid

"^^ synthesis

Among the first compounds found to be labeled by pho-
tosynthesis of C^*02 in algae were alanine, aspartic acid, and
several other amino acids (11). These compounds were
slowly labeled even in the dark when algae were exposed
to C^^02. They and malic acid were much more rapidly
labeled if the algae were photosynthesizing, or had been
photosynthesizing, just prior to the moment of addition of
C^^02. We recognized that these amino acids were therefore
products of photosynthetic reduction of CO2, even though
they could also become labeled by reversible respiratory re-
actions. Accelerated incorporation of C^^ into amino acids
in higher plants during photosynthesis has been noted in
this laboratory (23,24) and in many others (25-28). Nichi-
porovich (25) has presented and reviewed evidence that syn-
thesis of proteins in the chloroplasts of higher plants is
greatly accelerated during photosynthesis. This accelerated
protein synthesis appears to occur directly from the inter-
mediates of photosynthetic carbon reduction, since the pro-
teins were labeled when C^^02 was used but not when C^'*-
labeled carbohydrate was administered. Photosynthetically
accelerated synthesis of protein containing N^^ was also ob-

29

served when N^^H4+ was administered. Sissakian (29) has
reviewed evidence that protein can be synthesized in isolated
chloroplasts from nonprotein nitrogen, including peptides.

In experiments in this laboratory (30) it recently has
been possible to measure the proportion of the total carbon
fixed by Chlorella pyrenoidosa, which is directly incorporated
into certain key amino acids. These experiments show that,
during steady-state photosynthesis in bright light with an
adequate supply of inorganic nutrients, the synthesis of these
amino acids can account for 60 per cent of all the carbon
fixed by the algae and 30 per cent of the uptake of NH4+,
which is also measured. If the light is turned off, the NH4 +
uptake and C^* fixation into amino acids are both accelerated
for about 10 minutes and then drop to a very small fraction
of the rates in the light. Finally, these experiments indicate
clearly that in Chlorella pyrenoidosa there are at least two
pools of alanine, glutamic acid, aspartic acid, and serine, and
probably other amino acids as well. One of these pools,
especially in the cases of alanine and aspartic acid, is labeled
extremely rapidly after the introduction of C^*02 to the
algae. So rapidly are these compounds labeled, in fact, that
the site of their synthesis must be freely accessible to their
photosynthetically formed precursors, namely, phosphoenol-
pyruvic acid and PGA (see Figure 1). The studies of Tolbert
(22) and Moses et al. (31) indicate that the photosynthetic
pools are isolated from the extrachloroplastic region. We
conclude, therefore, that in Chlorella the more rapidly
labeled pools of amino acids are located at the site of photo-
synthetic carbon reduction, probably in the chloroplast.

The rates of flow of carbon through these pools of amino
acids as determined from kinetic labeling data with Chlorella
in a typical experiment are shown in Table 1.

The amino acids shown in Table 1 are those most prom-
inently labeled with C^^ during a few minutes of photosyn-
thesis. In addition, a nuinber of other amino acids become
labeled as time passes. The rates of labeling seem to indicate
that the carbon skeletons of these other acids are probably

30

Table 1
Rates of Flow of Carbon through Active Pools of Amino Acids

Calculated rate Equiv. NH4"'"
of synthesis R, uptake, Mmoles
Compound jumoles of carbon of NH4"'"

Alanine

2.67

0.89

Serine

0.49

0.16

Aspartic acid

0.89

0.22

Glutamic acid

0.98

0.20

Glutamine

0.32

0.13

Glycine^

0.04

0.02

Citrulline''

0.09

0.09

Threonine*

0.20

0.05

Total

5.44

1.69

Externally measured

uptake

17.0

2.6

% of total through these pools

32

65

* Not included in totals.

b

Figures are for carbamyl carbon only.

derived, for the most part, from the listed amino acids. How-
ever, the aromatic rings of the amino acids are synthesized by
another pathway.

In Table 1 we compare the rates of synthesis of carbon
skeletons that have been measured with the rate of uptake
of NH4 + . The rate of synthesis of any given amino acid
does not necessarily represent the rate of incorporation of
inorganic nitrogen into that amino acid, since it could be
formed by transamination from another amino acid. How-
ever, the total of the rates of synthesis of all "primary" amino
acids should account for the major fraction of the rate of
uptake of ammonia. By "primary" amino acids we mean
those amino acids whose carbon skeletons are not synthesized
from some other amino acid. Alanine, serine, and aspartic
acid are clearly primary amino acids, since their rates of
labeling reach a maximum as soon as the intermediates in

31

the carbon reduction cycle are saturated (about 3 minutes in
this experiment) and long before they themselves, or any
other amino acids, are saturated with radiocarbon (30). Prob-
ably glutamic acid is a primary amino acid also, but kinetic
data alone cannot prove this at the moment. Glutamine is
generally supposed to arise from glutamic acid, but there is
some evidence to indicate that it may arise as a primary
amino acid amide (32,30).

In any event, the rates of synthesis of alanine, serine, and
aspartic acid in reservoirs we believe to be closely associated
with the chloroplasts in Chlorella are great enough to permit
the following conclusions.

1 . An appreciable fraction of the carbon assimilated dur-
ing photosynthesis in Chlorella is used directly in the syn-
thesis of amino acids without the intermediacy of sugars or
any other class of compounds except acid phosphates and
carboxylic acids.

2. Since this amino acid synthesis accounts for a major
portion of the inorganic nitrogen uptake, these amino acids
must be used to a large extent in protein synthesis. However,
some important amino acids (i.e., glycine) are so slowly
labeled that they probably do not supply a major part of
the carbon for protein synthesis. Instead, the carbon skeletons
corresponding to these amino acids must be incorporated into
protein in some form other than as the free amino acid.

Before considering synthetic routes to specific amino
acids, we wish to reiterate our belief that photosynthetic re-
actions need not follow the same course as the better-known
synthetic reactions of other nonphotosynthetic organisms.
Also note that few if any enzymes involved in amino acid
synthesis have ever been isolated from chloroplasts. Thus
we are forced to suggest new and untested hypothetical paths.
Our guiding principles will be that chemical potential should
be used to drive the reactions rapidly in the forward direction
and that loss of carbon or reduction level should be avoided
wherever possible.

32

In Figure 6 are shown hypothetical pathways leading
from PGA to alanine, serine, aspartic acid, and malic acid.
These pathways differ somewhat from known enzymatic
pathways, in that, in each step leading to the amino acid,
ammonia reacts with a phosphoric acid ester.

The rapid incorporation of inorganic nitrogen into
organic compounds would be brought about by the large
negative free-energy change associated with each of these re-
actions. Thus these reactions, and not the reductive amina-
tion of ketoglutaric acid alone, would account for a major
portion of ammonia incorporation during photosynthesis.

COg

Reduction Cycle

H2C-0(P)

HC-OH
I
CO2"

PGA

H2C-OH H,C-OH

I I

HC-0® '^"^ HC-NH2 + HO (?)

CO2" CO2"

2-PGA

SERINE

-HOH

H2C

H2CH

" r^ NH4+ I ^

C-0(P) ». HC-NH2 + HO (P)

TPNH

C02'

CO2

HO(P)i

CO2"

HCH

I
HC-OH

I
CO2"

MALIC ACID

HOH

TPNH

C02~

I
HC

c-o(p)

COf

HHi

CO2-

ALANINE

I
HCH

TPNH HC-NH2+H0(£)

I
CO2-

ASPARTIC ACID

Figure 6. Hypothetical pathways of photosynthesis of alanine,
aspartic acid, serine, and malic acid.

33

This seems entirely reasonable when one considers that PGA
is both the immediate precursor in these reactions and the
primary product of carbon reduction during photosynthesis.
These amino acids could then supply ammonia via transami-
nase reactions for the synthesis of many other amino acids.
Holm-Hansen et al. (33) have demonstrated the presence of
a transaminase activity in spinach chloroplasts, which is very
effective in the transfer of amino groups from unlabeled ala-
nine to C^^-labeled pyruvic acid.

The three-carbon precursors to these amino acids are in
rapid equilibrium with PGA. PEPA becomes C^^-saturated
during photosynthesis in C^^02 in Chlorella almost as soon
as PGA itself. The proposed phosphoenoloxalacetate prob-
ably does not exist except in enzyme complexes. Thus, by
the time the PGA is C^*-saturated, these amino acids are
being labeled as rapidly as if they were formed directly from

It has been suggested that glutamic acid is formed dur-
ing photosynthesis by a carboxylation of y-aminobutyric acid
(34). Judging by our studies with Chlorella pyrenoidosa dur-
ing steady-state photosynthesis with C^''02, this reaction ap-
parently does not constitute a source of glutamate, since y-
aminobutyric acid does not become labeled, even by the time
the glutamic acid is 50 per cent saturated with C^* and long
after the rate of labeling of glutamic acid has passed its maxi-
mum. Clearly, a compound cannot be a precursor in a steady-
state system unless it is itself continuously regenerated. If
the reaction does occur at all, the glutamic acid so formed
could only be a shuttle for CO2, regenerating unlabeled y-
aminobutyric acid. Even so, such a carboxylation reaction
does not account for more than about I per cent of the car-
bon fixed in our studies of steady-state CO2 fixation by Chlo-
rella.

One possible route from PGA to glutamic acid would
begin with conversion of PGA to PEPA, followed by car-
boxylation of PEPA to give oxalacetic acid. Condensation
of oxalacetic acid with acetyl CoA would give citric acid,

34

thence aconitic acid, thence isocitric acid. Proceeding along
the Kreb's cycle, the next two steps are oxidation to oxalo-
succinic acid, followed by oxidation and decarboxylation
to give a-ketoglutaric acid. Finally, the reductive amination
would give glutamic acid. This pathway may be followed in
Chlorella pyrenoidosa in the synthesis of glutamic acid, par-
ticularly when the light is turned off. We suspect that it is
not the principal pathway during photosynthesis for two rea-
sons, one experimental and one theoretical. Experimentally,
the rates of labeling of the intermediate compounds such as
citric acid and ketoglutaric acid are too slow to permit them
to serve as precursors to the more rapidly labeled reservoir
of glutamic acid. Theoretically, the pathway is objectionable
to us as a photosynthetic route because it involves two oxida-
tions and a decarboxylation.

How else might glutamic acid be formed during photo-
synthesis? The availability of three-carbon and two-carbon
compounds suggests the possibility of a simple condensation.
Barker and co-workers (35-37) found an enzymic pathway in
certain microorganisms leading from glutamic acid to py-
ruvic acid and acetate via citramalate, mesaconic acid, and
/3-methylaspartate. The reverse of this pathway might operate
during photosynthesis also. However, we have been unable
so far to find significant amounts of radiocarbon in either /?-
methylaspartic acid or mesaconic acid in Chlorella which
were synthesizing glutamic acid from C^'*02. Moreover, a gen-
eral energy-conserving principle would suggest that PEPA
and not free pyruvic acid should be the three-carbon com-
pound that combines with the two-carbon fragment. As we
shall see in the discussion for the synthesis of aromatic rings,
it has been proposed that PEPA can condense with an alde-
hyde, erythrose phosphate, to give (eventually) phosphoshi-
kimic acid (38). Perhaps a similar reaction between PEPA
and glyoxylic acid could lead to a product such as y-hydroxy-
glutamic acid, which could be subsequently converted to
glutamic acid. Dekker (39) has reported the presence of an
enzyme in rat liver that converts y-hydroxyglutamic acid to

35

glyoxylate and another product, which may be alanine. The
presence of y-hydroxyglutamic acid in green leaves has been
reported by Virtanen and Hietala (40). The dehydration and
reduction of y-hydroxyglutamic acid to give glutamic acid
would be common types of biochemical reactions, analogous
to the formation of succinic acid from malic acid. However,
we have at present no experimental evidence for such a path-
way.

Threonine does not become labeled as rapidly as the
amino acids so far discussed, and it may well be secondary in
origin. That is, it may be an example of conversion of pri-
mary amino acids (aspartic acid, alanine, serine, and glutamic
acid) to other amino acids of their respective families, a proc-
ess that presumably occurs in photosynthesis.

The small amount of labeled glycine formed during
steady-state photosynthesis may come from either serine or
glyoxylic acid.

36

'^f' Carboxylic acids

Malic and fumaric acids

Malic acid and fumaric acid are rapidly labeled during
steady-state photosynthesis with €^^^02. These acids are
probably formed by reduction of the product of carboxyla-
tion of PEPA. In the steady-state experiment that yielded the
results shown in Table 1, about 5 per cent of the C^^ uptake
rate could be accounted for in the labeling of these two
acids. In that experiment very little of the radioactivity finds
its way into succinic acid. It would thus appear that, if malic
and fumaric acids are labeled by reductive carboxylation of
PEPA, either (1) the reaction sequence is highly reversible,
leading to exchange labeling, or (2) the malic and succinic
acids are converted to other compounds by as-yet-undeter-
mined paths.

The probability of labeling via exchange (1) may be
answered by a thermodynamic argument. Under the condi-
tions existing in the chloroplast during photosynthesis, the
actual free energy change accompanying the conversion of
PEPA, CO2, TPNH, and either ADP or IDP to malic acid,

37

TPN + , and ATP or IT? is probably at least -7 kcal. The
ratio of the forward reaction to the back reaction, given by

DT-i /forward rate'
V back rate

/back rate plus net rate\
\ back rate J

would thus be 10^ or greater. Since the rate of labeling of
malic acid is measurable and gives the net rate by a simple
calculation, the back reaction, and hence the exchange label-
ing, can be shown to be of negligible importance.

This type of calculation is of considerable importance in
in vivo steady-state kinetic calculations. Another example is
the conversion of malic acid to fumaric acid. In this case, the
actual free energy change is small; the two acids are essen-
tially in equilibrium with respect to C^*-labeling. Thus the
sum of the pools of the two acids can be treated from a
labeling standpoint as a single entity.

In any event, if malic acid is not labeled by exchange
and is not converted to succinic acid yet is being formed at
a rapid rate under steady-state conditions, it must undergo
some as-yet-unknown conversion. One possibility might be
that it is split to give glyoxylic acid and free acetate. The
actual free energy change for such a reaction under steady-
state conditions would be negative, whereas the reaction to
give glyoxylic acid and acetyl Co A would probably be posi-
tive and the latter reaction would not occur. Acetate could
be converted to acetyl phosphate with ATP and then to
acetyl CoA. The acetyl CoA thus formed could be used in
fatty acid synthesis and other biosynthetic reactions. The
glyoxylic acid could be used in the synthesis of glycolic acid,
glycine, and possibly, as suggested in the previous section,
glutamic acid.

The synthesis of labeled malic acid could occur via
condensation of glyoxylate with acetyl CoA, provided there
is some other route for the labeling of these two-carbon
acids (such as are suggested later). It is quite likely that malic

38

acid is so synthesized in the cytoplasm, outside the chloro-
plasts. Within the chloroplasts, however, the appearance of
C^^ in malic acid in the very shortest exposures to C^*02 and
in the pre-illumination experiments (see Figure 5) indicate
that it is, in part at least, a product of Ci-Cs carboxylation
and reduction.

Glycolic acid, acetic acid, and glyoxylic acid

Even if acetate and glyoxylate are formed from malic
acid, there are probably other more important synthetic
routes from the carbon reduction cycle to these compounds.
Benson and Calvin (41) found that barley seedlings sub-
jected to 30 seconds photosynthesis with C^^02, followed by
2 minutes light without CO2, formed large amounts of C"-
labeled glycolic acid. Calvin et al. (14) and Schou et al. (42)
degraded glycolic acid and phosphoglyceric acid obtained
from barley leaves and from Scenedesmus that had photo-
synthesized for a few seconds in the presence of C^*02 or
HC^^Os". The alpha and beta carbon atoms of PGA were
found to be always about equal to each other in radioactivity
and always less than the carboxyl carbon until such time
(1 to 5 minutes) as all three carbon atoms were completely
labeled. The two carbon atoms of glycolic acid were always
about equal to each other in labeling. When C^*H20H
— COOH was administered to the unicellular algae Scenedes-
mus during 10 minutes photosynthesis with V2 per cent CO2
in air or N2, a pattern of photosynthetic intermediates was
found similar to that obtained during photosynthesis with
C^*02. Moreover, upon degradation of the PGA we found that
less than 10 per cent of the radioactivity was in the carboxyl
carbon. Clearly, glycolic acid is incorporated for the most part
into normal intermediates of the carbon reduction cycle with-
out preliminary conversion to CO2, since so little C^* was
found in the carboxyl carbon of PGA. However, alpha and
beta carbon atoms of the PGA were found to be equally
labeled. Thus the pathway from glycolic acid to the alpha and

39

beta carbon atoms of PGA involves a randomization of the
label. This could mean that along this pathway there is a
symmetrical intermediate or that an intermediate is in rapid
reversible equilibrium with a symmetrical compound (see
Figure 7).

When Wilson and Calvin (19) studied the effect of CO2
depletion following a period of photosynthesis with C^^02
by algae, they found that the lowering of CO2 pressure re-
sulted in a great increase in the amount of labeled glycolic
acid. This increase in labeled glycolic acid was sustained for
at least 10 minutes. Upon application of 1 per cent CO2
again, the level of labeled glycolic acid declined.

Tolbert (22) found that glycolic acid formation from
C^^02 during 10 minutes photosynthesis in leaves of Sedum
alboresum is much higher at very low CO2 pressure than at
high CO2 pressure. As mentioned earlier, he also found that
glycolic acid is the predominant labeled compound excreted
into the medium by chloroplasts from Swiss chard photo-
synthesizing in the presence of HC^^Os-. He had shown
earlier (43) that glycolic labeled with C^* is excreted into
the medium by Chlorella photosynthesizing in C^^02. He
suggested that glycolate may function in ion balance with
HCO3- between cells and their medium or between chloro-
plasts and other cell compartments. He also proposed that
glycolate might be a carrier of "carbohydrate reserves" from
the chloroplasts to the cytoplasm.

Moses and Calvin (44) exposed photosynthesizing Chlo-
rella pyrenoidosa to tritium-labeled water for various periods
from 5 seconds to 3 minutes. Analysis was made by the usual
extraction, two-dimensional paper chromatography, and
radioautography. The greatest darkening of the film by far
occurred where it was in contact with the glycolic acid area
of the chromatogram. This result, which we shall discuss
later, seems to agree with Tolbert's suggestion that the gly-
colic acid acts as a carrier of hydrogen.

During normal photosynthesis (Figure 2), two-carbon
moieties (carbon atoms number 1 and 2 from a keto sugar

40

phosphate) are transferred during a reaction similar to that
catalyzed by transketolase (45,46) to an aldo-sugar phosphate,
producing a new ketose phosphate, two carbon atoms longer
than the starting aldose. Other enzymes have been found in
nonphotosynthetic organisms which convert the carbon
atoms number 1 and 2 of a ketose phosphate to acetyl phos-
phate, leaving the remainder of the sugar as an aldose phos-
phate. One of these is phosphoketolase (47), which is specific
for xylulose-5-phosphate, while another is fructose-6-phos-
phate ketolase (48), which can act on either fructose-6-phos-
phate or xylulose-5-phosphate. These enzymes require thia-
mine pyrophosphate, inorganic phosphate and, in some cases,
Mg+ + . Stimulation by Mn++ or Ca++ in place of Mg+ +
could sometimes be observed, whereas levels of Mn above
10~^ were inhibitory.

Breslow has proposed a mechanism for the role of thi-
amine pyrophosphate in these reactions (49,50). In his mech-
anism, some of which forms the basis for part of Figure 7,
the hydrogen at position 2 of the thiazole ring is an active
hydrogen which can dissociate from the acidic carbon at that
position to give a carbanion. This carbanion adds to the Qar-
bonyl carbon of the ketose (somewhat analogous to cyan-
hydrin addition). The bond between carbons 2 and 3 of the
ketose breaks, with the electron pair going to the reduction
of carbon 2 of the ketose, to give a glycolaldehyde-thiamine
pyrophosphate. The remainder of the sugar becomes an
aldose. Reversal of this reaction path, with a different aldose,
completes the transketolase reaction.

Alternatively, glycolaldehyde-thiamine pyrophosphate
may eliminate the elements of water (OH~ from the beta
carbon and H+ from the alpha carbon of the glycolaldehyde
moiety) to give the enol form and thence the keto form of
acetyl-ThPP. This compound can undergo phosphoroclastic
cleavage to give acetyl phosphate and thiamine pyrophos-
phate (ThPP).

The mechanisms find support in the demonstration by
Breslow that the hydrogen atom on the number 2 position

41

CHjOH

CHOH

CH^OPO^

CC-D-GLYCEROL
PHOSPHATE

CH.

0
,3(CH,^4-C0A

9=0

(9H0H)n
CH^OPO^

HOPO^

-CfeC-CHg-C-CoA.
MALONYL COA

CHj-C-CHg-C-Coft
ACETOACETYL CoA

OH 0
CHj-C-CHg-C-CoA

^\ 4[H]

MEVALONATE
OH

CH3-9-cH2-ay)H

CH2

/ \

SJERODS CAfiOTENaDS

Figure 7. Pathways from carbon reduction cycle to acetyl phos-
phate and glycolic acid. For details of the carbon reduction cycle,
see Figure 2.

of the thiazole ring does exchange rapidly in D2O (49). In
support of an analogous mechanism for the role of ThPP
in the oxidation of pyruvate, Krampitz and co-workers (51,
52) synthesized the postulated intermediate, an acetaldehyde-
ThPP compound with the acetaldehyde bonded to the num-
ber 2 carbon atom of the thiazole ring as an alpha hydroxy-
ethyl group. This compound was found to be active in the
reactivation of carboxylase and also to be capable of non-
enzymatic reaction with acetaldehyde to give acetoin. The
postulated mechanism for the oxidation of pyruvic acid thus
begins with a reaction between pyruvate and ThPP to give
addition of the carbonyl carbon to the thiazole-ring-position
number 2. Concurrently or immediately following this addi-

42

tion, decarboxylation occurs to give acetaldehyde-ThPP.
This compound reacts with oxidized lipoic acid to give acetyl
dihydrolipoic acid, which in turn reacts with CoA to give
dihydrolipoic acid and acetyl CoA (53-56).

Wilson and Calvin (19), following their observation of
glycolate accumulation at low CO2 pressure, suggested that
the glycolyl moiety transferred by transketolase is the source
of glycolic acid. We should now like to suggest specifically
that the glycolaldehyde-ThPP compound formed in the first
step of the transketolase or phosphoketolase reactions may
undergo oxidation to give glycolyl CoA and, eventually,
glycolate. This oxidation need not follow a pathway exactly
analogous to the oxidation of acetaldehyde-ThPP, but we
have shown it so in Figure 7.

As mentioned earlier, during photosynthesis glycolate
can be converted to the alpha and beta carbon atoms of PGA
via carbon atoms 1 and 2 of the pentose in the carbon reduc-
tion cycle. Thus it appears that the pathway from pentose
phosphate to glycolate and glyceraldehyde phosphate should
be reversible. The incorporation of glycolate via such a path-
way would require an energy input, probably in the form of
an activation by ATP. Finally, some state in the incorpora-
tion pathway should involve equilibration with a symmetric
intermediate because administration of glycolate-2-C^* to
photosynthesizing plants leads to PGA labeled equally in
the alpha and beta carbon atoms. We have indicated one
such symmetric compoimd and there may be other possi-
bilities.

The formation of glycolyl CoA and reduced lipoic acid
as shown in Figure 7 are hypothetical. If glycolyl CoA were
formed, it seems likely that it would be an important inter-
mediate in paths as yet unknown. In any event, if there is
any conversion of carbon atoms number I and 2 of ketose
to glycolic acid during photosynthesis, then an oxidation of
the glycolyl fragment is required so that some cofactor, al-
though not necessarily lipoic acid, must be reduced.

Let us now attempt to explain the observation that

43

labeled glycolate accumulates during photosynthesis with
C^*02 when the CO2 pressure is reduced.

1. Enzyme systems present in chloroplasts can bring
about the oxidation of glycolate to glyoxylate with oxygen
and the reduction of glyoxylate to glycolate with DPNH
(57). If some steady-state relation between these two acids
exists, it might well be shifted toward more glycolate at low
CO2 pressures by the increase in the ratio of DPNH/DPN +
that would result from the decreased utilization of TPNH
for the carbon reduction cycle. Moreover, the oxidation of
glycolate by O2 must in fact be limited in rate during photo-
synthesis, or glycolate would not be seen at all. Possibly gly-
colate is more effectively oxidized by some intermediate hy-
droxy 1 or peroxide involved in the liberation of oxygen
following the splitting of water during the primary act in
photosynthesis. If so, such an intermediate oxidant may de-
crease in concentration at low CO2 pressure because of re-
combination with primary reductant that would build up,
again as a result of decreased utilization by the carbon re-
duction cycle. A decrease in the oxidant concentration would
reduce the oxidation of glycolate.

2. Low CO2 pressure might result in higher pH inside
the chloroplasts. The phosphoketolase reaction, leading to
acetyl CoA and involving the removal of OH" from gly-
colaldehyde-ThPP, might be blocked, and the oxidation of
the glycolaldehyde-ThPP to glycolyl CoA might be favored.

3. If glycolyl CoA is formed and is a biosynthetic inter-
mediate, the reactions in which it is used might require CO2
analogous to the conversion of acetyl CoA to malonyl CoA
in fatty acid biosynthesis. Low CO2 pressure could thus lead
to an increased concentration of glycolyl CoA and permit its
more rapid hydrolysis to glycolate.

Tanner and co-workers (58,59) have recently proposed
a direct route from CO2 to glycolic acid during photosyn-
thesis. According to his scheme, CO2 is reduced by TPNH
and MnCl~ to the radical CHO-. Two of these CHO- radi-
cals are then condensed to give glyoxal, thence glycolic acid.

44

This glycolic acid is then oxidized by 2 molecules of MnCl-
(OH)2 (produced in the first step) to give glyoxylic acid.
According to Tanner, the greater labeling of glycolic acid
at low CO2 pressure during photosynthesis with C^^02 is
due to the first step being first order with respect to the utili-
zation of CO2 and the production of trivalent manganese,
whereas the second step is second order with respect to the
utilization of trivalent manganese.

Whether or not Tanner's suggested route from CO2 to
glycolic acid will be borne out by experiment remains to
be seen. In all our experiments with C^*02, labeled glycolic
acid has been a relatively minor product of the photosyn-
thesis, except in those cases where the CO2 pressure has been
permitted to drop to a very low level. Glycolic acid is some-
what volatile, but it is a curious characteristic of this com-
pound on paper chromatograms that, although 20 to 85 per
cent may evaporate from the paper during development of the
chromatogram, the remainder disappears only very slowly
from the papers. This statement is based on measurement
of radioactivity following chromatography of synthetic C^*-
labeled glycolic acid. Thus it would seem that if a pathway
leading directly from CO2 to glycolic acid (that is, with no
isolable intermediates) were quantitatively important, we
should have seen much more labeled glycolic acid following
short periods of photosynthesis with C^^02, It could be that,
under normal conditions of photosynthesis (say with 1 per
cent CO2 in air), the reservoir size or concentration of gly-
colic acid is very small, so that it would not appear to be
strongly labeled, even though carbon from C^^02 enters it
very rapidly.

However, Moses and Calvin (44) conducted parallel
experiments (3 minutes photosynthesis by Chlorella in the
presence of C^*02 in one case and T2O in the other). The
tritium-labeled glycolic acid accounted for more than 50
per cent of the darkening of the radioautograph in the sub-
sequent analysis by chromatography, whereas in the parallel
experiment the glycolic acid contained less than 5 per cent

45

of all the C^* found in compounds on the chromatograph.
Thus the incorporation of hydrogen into nonexchangeable
positions on glycolic acid seems to occur at ten times or
more the rate of incorporation of C^* into the same com-
pound. The simplest interpretation is that glycolic acid
plays a much more important role in the transport of hy-
drogen or reducing power than it does as an intermediate in
carbon-compound formation from CO2. If any carbon di-
oxide is reduced directly to glycolic acid during photosyn-
thesis by Chlorella, it would seem to be a minor part of the
total.

A special role for glycolic acid in hydrogen transport is
suggested by a combination of experimental findings from
several laboratories. To Moses' finding of extremely rapid
tritium labeling of glycolic acid and Tanner's implication
of the role of glycolic acid with the requirement for man-
ganese, we may add Delavin and Benson's report (60) of the
light stimulation of the oxidation of glycolic acid with O2
to glyoxylate and peroxide in isolated chloroplasts. Further,
we must mention that manganese is thought by Kessler (61)
to play some part in the formation of peroxide or O2 from
water during the early stages of photosynthesis. Some form
of peroxide is commonly postulated as an intermediate be-
tween water and O2 during photosynthesis, and it may be
that the plant has some mechanism for conserving the chem-
ical potential energy that would be lost if peroxide were
permitted to decompose to water and oxygen by a catalase
mechanism.

The decrease in labeled glycolate in algae grown in
Mn++ -deficient media (58,59) may be due to (1) some in-
crease in the level of an intermediate in the oxygen-evolution
pathway which is also capable of oxidizing glycolate to gly-
oxylate (presumably Mn++ might be required for the break-
down of this oxidant to O2); (2) a decrease in reduced pyri-
dine nucleotide concentration, owing to impairment of the
oxygen-evolving pathway; or (3) some enzymic requirement

46

for Mn + + in the biosynthetic pathway from glycolaldehyde-
ThPP to glycolate.

Points (1) and (2) are related to the mechanisms sug-
gested earlier for the effect of low CO2 pressure on glycolate
concentration.

Acetate

As shown in Figure 7, acetyl phosphate can be formed
from the carbon reduction cycle via the phosphoketolase
pathway. This involves dehydration of the ThPP-acetalde-
hyde compound derived from carbon atoms 1 and 2 of ketose
phosphates. This route is especially attractive as a photo-
synthetic pathway, since it conserves chemical energy and re-
quires no oxidation or decarboxylation. Known enzyme sys-
tems would readily convert the acetyl phosphate to acetyl
CoA for fatty acid photosynthesis.

Another pathway from the carbon reduction cycle to
acetyl CoA could be via oxidative decarboxylation of pyruvic
acid. This reaction is of the type we have earlier viewed as
unlikely in photosynthesizing chloroplasts on grounds of
economy. However, this economy takes on a different aspect
if one considers the rapid formation of alanine, which we
believe might be a reductive amination of phosphoenolpyru-
vic acid derived from the carbon cycle (30). Our experiments
indicate that about one-third of all NH4+ uptake occurs
via this route. The resulting alanine must be used to a
considerable extent in transamination reactions, resultine in
the production of pyruvic acid. Although pyruvic acid is not
labeled soon enough after the introduction of C^^02 to photo-
synthesizing plants to permit us to consider it a precursor to
alanine, it does become slowly labeled at later times. Thus
pyruvic acid could be a product of transamination from ala-
nine. The slow labeling of pyruvate may be because alanine
has a very large reservoir, which does not saturate with C^*
for some minutes. Once formed, the pyruvic acid cannot

47

easily be converted back to PEPA. Rather, it must either go
to malic acid via reductive carboxylation or be oxidized to
acetyl CoA and CO2.

The light-dark transient effect in C^^02 uptake during
photosynthesis has often been observed (16,20). When the
light is turned off, following a period of photosynthesis of
algae with C^^02, labeled glutamic acid and citric acid ac-
cumulate. One explanation of this effect has been given,
based on the proposed formation of acetyl CoA by pyruvic
acid oxidation. Lipoic acid in its oxidized form is required
to accept the electrons in this oxidation. It was suggested
that while the light is on this cofactor is kept mostly in its
reduced state, dihydrolipoic acid. The reduced cofactor could
not promote pyruvic acid oxidation. When the light is turned
off and reducing power is no longer generated, the oxidized
form of lipoic acid would be made, and the oxidation lead-
ing to acetyl CoA would occur. Subsequent reactions, via the
glyoxylate cycle, would then produce citric and glutamic
acids.

However, if acetyl phosphate is formed by phosphoke-
tolase during photosynthesis, a different explanation can be
made. If we suppose that acetyl phosphate is still formed via
phosphoketolase just after turning off the light, it will tend to
accumulate. No reducing power or ATP is available for syn-
thesis of fatty acids in the dark inside the chloroplasts. There-
fore, acetyl phosphate will break down to free acetate, which
will diffuse out of the chloroplast into the cytoplasm. There
it will be used, via the glyoxylate cycle, in the synthesis of
glutamic acid (62).

48

l8>

^^^ Carbohydrates

M onosaccharides

The carbon reduction cycle (Figure 2) includes as in-
termediate compounds the following sugar phosphates: 3-
phosphoglyceraldehyde, dihydroxyacetone phosphate, fruc-
tose-1,6-diphosphate, fructose-6-phosphate, erythrose-4-phos-
phate, sedoheptulose-l,7-diphosphate, sedoheptulose-7-phos-
phate, xylulose-5-phosphate, ribulose-5-phosphate, ribose-5-
phosphate, and ribulose-l,5-diphosphate. Besides these com-
pounds, glucose phosphates are found to be very rapidly
labeled in all plants in which we have studied the photosyn-
thesis of carbon compounds from C^^02. When characterized,
both glucose-6-phosphate and glucose- 1 -phosphate have been
found. Other sugars found to be labeled somewhat more
slowly in these experiments and identified as the free sugars
following hydrolysis of the sugar monophosphate area include
mannose and galactose.

In virtually all the studies of the labeled products of the
photosynthesis of carbon compounds from C^'*02 there has
been found a striking absence of unphosphorylated mono-
saccharides (14). This is hardly surprising, since photosyn-

49

thesizing chloroplasts form phosphorylated sugars as inter-
mediates in the carbon reduction cycle, since there is an abun-
dance of ATP in the chloroplasts and since most known
transformations of monosaccharides require phosphorylated
forms of the sugars. Transformation of the phosphorylated
sugars to the free sugars would for the most part result in
a waste of chemical energy, for the sugar would then usually
have to be phosphorylated again in reactions requiring ATP
or UTP. Only when it becomes necessary to form a mole-
cule that can be transported through the chloroplast mem-
brane is it likely that a free sugar of relatively small molec-
ular weight such as sucrose would be produced.

A listing of various enzyme systems that appear to be
responsible for the carbon reduction cycle has been delayed
until now, since many of these biochemical steps are of in-
terest in a discussion of carbohydrate synthesis. In Table 2
there are listed the enzymes reported in the literature which
appear to be responsible for steps of the carbon reduction
cycle (Figure 2). Table 3 lists other enzymes which could
account for subsequent steps in the synthesis of carbohydrates
found to be labeled following relatively short periods of
photosynthesis of algae with C^*02.

We wish to emphasize that the finding of an enzyme in
plant tissue does not, of course, prove that that particular re-
action goes on in the photosynthesizing chloroplast either at
all or in precisely the same way that it has been found to
occur in vitro. Moreover, we would not consider the isolation
of an enzyme with high catalytic activity a necessary condi-
tion for believing that a given reaction may occur in vivo.
The organization of the intact chloroplast inside the living
cell and replete with all necessary natural cofactors and en-
zymes is such that some steps which occur in vivo may prove
extremely difficult to demonstrate in cell-free systems. None-
theless, the isolation of a cell-free system, capable of carrying
out a reaction that has been suspected on the basis of in vivo
studies, is important corroborative evidence.

The various enzymes listed in Tables 2 and 3, if present

50

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in chloroplasts, could account for virtually all the monosac-
charide phosphates found to be significantly labeled with C^"*
following a period of photosynthesis with C^*02 for several
minutes in algae. Presumably there is present also another
phosphohexose isomerase which catalyzes the conversion of
fructose-6-phosphate to mannose-6-phosphate.

Among the enzyme systems listed in Table 3 are several
that utilize sugar nucleotides in the biosynthetic conversion
of sugars. Such systems have been widely studied and have
been discussed and reviewed elsewhere (88-90). Hassid and
co-workers have widely studied the interconversions of sugars
by these systems in higher plants and have summarized the
interrelations of many of these systems in plants (91). Certain
of these systems, which appear in Table 3, are particularly
active in the early labeling of sugars in plants photosynthe-
sizing with C^*02 and must be mentioned here, if only briefly.

Buchanan et al. (15) identified uridine diphosphate glu-
cose (UDPG) and uridine diphosphate galactose (UDPGal)
in algae and found that the hexose moieties of these com-
pounds were labeled with C^^ during short periods of 0^^*02
photosynthesis even before sucrose. Thus the galactose found
to be labeled in some experiments may be formed by the
UDPG-UDPGal system.

Disaccharides and polysaccharides

As already indicated, when Chlorella pyrenoidosa photo-
synthesizes in the presence of C^*02, sucrose is the first free
sugar to be labeled to any extent. Benson (92) found that the
radiocarbon in the fructose moiety of the sucrose, following
photosynthesis of C'^02 by Chlorella, Scenedesmus, and soy-
bean leaves, was greater than the radioactivity in the glucose
moiety. Moreover, the difference between fructose and glu-
cose became greater as the time of photosynthesis was de-
creased. 1 he prior labeling of the fructose indicated that the
glucose phosphate used in the synthesis of sucrose is formed
from fructose phosphate.

53

A study of the phosphorylated products of sfiort-term
photosynthesis in C^^02 led to the discovery of a sucrose
phosphate (93). The "hexose monophosphates" produced
during photosynthesis in C^'^02 were treated with an inver-
tase-free phosphatase preparation and subjected to paper
chromatography. Although in most cases there were only
minute traces of sucrose formed by this treatment, in sugar
beet (5 minutes in Ci''02) there was an appreciable quantity.
It was identified by cochromatography and enzymic hydroly-
sis to glucose and fructose.

When this "hexose monophosphate" sample was sub-^
jected to chromatography in ^butanol: picric acid: water, ra-
dioactive areas corresponding to glucose-6-phosphate, fruc-
tose-6-phosphate, sedoheptulose and mannose phosphates,
and sucrose phosphate were obtained. The sucrose phosphate
gave sucrose on phosphatase treatment, and on acid hydrolysis
glucose and fructose phosphate were produced. The latter
did not cochromatograph with fructose-6-phosphate.

It appeared that in sucrose synthesis in green plants
there are two possible mechanisms. Glucose- 1 -phosphate
might react with fructose- 1 -phosphate to give sucrose phos-
phate, which would be dephosphorylated to sucrose. Alter-
natively, sucrose phosphate synthesis might be envisaged to
occur through uridine diphosphate glucose (15), which be-
comes labeled shortly before sucrose in kinetic experiments
with €^^^02 (18). The uridine diphosphate glucose may be
formed from glucose- 1 -phosphate by a UDPG pyrophosphory-
lase (reaction 15, Table 3). This pathway is shown in Figure
8 along with other pathways that may very likely occur dur-
ing photosynthesis of carbohydrates from CO2.

Leloir and Cardini (85) have isolated from wheat germ
what appears to be two systems, one that catalyzes the reac-
tion of fructose plus UDPG to give sucrose plus UDP, and
a second that catalyzes the reaction UDPG plus fructose-6-
phosphate to give sucrose phosphate plus UDP. Burma and
Mortimer (94) have reported that with excised sugar beet
leaves and leaf homogenates radioactive UDPG and sucrose

54

-FIP-

CO2 Reduction-
Cycle

F6P

Sucrose
Sucrose P

G6P

UTP

Starch

GIP:

r:UDPG=::iUDPGal^;=rGal IP

PP

Oligosaccharides
Polysaccharides

Figure 8. Biosynthetic pathways for photosyn-
thesis of carbohydrates.

were formed when radioactive glucose- 1 -phosphate, fructose-
6-phosphate, and UTP were added. They propose a mecha-
nism identical to that postulated by Buchanan except for the
choice of fructose-6-phosphate as the precursor instead of
fructose- 1 -phosphate.

Not much is known about the formation of other poly-
saccharides. There is a rapid labeling of unidentified polysac-
charides during photosynthesis with C^^Oo. On the usual two-
dimensional chromatogram, developed as described earlier,
these compounds form what appears to be a homologous series
of polyglucoses extending from the origin nearly to sucrose.
The compound of this series closest to sucrose has been hy-
drolyzed and found to contain only glucose.

55

Fats

During photosynthesis by unicellular algae, it is not
uncommon for as much as 30 per cent of the carbon dioxide
taken up to be incorporated into fats. In Scenedesmus, for
example, after 5 minutes in light in the presence of C'*-
labeled carbon dioxide, 30 per cent of the fixed radioactivity
is found in lipid materials. This incorporation of radiocarbon
is greatly in excess of the rate of any synthesis that could
take place in the dark and is an indication of the stimulation
of fat production in the light. Fat synthesis requires a greater
number of equivalents of reducing agents than does synthesis
of carbohydrate or protein. Moreover, the composition of
the chloroplasts includes a very high proportion of fat ma-
terial. Since there is an abundance of reduced cofactors and
ATP in the chloroplast, and since the end product, fat, is
needed in the chloroplast, it is likely that much fat synthesis
takes place in the chloroplast and is therefore to be consid-
ered photosynthetic.

Fatty acids

All the well-known biosynthetic pathways to fatty acids
require as a starting material acetate or acetyl CoA. We have

56

already suggested under "Carboxylic Acids" four ways in
which acetate, or acetyl CoA, could be made. These were: (1)
splitting of malic acid to glyoxylate and acetate, (2) reduction
of glycolic acid to acetate, (3) oxidation of pyruvic acid to
acetyl CoA, and (4) dehydration and phosphoroclastic split-
ting the postulated glycolyl-enzyme complex from transketo-
lase reaction of the carbon reduction cycle to give acetyl phos-
phate. We favor the last way as being the most likely. How-
ever, if only the first three of these pathways are available,
the third is probably the most important.

However the acetate is formed, it is rapidly converted
to fats in the light in algae. Experiments with Scenedesmus
photosynthesizing in the presence of acetate- 1-C^^ and C^^02
(14) demonstrated a light-accelerated incorporation of ace-
tate into fats. A similar light-enhanced incorporation of ace-
tate-2-C^* into lipids by Euglena was found by Lynch and
Calvin (95). Sissakian (96) demonstrated the synthesis of
higher fatty acids from labeled acetate in chloroplasts from
sunflower plants. The utilization of free acetate in the light
by chloroplasts is to be expected, since there is an abundance
of ATP in the photosynthesizing chloroplasts for the conver-
sion of acetate to acetyl phosphate and thence to acetyl CoA.

The scheme of fatty acid synthesis proposed by Wakil
and Ganguly (97) for the formation of fatty acids from
acetyl CoA in animal tissues has been widely accepted. A
similar pathway may exist in photosynthetic tissues. This
pathway is incorporated in the hypothetical scheme of fat
photosynthesis shown in Figure 9. Wakil (98) and Wakil and
Ganguly (99) report that the first step in the synthesis from
acetyl CoA is a carboxylation to give malonyl CoA. This step
requires biotin and ATP, as well as Mn+ + . Malonyl CoA and
acetyl CoA then condense to give acetoacetyl CoA, which
then undergoes a series of reductive steps to give eventually
butyryl CoA and carbon dioxide (97).

Although the work of Ganguly and Wakil has been with
animal tissues, it appears from the studies of Stumpf and
co-workers (100-103) that similar systems of fatty acid syn-

57

c

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i-l

^

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^

X2

1

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K

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5

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thesis exist in plant tissues. The early stages of fat synthesis
may well be similar in photosynthesizing chloroplasts to those
known for other plant tissue and animals. The later stages
and the fat products formed during photosynthesis in chloro-
plasts are very likely different, since the chloroplast in all
likelihood requires specialized fats for its operation. Benson
and co-workers have identified a number of interesting com-
pounds of glycerol phosphate and fatty acids as products of
fat formation in green tissues. According to these workers,
phosphatidyl glycerol is a major component of plant phos-
pholipids. Moreover, they state that active transphosphatidyl
action is observed during photosynthesis (104-106).

Glycerol phosphate

Alpha-D-glyceryl-1 -phosphate is presumably formed in
chloroplasts during photosynthesis by direct reduction with
TPNH of dihydroxyacetone phosphate. This compound
could then be further converted to the polyglycerol phos-
phates reported by Benson. The various glycerol phosphates
would then presumably react with fatty acetyl CoA to pro-
duce fats. Some of these postulated biosynthetic routes are
shown in Figure 9.

59

l8f

"^J^ Pigments

Of major importance among the biosynthetic pathways
of the chloroplast must be those leading to photosynthetic
pigments. Akhough some of these may vary from one organ-
ism to another, all organisms must be capable of making at
least one of the chlorophylls, carotenoids, and hematin pig-
ments. During photosynthesis the simple precursor molecules
for these synthetic paths are available from the carbon reduc-
tion cycle, whereas the reduced pyridine nucleotides and
ATP are of course at high levels in the chloroplast.

Carotenoids and phytol

The starting point for the synthesis of carotenoids and
phytol, as well as steroids and terpenes, is acetyl CoA. In the
previous sections we discussed routes from the carbon reduc-
tion cycle to acetyl CoA. These are shown in Figures 7 and 9.

The biosynthetic paths to terpene compounds have been
much clarified in recent years by work from the laboratories
of Lynen (107), Bloch (108), Folkers (109), and Popjak (110).
Successive condensations of acetyl CoA give acetoacetyl CoA
and then y8-hydroxy-^-methyl-glutaryl (or crotonyl)-CoA

60

(HMG-CoA). The HMG-CoA is then reduced to give meva-
lonic acid (Figure 9). Further steps along the biosynthetic
path are shown in Figure 10. Pyrophosphorylation and de-
carboxylation of mevalonate give isopentenyl-pyrophosphate,
the biological isoprene unit.

According to Lynen, isopentenyl-pyrophosphate units
then condense to give, successively, Cio, C15, and C20 com-
pounds, as shown in Figure 10. Hydrogenation of the C20
compound could presumably lead to phytol, an alcohol that
forms the phytyl tail of chlorophyll. Dimerization of the C15
compound, farnesyl pyrophosphate, gives squalene, the pre-
cursor for steroids. We might expect the C20 compound,
geranylgeranyl pyrophosphate, to undergo a similar conden-
sation to give C40 compounds, which could in turn be con-

M£VAlJONAT£
S
i

HgC-OPjHi'
ISOPENTENYL-PrROPHOSPHATE
(IPP)

GERANYL - PYKOPHOSPHATE

^'^^^..-^CHe-

— SOUALENE

FARNESYL-PrmPHOSPHATE \

STEROIDS

>'VsX-sA.^H2

■oPfeHe'

PHYTOL •^?—GERAUYL-GERAf/rL- PYROPHOSPHATE

PHYTOENE

SUCCESSIVE DEHYDRO-
GENATICNS. RING
CIXSURE. BMEFSZOKHS,
ETC.

CC ANDfi CAROTEte, WTEIN. VKX-AXANTHIN. ETC

Figure 10. The biosynthesis of carotenoids.

(* For details see Figures 7 and 9.)

61

verted to carotenoids. Stanier (HI) has reported evidence
indicating that the initial compound in this series is phytoene
or tetrahydrophytoene (see Figure 10).

Present evidence indicates that conversion of the C40
compound formed from the condensation, to carotenoids,
involves a number of dehydrogenations, and finally ring
closure at the ends of the molecule. The various oxygen-con-
taining carotenoid compounds are probably formed by oxida-
tions, hydrations, etc. The structures of a great many of these
compounds, both intermediates and end products, have been
established in the laboratories of Karrer (112), Zechmeister
(113), Inhoffen (114), Weedon (115), and others.

Chlorophyll and heme

The pathways to porphyrin compounds have been re-
cently reviewed by Granick (116,117), Shemin (118), Rim-
ington (119), and Bogorad (120). Some of the key steps from
these paths are shown in Figure 11. Glycine and succinate
formed from the carbon reduction cycle are the starting com-
pounds for the syntheses of these pigments. Glycine may be
formed from serine, which in turn is probably synthesized
from 2-phosphoglycerate, formed from the 3-phosphoglycerate
of the cycle (see the section on Amino Acids). Alternatively,
glyoxylate may be transaminated to give glycine. The deriva-
tion of this glyoxylate from the carbon reduction cycle is not
known for certain, but is probably related to the formation
of glycolic acid (see the section on Carboxylic Acids). Thus
glycolate formed by oxidation of the glycolyl fragment from
the sugar phosphate transketolase system could be further
oxidized to glyoxylic acid. A hypothetical split of malate
could lead to acetate and glyoxylate.

If the chloroplast contained isocitritase, both succinate
and glyoxylate could be formed by the same reaction on iso-
citrate. The isocitrate would in this case come from acetyl
CoA and oxalacetate condensation, via citrate. Oxalacetate

62

HOOC~CHo~'CHo"*C~CHp
0 NH2
S AMINO LEVULINIC ACID

CO2H
GLYCINE

COjH
CH2

I

CO2H
CHp

CH2.
C=0
I
,CH2

HgC

0 NH,

NH2

CC^HCHg

Ch^CHgCOgH

^.

COgHCHg^^^y^CHgCOgH

f^

CH2

ccyn

CH2

CH,

"^

CH2f^2(j;H2

CH?
I

NH2
PORPHOBILINOCeN (PBG)

CH2
UROPORPHYRINOGEN M

I DECARBOXYLATIONS
I DEHYDROGENATIONS

PROTOPORPHYRIN - 9
F. + +

HEME

Mg'

RING 3Z:
.* FORMATION

• Mg PROTOPORPHYRIN -► — —

Vi = -CH=CH2

Pr = -CH2-CH2-CO2H

Mt' -CH3

PHYTOL'

CHLOROPHYLL

Figure 11. The biosynthesis of porphyrins.

is formed from the cycle by carboxylation of phosphoenolpy-
ruvate, derived from phosphoglycerate.

Another, and perhaps more likely route to succinate is
via reductive carboxylation to form malate, dehydration, and
reduction of malate to give succinate.

63

As shown in Figure 11, condensation of glycine with suc-
cinic acid gives S-amino levulinic acid, which in turn con-
denses with itself to make a substituted pyrrole ring (por-
phobilinogen). Condensations and isomerizations, the exact
mechanisms of which are not known, lead to the formation
of the tetrapyrrole structure of uroporphyrinogen(III) from
four porphobilinogen molecules.

The conversion of uroporphyrinogen to protoporphyrin
requires a number of decarboxylations of the substituent acyl
groups, oxidation of two of these groups to vinyl groups, and
dehydrogenation and aromatization of the pyrrole rings and
the methylene bridges connecting them.

Protoporphyrin-9 is an important branching point: in-
corporation of Fe++ leads to heme and thence to the various
hematin pigments, whereas incorporation of Mg++ ion leads
ultimately to the synthesis of the chlorophylls. The latter
pathway must first accomplish the formation of the fifth
ring and the partial saturation of one of the pyrrole rings.

Finally the phytol alcohol, probably formed as shown
in Figure 10, is attached to the pigment molecule as a phytyl
group, and the synthesis of chlorophyll is complete. At some
time, before or after this step, the alterations needed to make
the various forms of chlorophyll, and to incorporate it into
the structure of the photosynthetic apparatus are completed.

64

'^tf' Aromatic nuclei

The shikimic acid pathway for the biosynthesis of aro-
matic compounds, including amino acids, from carbohydrates
has been well established by the work of Davis (38) and his
collaborators, who used biochemical mutants of E. coli.
Without going into the details of this pathway, we may point
out that the starting materials are phosphoenolpyruvate,
which is readily available as a photosynthetic intermediate,
and D-erythrose-4-phosphate, which is also an intermediate of
the carbon reduction cycle. Presumably, therefore, the syn-
thesis of aromatic amino acids in photosynthesizing plants
would follow a pathway similar to the shikimic acid path-
way. The first step in that pathway is the condensation of
phosphoenolpyruvate with erythrose-4-phosphate to give a
seven-carbon compound which has been identified as 2-keto-
3-deoxy-D-araboheptonic acid-7-phosphate. This intermediate
subsequently undergoes ring closure to give dehydroquinic
acid. Rearrangements via a number of additional steps gives,
eventually, phenylalanine and tyrosine. Higuchi (121) has
summarized some of the reasons for believing that the shi-

65

kimic acid pathway does occur in higher plants. For example,
shikimic acid is of widespread occurence, and some of the
enzymes of the pathway have in fact been found in higher
plants. Neish (122) has further reviewed evidence for the shi-
kimic acid pathway in plants.

66

WP

Other
biosynthetic

^^^ products

As we learn more about the capabilities of the chloro-
plast to form compounds from carbon during photosynthesis,
we come closer to the conclusion that the chloroplast, as it
exists in the living, undisturbed cell, is a self-sufficient factory
capable of producing essentially all the materials required
for its replenishment. Thus it appears to be able to make
all kinds of sugars, polysaccharides, protein, fats, pigments,
enzymes, and cofactors. In addition to this, it produces for
export to the cytoplasm reserves of organic compoimds.
These are probably sugars, glycolic acid, and other neutral,
relatively small, molecules which can be readily transported
through the chloroplast membrane. Until more is known
about the development and formation of chloroplasts, we can-
not say just when it gains this complete synthetic ability. No
doubt there are early stages in the development of chloro-
plasts in which it must be built from cytoplasmic materials
derived in turn from already-functioning chloroplasts. There
is no reason to suppose the chloroplast functions without nu-
clear control, even though it does not appear to have a nu-
cleus of its own. Presumably it is possible for RNA mole-
cules to move in and out of the chloroplast in some way. It

67

cannot be said at the moment whether or not the chloroplast
is capable of synthesizing nuclear material. It would seem
likely, however, that the chloroplast can synthesize pu-
rines and pyrimidines, coenzymes, and nucleotide materials
needed for the continued functioning of the chloroplast as
a self-sufficient biosynthetic factory. If, as we now think,
protein synthesis and enzyme synthesis occur in the chloro-
plast, then either the chloroplast must obtain a store of RNA
molecules at its initial construction or else such molecules
must be able to travel back and forth from the chloroplast
to the cytoplasm.

In conclusion, we should say that the point of view of
the ability of the chloroplast to carry out photosynthetic
formation of many compounds is a departure from the view
held only a few years ago. It was then thought that the primary
function of photosynthesis was to form carbohydrate only.
This carbohydrate was then thought to be used by the cyto-
plasm in the synthesis of all other compounds. Of course, the
chloroplast must stipply the carbohydrate and reducing power
for the cytoplasmic synthesis. It now appears that chloroplasts
also synthesize a complete spectrum of biochemical products,
all of which might reasonably be considered to be photosyn-
thetic products. Finally, as we learn more about the photo-
synthetic paths to these products, we are impressed not
merely by their complexity but much more by the economy
with which both energy and material are utilized.

68

^^^ References

1. Bassham, J. A., A. A. Benson, L. D. Kay, A. Z. Harris, A. T.
Wilson, and M. Calvin, /. Am. Chem. Soc, 76, 1760 (1954).

2. Bassham, J. A., and M. Calvin, The Path of Carbon in Pho-
tosynthesis, Prentice-Hall, Inc., Englewood Cliffs, N.J., 1957.

3. Bassham, J. A., and M. Kirk, Biochim. Biophys. Acta, 43,
447 (1960).

4. Norris, L. T., R. E. Norris, and M. Calvin, /. Exptl. Botany,
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6. Axelrod, B., and R. S. Bandurski, /. Biol. Chem., 204, 939
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7. Arnon, D. I., Science, 116, 635 (1952).

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69

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23. Aronoff, S., A. A. Benson, W. Z. Hassid, and M. Calvin,
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25. Nichiporovich, A. A., "Tracer Atoms Used to Study the
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70

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75

Reprints

EXPERIENTIA VOL. Vni/12. 1952 - p. 445

VERLAG BIRKHAUSER, BASEL/SCHWEIZ

The Path of Carbon in Photosynthesis'

(XX. The Steady State)
By M. Calvin and P. Massini^ Berkeley, Cal.

Photosynthesis, the process by which green plants
are able to capture electromagnetic energy in the form
of sunlight and transform it into stored chemical energy
in the form of a wide variety of reduced (relative to
carbon dioxide) carbon compounds provides the only
major soufce of energy for the maintenance and pro-
pagation of all life. For this and other reasons, the
study of the nature of this process has been a very
attractive area for many years and a wide variety of
scientific interest and backgrounds have been brought
to bear upon it. These range from the purely biological
to the strictly physical with the biochemical and phy-
sicochemical area lying between. Important contri-
butions to the understanding of the phenomenon have
come from all these areas, but in spite of the enormous
amount of work and study that has gone into the prob-
lem, relatively little is known, or rather understood,
about the fundamental character of the process even
today. It is perhaps pardonable that one engaged in
studies in this area tends to the conclusion that most of
the knowledge has been acquired in the relatively recent
past. Discounting that tendency, it still seems fair to
say that we have only just begun in the last decade or
so to gain some understanding of the intimate details
by which the basic process represented in the overall
reaction

CO, + H,0

-Hhti

-> 0,+ {CH,0),

- Energy

has come to be understood. The recognition of this
overall reaction as written, to represent the basic nature
of the process of photosynthesis, and, further, that its
reversal represents the basic reaction of respiration is,
of course, an old one.

As a result of more recent study, it has been possible
to separate the process of photosynthesis into two dis-
tinct and separate parts. The general features of this

^ The work described in this paper was sponsored by the U.S.
Atomic Energy Commission.

■ Radiation Laboratory and Department of Chemistry, Univer*
sity of California, Berkeley. Fellow of the Swiss Foundation,
iStiftung fiir Stipendien auf dem Gebiete der Chemie», 1951-1952.

separation may be represented in the following chart
(Fig. 1). The essential feature of the separation is the
independence of the photochemical part of photosyn-
thesis from the carbon dioxide reduction part. We shall
not here even try to outline all of the various forms of
evidence which have been adduced in support of such
a scheme but only to point out additional bits which
have been added in recent years and particularly those
which stem from our own work'.

ICHgO)

C02

L.

Fig. 1.

The scheme itself is an outgrowth of proposals of
some fifteen years ago by Van Niel* resulting from his
studies of the comparative biochemistry of photosyn-
thesis. More recently, the photochemical apparatus has
been shown to be separable from the rest of the plant
by the experiments of Hill'.

He was able to make preparations of chloroplasts
and chloroplastic fragments which, upon illumination
in the presence of suitable oxidizing agents other than
carbon dioxide, were able to evolve molecular oxygen.
Still more recently, Ochoa an others* were able to
demonstrate that these same preparations were capable
of using coenzyme I and II (D.P.N, and T.P.N.) as

' M. Calvin and A. A. Benson, Science 107, 476 (1948). - A. A.
Benson and M. Calvin, Cold Spring Harbor Symp. quant. Biol. tS,
6 (1948). - M. Calvin and A. A. Benson, Science lOS, 140 (1949).

» C. 3. Van Niel, P*o/osy>i(A«si5 in P/anb, Chapter 22 (Iowa State
College Press, Ames, Iowa, 1949), pp. 437-495.

' R. Hill, Nature 139, 881 (1947); Proc. roy. Soc. (London) [BJ
127, 192 (1939).- R. Hill and R. Scarisbrick, Nature H6. 61 (1940).

• W. VisHNlAC and S. Ochoa, J. Biol. Chem. 19S, 75 (1952). -
D. I. Arnon, Nature H7, 1008 (1951). - L. J. Tolmach, Arch, Bio-
chem. Biophys. 33, 120 (1951).

79

446

u viN and r. Massisi: TIip Tnth of C.nrl.nn in Photosynthesis

IEXPERIENTI»V0I..VI 11/12]

suitable oxidizing agents leading to the evolution of
oxygen. Furthermore, the experiments of Ruben*
showed that the molecule of oxygen evolved in photo-
synthesis had its proximate origin in the oxygen of
the water molecule and that the oxygen atom associ-
ated with the carbon dioxide must first pass through
water before arrivingatgaseousoxygcn. From the chart
it may be seen that the ultimate result, then, of- the
photochemical reaction initiated by the absorption of
light by the chlorophyll molecule is the division of the
water molecule into an oxidized part which ultimately
leads to molecular oxygen and some reduced parts
represented in the chart by [H],

This reduced part [H] we have called "reducing
power" because as yet it is not possible to state specifi-
cally what form or forms it may be in. This reducing
power is capable of reducing carbon dioxide in the
absence of light; that is to say, that the reduction of
carbon dioxide itself is a dark reaction. This was indi-
cated first in the earlier experiment of McAlister" in
which he was able to show that following a period of
photosynthesis a number of plants continued to absorb
carbon dioxide for a short period (seconds to minutes)
after cessation of illumination. We were able to demon-
strate this in an even more direct and uneciuivocal
fashion and generalize it for all plants so far tried when
we were able to show that not only did all of these
plants absorb (|uantities of carbon dioxide in the dark
after illumination but that the products formed in the
dark were (jualitatively and under certain conditions
quantitatively similar to those formed in a fairly com-
parable light period'. The method used for this demon-
stration was the same as those to be described later in
the review. The lifetime in the dark of this reducing
power which is generated by light is also of the order
of seconds to minutes and almost certainly corresponds
to a concentration of one or more definite chemical
species. It is quite conceivable, as mentioned earlier,
that some of it might be in the form of reduced coen-
zymes.

Very recently it has been reported* that both the
higher plants and isolated chloroplasts emit a chemi-
luminiscence following cessation of illumination. This
chemiluminiscence has a decay time which corresponds
very closely to that which we have observed for the
reducing power. In fact, it would seem almost surely
to represent the reversal of the conversion of electro-
magnetic into chemical energy, namely, the transfor-
mation of at least some of the chemical energy stored
in the reducing power into the electromagnetic energy
of luminiscence. Furthermore, the luminiscence is re-

' S. UruKN, M. Randaij., M. 11. K\men, .ind J. Hvor. J. A\\\.
Chcin. Soc. C3, 877 (1941).

' K. U. McAr.isTER and J. MvF.RS, J. Smithsonian Insl. I'uM
(Misc. Coll.) c, aa (1940).

' M. Calvin, J. Chcui. Uducation J6, 030 (lOl'J).

* B. L. STREHi.tR and W. .\rnoi.u, J. Gen. Physiol. 34, sua
(lull). - H. I.. STRtHUF.R, .^rch. BiochoiTi. Hiophys. 34, M9 (19:.l)

duced by the presence of carbon dioxide in those cases
in which the carbon dioxide fixing system is still pre-
sent. However, when the carbon dioxide system has
been removed, as is true in the case of chloroplasts, the
luminiscence becomes independent of carbon dioxide.
While it thus appears that the unique problem of
photosynthesis lies in the right hand half of thechart
of Figure 1, the present discussion will be limited to
the other side of the chart, that is, the path through
which carbon passes on its way from carbon dioxide to
all the raw materials of the plant. It is essentially a
study of what we now believe to be entirely dark
reactions and might best be characterized as phyto-
synthesis. This area not only has a great interest for its
own sake but would almost certainly cast some light
upon the nature of the reducing agents which arrive
from the photochemical part of the reaction and drive
the carbon cycle toward reduction. The reason for this
particular interest lies in the fact that we have, in recent
years, come into possession of a tool which is especially
suited for this study, namely, labeled carbon atoms in
the form of a radioactive isotope of carbon, O*. All of
the results that will be described later were made
possible through the use of this labeled carbon dioxide.
With such a labeled molecule available, the design of
an experiment for determining the sequence of com-
pounds into which the carbon atoms of carbon dioxide
may pass during the course of their incorporation in the
plant is, in its first phase, a straightforward one.

./

CO2

We may visualize the problem in terms of the chart
in Figure 2 in which the green leaf is represented
schematically as a closed opaque container into which
stream the raw materials of photosynthesis, namely,
carbon dioxide, light and water containing the neces-
sary mineral elements. From this container are evolved
the products of photosynthesis- oxygen gas and the
reduced carbon compounds constituting the plant and
its stored reserves. Heretofore, it has been possible to
study in a quantitative way the nature of the process
going on inside the opaque container only by varying
external conditions and noting variations in the final
products. Although there has been no serious doubt
that the formation of sugar did not take place by the
aggregation of six molecules of carbon dioxide, six

80

ri5. XII. 1952]

M. Calvin and P. >[assini: The I\itli u( Ciiboii in Pliolusyntheais.

447

molecules of water and the requisite number of light
quanta into a single unit followed by the rearrangement
into hexose and molecular oxygen, no specific infor-
mation was available as to the compounds which might
act as intermediates. Assuming that such a chain of
intermediates exists, it is quite clear that by setting up
some photosynthetic organism, leaf or other suitable
material, in a steady state of photosynthesis in which
the various ingredients are being absorbed and pro-
ducts formed in some uniform manner and injecting the
labeled carbon dioxide into the entering carbon dioxide
stream, we should find the label appearing successively
in time in that chain of intermediates. This can be
observed by stopping the entire process after a suitable
lapse of time and examining the incorporated labeled
carbon to determine the nature of the compounds into
which it has been built. It is also clear that in addition to
the identity and sequence of the compounds into which
the carbon is incorporated, we may also determine the
order in which the various carbon atoms within each
compound acquire the label. With this type of infor-
mation at hand it should be possible to reconstruct the
sequence of events from the time of entry of the carbon
atom into the plant as carbon dioxide until it appears
in the various more or less finished products of the plant .

Fig. 3.— .\lg.ic I'l.iiU.

While photosynthetic experiments have been done
with a vide variety of plant materials, the major ki-
netic work has been carried out with suspensions of
unicellular green algae. The reason for this lies in the

I'ig. 4. — "Lollipop".

fact that these algae may be obtained in a reproducible
biological form relatively easily and in any amount.
They are grown in the laboratory in a continuous cul-
ture arrangement shown in Figure 3. The algae maybe
harvested from these flasks daily or every other day,
depending upon the type of material desired. Such
cultures have been maintained in a continuous fashion
over periods extending beyond several months. Most
of our experiments have been performed with the uni-
cellular green algae Chlorella or Scenedesmus. After
harvesting the algae are washed with distilled water
and resuspended in the medium in which the experi-
ment is to be done. This suspension is placed in a flat
vessel called a "lollipop", a photograph of which is
shown in Figure 4. A stream of air containing carbon
dioxide is passed through the algae while they are
being illuminated so as to achieve a steady state of
photosynthesis.

In order to begin the experiment the air stream is
interrupted and the labeled bicarbonate is injected into
the algal suspension. After the preselected period of
time, the algae are killed by opening the large stopcock
at the bottom of the flask, allowing the algal suspension
to fall into alcohol in order to stop the reaction and
extract the photosynthesized material. Although a
variety of killing and extracting procedures have been
tested, most of the experiments were performed by
dropping the algae into alcohol so as to result in an
80% alcohol solution. The total amount of carbon
fixed is then determined by taking an aliquot of this
entire suspension, evaporating it to dryness on an alu-
minum disk and counting it on a Geiger counter'. The
fraction soluble is determined by either filtering or
centrifuging the suspension and then recounting the
clear supernate or filtrate.

The distribution of the fixed radiocarbon among the
various compounds must now be determined. Since in

' M. Calvin, C. HtlutLUERGER, J. C. Reid, Lt. M. Tolbert, and
P. E. Yankwich, Iwtopic Carbon (John Wiley & Sons, Inc., New
York, 1940).

>

81

jij

i .

JB

448

M. CvLViN and P. Massini: The Path of Carbon in Photosynthesis

[ExperientiaVol.VIII/12]

relatively short periods of time most of the fixed radio-
activity is found in the soluble components, the prob-
lem is one of analyzing for the distribution in the
soluble fraction. This has been done by an application
of the method of paper chromatography introduced
and developed for amino acid analysis by Consden,
Martin, and Synge'. It has since been applied to a
wide variety of compounds and no detailed description
of it will be given here. The unique extension to our
work lies in the ability to locate particularly those
compounds which contain the radioactive carbon atoms
on the paper by means of a radioautograph of the
resulting paper chromatogram obtained by allowing an
X-ray film to remain in contact with the paper for a
suitable period of time. Those areas of the paper which
are occupied by radioactive compounds will, of course,
expose the X-ray film. Such a map of the disposition
of the radioactive compounds contained in an extract
is shown in Figure 5. The chemical nature of the com-
pounds defined by the exposed areas can be inferred
from the position occupied by a compound with re-
spects to the origin of the chromatogram. More precise
determination of the chemical character is assisted by
chemistry performed on the material eluted from the
spot defined by the radiogram and rechromatography.
Final identification, however, is usually dependent on
the co-chromatography of the unknown, or questioned,
radioactive material eluted from the paper with an
authentic specimen of the suspected compound and
the demonstration of the complete identity of the car-
rier material as determined by some visible test on the
paper with the pattern of radioactivity in the co-chro-
matogram. The amount of radioactivity incorporated
in these compounds can be determined quite accurately
by using the X-ray film as a means of defining that
area of the paper containing the compound, thus per-
mitting the particular spot to be cut out from the larger
and eluted from the paper and mounted on a plate to
be counted.

*f.^i=n8s»i:':r.--i' T.",i»W:'

OMVOMTtMCTONK mOIMWTf

wWBafSBSWBBSr'"^'^'-'

\ PHO*n(0«4.Y0tllATt

.^

.y

mtUlOM mtOfHATE

mtOM MOMHATI
MRMTOM mOVMTl ft MMMOtC mOWlun' f

•LUOOM nWtMUTC ft ItlMMPTULOte MQtPHATI

m

•uftULOtc wmowtun • niiatB emioftnuTff

Fig. 5,

-Radiogram of a paper chromatogram from 10 s C**0, fixation
ID the light by Scenedesmus.

* R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J.
28, 224 (1944). - A. A. Benson, J. A. Bassham, M. Calvin, T. C.
GooDALE, V. A. Haas, and W. Stepka, J. Am. Chem. Soc. 72, 1710
(1950).

ao

re

'2

iB

?'

PG«

6.4

,'

3

,

t^o

•

h

1

PSS

/

2° C

f«

1

»«

1

k

/

44

s

/

o

^">

t

t

1'^

8,'

:^

'

%

ij8

/

i*

/

20

>

o

^

O onotphotc

16

o

/

12

0/

J

06

/

:^

04

— • a —

-^

Malic

T8 U,

60

8C

Fig. 6,

— Behavior of radioactivity in specific compounds in extracts
Scenedestnus , exposed to radioactive carbon dioxide at 2*'C.

A much simpler means would be to count the spot
right on the paper with a Geiger counter. The fraction
of the total amount of radioactivity in the spot which
is thus registered by the Geiger counter is fairly con-
stant for all compounds for any given chromato-
graphic system. Thus, for most purposes it is sufficient
simply to expose the paper to X-ray film in order to
determine just where the radioactive spots are, and
then having so defined them, to count them right on
the paper for quantitative comparison, by the Geiger
counter. It is clear from Figure 5 that the variety of
products synthesized at room temperature by Scene-
desmus (as well as by all other plants tried) is great,
even in a very short time such as ten seconds. But even
so, it is clear that the predominant compound as the
time gets shorter is phosphoglyceric acid.

This is even more strongly demonstrated when the
experiment is carried out at reduced temperature, for
instance 2°C, so as to slow down all of the reactions
and enable us to see more clearly the earliest products.
Figure 6 shows a plot of the concentration of radio-
activity per unit of algae for three of the major early
compounds, formed at 2°C. On such a plot as this, it
is clear that those substances which are formed directly
from carbon dioxide with no appreciable intermediates

82

115. XII. 1952]

M. Calvin and P. Massini: The Path of Carbon in Photosynthesis

449

TabU I
C^* Distribution in Photosynthetic Products of Barley and Scenedismus

Conditions^

Glyceric Acid

GlycoHc Acid

Hexose

-COOH

— CHOH

— CHjOH

—COOH

— CHjOH

C3,4

C 2,5

C 1,6

Barley
Preillum :
2 min dark . . .

4 s PS

15 s PS

15 s PS

30 s PS

30sPS»

40 s PS

60s PS

Scenedesmus

5 s PS

30 s PS

30 s PS'MI' . . .

60 s PS»

60sPS«MI. . . .

60 s PS»

60s PS'MI. . . .

96

87
56
49

75
443

95*

73
51
48

43

2-6
6-5
21

25

6

30

2-5

12
24
24

27

1-7
6-8

23

26

9

25

1-2

15

25
28

30

48-5
50 ±5

48

47

51-5

50 ±5

52
53

52
87'

25
7

24
6

' Experiments are steady-state photosynthesis, 10,000 footcandles unless otherwise stated. ' 1000 footcandles. ' Alanine obtained from this
extract was 48% carboxyl-labeled. * Under the same conditions, Chlorella produced phosphoglycerate latelcd 93%, 3% and 2%,
respectively. ' In this extract, malic acid was labeled 6*5% and aspartic acid 4% in the non-carboxyl carbons. ' 3000 footcandles.

" Malonate inhibited.

lying between them and carbon dioxide will be the only
ones that will show a finite slope ; all others should start
with a zero slope. A finite slope is certainly the case for
phosphoglyceric acid and possibly for malic acid, in-
dicating at least two independent catrbon dioxide fixing
reactions, one leading to a three-carbon compound and
the other producing a four-carbon compound'.

Since the hexose phosphates appear extremely early
in all of these photosynthesis experiments and because
of the known close relationship between the hexose
phosphates and phosphoglyceric acids in the glycolytic
sequence, it seemed most reasonable to suppose that
these hexose phosphates were formed from the phos-
phoglyceric acid by a combination of the two three-
carbon fragments derived from phosphoglyceric acid in
an overall process very similar to, if not identical with,
the reversal of glycolysis.

One means of testing this suggestion would be a com-
parison of the distribution of radioactivity in the three
carbon atoms of glyceric acid with those in the hexose
as shown in Table I. It thus appears that the hexose is
indeed formed by the combination of two three-carbon
molecules derived from the glyceric acid in such a
manner that carbon atoms three and four of the hexose
correspond to the carboxyl-carbon of the glyceric acid ;
carbon atoms two and five with the alpha-carbon ; and
carbon atoms one and six with the beta-carbon of the

* E. J. Badin and M. Calvin, J. Am. Chem. Soc. 7^, 5266 (1950).
- S. Kawaguchi, a. a. Benson, M.Calvin, and P. M. Hayes, J.Am.
Chem. Soc. 7i. 4477 (1952).

glyceric acid. This correspondence is maintained when
the distribution in these two compounds (glyceric acid
and hexose) is compared for a wide variety of different
times.

With this clear cut indication of the similarity be-
tween the path of hexose synthesis and the known path
of its breakdown, another means of testing how closely
this parallelism might be followed suggests itself. The
hexose derivative which is last in the sequence of
changes prior to the breakdown of the carbon skeleton
during glycolysis is the fructose-l,6-diphosphate.
Correspondingly, then, it presumably would be the
first hexose derivative to appear in the reverse direction.
If this is the, case and, furthermore, if the hexose deriv-
ative reservoirs involved in sucrose synthesis are more
or less isolated from those involved in storage and gly-
colysis, the radioactivity should appear in the fructose
half of the sucrose molecule prior to its appearance in
the glucose half. This is indeed the case'. However,
sucrose does not seem to be formed by the simple re-
versal of the sucrose phosphorylase system which was
described for certain bacteria^, since for this to be the
case, free fructose would have to be apparent in the
photosynthesizing organism, whereats it is never so
found, nor has the enzyme itself ever been isolated
from any green plant.

* S. Kawaguchi, A. A. Benson, N. Calvin, and P. M. Hayes,
J. Am. Chem. Soc. 7i, 4477 (1952).

' W. Z. Hassid, M. Doudoroff, and H. A. Barker, J. Am. Chem.
Soc. se, 1416 (1944). - M. Doudoroff, H. A. Barker, and W. Z.
Hassid, J. Biol. Chem. ISS, 725 (1947).

83

450

M.C\uviN ami 1'. Massini: Tlie TjIIi ot Carbon in I'hotosyulhesis

[Exi'tKltNlIA\'0L.\'III/I2J

The recent identification' as uridine diphospho-
glucose (U.D.P.G.) of the spot which had been previ-
ously* called «the unknown glucose phosphate spot»
has lead to another suggestion as to the mode of for-
mation of sucrose. Glucose-labeled U.D.P.G. appears
very early in the sequence of compounds formed. Fur-
thermore, it has been possible to demonstrate the pres-
ence in the hexose monophosphate area of a sucrose
phosphate by using a carefully selected phosphatase,
containing no invertase, in the treatment of this entire
phosphate area'. We have suggested, therefore, that
U.D.P.G. may be involved in sucrose synthesis in a
manner similar to that of glucose-1-phosphate in the
numerous phosphorylase reactions, with the difference,
however, that the acceptor of the glucose moiety would
be some phosphate of fructose, thus producing a sucrose
phosphate. Recent work by Putnam and Hassid' gives
further support to the idea that only phosphorylated
derivatives of glucose and fructose are involved in
sucrose synthesis in higher plants. They found that in
sucrose synthesis, from labeled glucose in leaf punches,
no free fructose was formed, although the sucrose be-
comes equally labeled in both the glucose and fructose
portions. Conversely, when labeled fructose is used, no
free labeled glucose appears, while the sucrose is uni-
formly labeled in both moieties.

It is possible that compounds of the U.D.P.G. type
could be concerned in the transformation of sugars and
the subsequent incorporation into polysaccharides.
Uridine diphosphate would thus serve as a carbon
carrier in the same way that pyridine nucleotides and
flavonucleotidcs are involved in hydrogen transfer ; the
adenylic acid system in phosphate transfer ; and coen-
zyme A in the transfer of acetyl groups. There is already
some evidence for the existence of other members of
the uridine diphosphate group from our own work, as
well as that of others*.

We may now turn our attention from the fate of the
glyceric acid to the problem of its origin. An exami-
nation of Table I indicates quite clearly that the first
position in the glyceric acid to become labeled is the
carboxyl group. As time proceeds, the other two carbon
atoms in the glyceric acid acquire radioactivity and it

' J. G. Ulchana.n €t at., in press. - J. G. Blchanan, J. A. Uass-
HAM, A. A. UtNSON, D. F. Bradley, M. Calvin, L. L. Dals, M.
Goodman, P. M. Hayls, V. H. Lynch, L. T. Norris, and A. T.
Wilson, Phosphorus Metabolism, \'ol. II (Johns Hopkins Press,
Baltimore, Maryland. 1952), in press.

' S. Kawaclchi, a. a. Benson, N. Calvin, and P. M. Hayls,
J. Am. Chem. Sor. 71, AV7 (1052).

' J. G. Buchanan, J. A. Bassham, A. A. HtNSON, V. V. Uradllv,
.M. Calvin, L. L. Dals, M. Goodman, P. M. Hayes, V. H. Lynch,
L. T. Norris, and A. T. Wilson, Phospliorus Metabotism, \'ol. II
(Johns Hopkins Press, Baltimore, Maryland, 1902), in press. - J. G.
Buchanan, in press.

• E. W. Putnam, Thesis (University of California, Berkeley. l'J5'.>).

* R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Paladim.
J. Biol. Chem. IS4, 333 (1950). - A. C. Paladini and L. F. Leloir,
Biochem. J. 51, 126 (1951). - J. T. Park, J. Biol. Chem. 1S4, 885
(1952).

appears that they acquire it at equal rates, at least
within the present accuracy of the experiments.

It thus appears that the most rapid reaction which
carbon dioxide can undergo at least at high light in-
tensities, is a condensation with a Cj fragment leading
directly to phosphoglyceric acid. An examination of the
chromatograms of a very short photosynthetic period
shows glycine and glycolic acid as the only two-carbon
compounds present. The distribution of radioactivity
among the carbon atoms of these two compounds is
always equal and the same and corresponds very well
with that in the alpha- and beta-carbon atoms of the
glyceric acid, as may be seen from Table I. This sug-
gests that glycolic acid either is in the direct line for
the formation of the Q carbon dioxide acceptor, or is
very closely related thereto.

The question now arises as to the source of this Cj
carbon dioxide acceptor. There are, of course, only two
possibilities for its origin. Either it results from a one-
plus-one combination or it must result from the split-
ting of a four-carbon compound or a larger one. In order
for it to result from the combination of two one-carbon
fragments there must exist as an intermediate some
one-carbon compound more reduced than carbon diox-
ide which, in turn, may combine either with itself or
with carbon dioxide. Furthermore, the reservoir of
this one-carbon intermediate would have to be vanish-
ingly small since all attempts to find labeled, reduced,
one-carbon compounds, such as formic acid or formal-
dehyde, in the early stages of photosynthesis have
failed and, in addition, the resulting two-carbon frag-
ment is very nearly equally labeled in both carbon atoms.
One would also expect that these one-carbon com-
pounds would tend to disappear under conditions of
low carbon dioxide concentrations leading to the disap-
pearance of the two-carbon condensation product re-
sulting from them. This leads us to the supposition that
the formation of glycolic acid would be expected to
drop off under conditions of low carbon dioxide con-
centration which is the reverse of what is observed.

We are thus left with the following possibility for the
C'a compound -the cleavage of some C4 or larger struc-
ture. The fact of the early appearance of label in malic
acid, taken together with the lack of any appreciable
amounts of label in the compounds of the tricarboxylic
acid cycle', led us to the supposition that malic acid
was either a precursor to, or very closely related to, a
four-carbon compound which could be split to produce
the required two-carbon fragment.

In the course of the search for the two-carbon ac-
ceptor, and its immediate precursors, two new com-
pounds were identified as early products of carbon
dioxide incorporation which seem to have little to do
with the direct synthesis of hexoses and, therefore, had
a very likely function in the regeneration of the two-

1 A. A. Benson and M. Calvin, J. Exptl. Botany ;, 63 (1950).

84

[15. XII. 1952)

M. Calvin and P. Massini: The Path of Carbon in Photosynthesis

451

carbon acceptor. These were the phosphates of the
seven carbon sugar sedoheptulose and of the five car-
bon sugars ribulose, ribose and arabinose'.

The question immediately presents itself as to the
relation between these two compounds along the path
of carbon assimilation, not only with each other but
with the precursors which are already known and the
possible products that might be formed from them.
The attempt to answer this question focuses our at-
tention once again upon some of the shortcomings and
limitations of the method of observation that we are
using and the nature of the exjjeriment which we are
performing. Our initial hope of determining the se-
quence of intermediates by a simple observation of a
sequence of compounds into which radioactivity has
been incorporated in steady state experiments is now
complicated by the uncertainty as to the amount of
compound present during the steady state. It is easy
to visualize a situation in which the actual amount of
intermediate present during the steady state is so small
as to escape observation by our methods, or perhaps
even to be so unstable as to be lost by our methods of
observation. This complete failure of a compound to
appear on a chromatogram, although it might con-
ceivably be an intermediate, is, of course, an extreme
case. The more usual situation is one in which most of
the intermediates are present but in varying concen-
trations in the steady state. Under such conditions a
single or even several observations of the relative
amount of radioactivity incorporated into a variety of
compounds would not necessarily be any real criterion
of the relative order of these compounds in the se-
quence of events.

In order to achieve the full value of the method of
observation then, it becomes necessary to perform
rather extended kinetic experiments in which the ap-
pearance of radioactivity in all compounds is plotted
as a function of time at sufficiently short intervals to
enable a rather accurate and detailed curve to be
obtained. Furthermore, the distribution of radioacti-
vity among the atoms within each compound should
also be determined as a function of time. The validity
of any proposed sequence of events could then be de-
termined by a comparison of the calculated appearance
and distribution curves with those actually observed.
In order to calculate such appearance curves, as well
as the distribution curves amongst the atoms in each
compound, one can set up a system of linear differen-
tial equations based upon the following model ;

CO,

-+ B

(1)

where COj represents the entering carbon dioxide;
.4, B, etc. represent intermediates involved in carbon

' A. A. Bt.MSO.s, J. A. BASSIIA.M, .M. Calvin, A. G. Hall, H. E.
HiRSCii, S. Kawaguchi, V. H. Lynch, and N. E. Tolbert, J. Biol.
Chcm. 196, 703 (1952).

dioxide assimilation; S represents more or less final
storage product; /? is a measure of the total rate of
carbon dioxide assimilation in the steady state ex-
pressed in moles of carbon per minute.

The rate of change of the specific activity of a single
carbon atom in A , given by X^ , is then expressed by
Equation (2). (The specific activity of the entering
carbon dioxide is here taken as unity. [A], the concen-
tration of the compound A, is independent of time.)

^ = w (1-^^)-

(2)

The specific activity of the corresponding atom in
compound B is given by an exactly similar Equation
(3),

dXo R

dt

[B]

{X,-X^).

(3)

Equations of identical form may be written for every
atom of every compound that might be considered an
intermediate. These equations may be solved expli-
citly by means of a differential analyzer provided two
parameters are known. These are the total rate of entry
of carbon into the system during the steady state, R,
and the steady state concentration of each atom which
might be considered as lying along the path of carbon
assimilation [,4], [B], etc.

It is clear that if such compounds (whose prime func-
tion it is to serve as carbon carriers between the en-
tering carbon dioxide and the final storage products in
the plant) do indeed exist in biological systems they
would very soon become saturated with radioactivity.
By this is meant that the amount of radioactivity ob-
served in that particular compound would very soon
reach a maximum value and remain that way. The
reason for this is that by definition the amount of these
intermediate compounds is not changing, and also is
small compared to the total amount of carbon the plant
assimilates during the experiment. Since all of the
carbon, or at least most of it, must pass through these
reservoirs of intermediates they will very soon acquire
the same specific activity as the entering carbon di-
oxide. In contrast to this, those materials which are
not functioning as simple intermediates but rather are
functioning as storage reservoirs, or are very distant
from the immediate photosynthetic intermediates, will
not acquire radioactivity as rapidly, or if they do they
will not become saturated as rapidly as those which are
directly mvolved in the path of carbon assimilation.
The amount of radioactivity found in those compounds
which saturate in a relatively short time now provides
a relatively easy method of determining the size of the
functioning reservoirs of these compounds which are
directly engaged in the path of carbon assimilation. A
simple measurement of this amount compared to the
specific activity of the entering carbon dioxide will
provide a measure, in moles per unit volume of the

85

452

M. Calvin and P. Massini: The Path of Carbon in Photosynthesis

[ExperieniiaVol.VIII/12]

biological material, of the compound in question.
Furthermore, having once achieved a relatively uni-
form label in these photosynthetic intermediates, it
becomes possible to follow the behavior of the reservoir
size as a function of change in external variables, for
example, light intensity. We have chosen to include in
this review a more or less detailed description of just
this determination of the effect of light intensity upon
reservoir sizes as a means of describing the general ex-
perimental technique which is involved.

Steady state and reservoir sizes— Methods and results

The apparatus used for these experiments was con-
structed to permit the algal suspension to be left under
controlled external conditions (illumination intensity,
temperature, carbon dioxide and oxygen concentration)
while photosynthesizing for at least one hour. Further-
more, it was required that the change, natural to radio-
active carbon dioxide, which was to be circulated in a
closed system, and the withdrawal of several samples
at given time intervals be accomplished with a mini-
mum of change in these conditions.

The apparatus consisted of :

(a) A square illumination vessel A (Fig. 7) made out
of Lucite (polyacrylic plastic), 49 cm high, 11 cm wide
and 0-7 cm thick (inside dimensions). The bottom was
provided with a gas inlet tube with five small holes to
allow good contact between gas and liquid and a drain
tube closed with a screw clamp. The top of the vessel
was provided with a gas outlet tube. A water-alcohol
mixture from a constant temperature bath was allowed
to flow over the outer surfaces of the vessel in order to
control the temperature of the suspension.

Fig. 7.— Diagram of the assembly for steady state photosynthesis.
(For explanation of the letters, see text.)

[b) Two illumination banks (represented by B), each
with four fluorescent tubes (General Electric, quality
white, 20 W each), providing an almost uniform illumi-
nation over the whole surface of the vessel, of 7 x 10*
ergs. /cm ^ (roughly 700 footcandles).

Kig. S.— .\sscnibly for steady state photosynthesis. (For explanation
of the letters, sec text.)

(c) An ionization chamber C, connected to a record-
ing vibrating reed electrometer, to record the activity
of the gas leaving the vessel continually during the run.

{d) Three gas traps D, to permit the addition of a
known amount of radioactive carbon dioxide to the
system, and trap the remaining radioactivity after
the run.

(e) A flask E, of 5 1 volume, containing a mixture of
1% radioactive carbon dioxide in air. The reservoir
contained so much carbon dioxide that the algae assi-
milated no more than 20% of it during a run.

(/) A gas circulating pump F of the rubber tubing
type, and a flow meter G.

(g) A system of four-way stopcocks H, which per-
mitted the vessel to be flushed with a mixture of 1 %
ordinary carbon dioxide in air, from the cylinder I. The
assembly is shown in Figure 8.

In a typical experiment, 2 cm' (wet packed) of one-
day old Scenedesmus, washed and resuspended in 200
cm' of deionized water, were placed in the vessel and
aerated with the ordinary gas mixture for at least one-
half hour, while the mixture of radioactive carbon
dioxide circulated in the gas system for thorough
mixing, without passing through the vessel. The sus-
pension was kept at 24 °C. After this time, during which
a steady state of photosynthesis had been reached, the
radioactive mixture was passed through the vessel in
place of the ordinary gas mixture, by a manipulation

86

[13, XII. 1952J

M. Calvin and P. Massini: The Path of Carbon in Photosynthesis

453

I ^ SCCNCKSMUS

Fig. 9. — Radiogram of a paper chromatograra from lu min. 0^*0^
fixation in light by ScCTi^iitfSmui. I % suspension, 1% Ct)j in air; light
intensity 7 x 10* ergs./cm^-s. D ll.A.P. ;dihydroxyacetone phosphate;
P.E.B. :phosphoeno]pyruvir acid; P.M.P.:pentose monophosphates;
P.Go.A.:phosphoglycolicacid: P.G.A.:phosphoglycericacid; H.M.P.:
hexose monophosphates; U.P.rpentose and hexose diphosphates.

of the pair of stopcocks at H, and samples of 20 cm' of
the suspension withdrawn at intervals of five or ten
minutes. These samples were dropped into 80 cm' of
alcohol of room temperature, to make an e.xtraction
in 80% alcohol. After 30 min of photosynthesis, the

lights were turned off and the suspension allowed to
remain in the dark for a period of 5 min, during which
time again several samples were withdrawn, and treated
in the same manner. In one experiment another light
jseriod followed the dark period.

The samples were shaken for 1 h and centrifuged.
The residue was re-extracted in 50 cm' of 20% alcohol
at room temperature, centrifuged, and re-extracted
again with 20 cm' of water. The extracts were concen-
trated together to 0-5 cm'.

An aliquot of the concentrate equivalent to 30 /il of
packed cells was evaporated on a corner of a filter paper
(Whatman #1), and the chromatogram run with water-
saturated phenol in one direction and n-butanol-
propionic acid-water in the other. The chromatograms
were exposed to X-ray film for about two weeks'. The
labeled compounds appeared on it as black spots.
Figure 9 shows the radiogram for ten minute photo-
synthesis of Scenedesmus. The amount of radioactivity
contained in the different compounds was determined
by counting the corresponding spots on the paper di-
rectly with a large-area Geiger-MCller tube with
thin mica window. The compounds were identified by
a combination of the following criteria: (a) Their posi-
tion on the paper; (b) the spot was cut out, eluted from
the paper with water and run again in suitable solvents,
together with such an amount of the suspected com-

LIGHT

TIME (min.) OF EXPOSURE TO

Fig. 10. — C'*0, fixation by Scenedesmus. 1 % suspension, 1 % COg in
air, light intensity 7 x 10* ergs./cm*-s.

JO
DARK 0 2
TIME (Kiln.) OF EXPOSURE TO (?*0j

Fig. 11.— Behavior of radioactivity in specific compounds in the ex-
tract from the experiment of Figure 10.

pound that it could be detected by a specific spraying
reagent. The black spot on the film had to coincide
accurately with the color reaction ; (c) the eluted spot
was chemically transformed {e.g. treating the sugar
phosphates with phosphatase) and the resulting com-
pound cochromatographed with carrier detectable by
spray.

Figure 10 shows the total and the extracted amounts
of radiiKarbon fixed by 1 cm' cells during 30 min of

' M. Calvin (. Chem. Education ?6, 639 (1949).

87

454

M. Calvin iiud P. M.\ssl^•l: Tlic Path of Carbon iu Photosynthesis

[F.xi'ER1f.niiaVol.V1II/121

SUCROSE

A 6 MALIC ACIO

O-O GLUTAMIC ACIO
D- -Q CITRIC ACIO

labU 11

Steady State Concentrations of Some Compounds Involved iu the

Photosynthesis Cycle. Scenedesmus, experimental conditions as in

Figure Iu

TIME (mtoj OF EXPOSURE TO C'*0,

Fig. 12. — Behavior of radioactivity in specific compounds in the ex-
tract from an experiment done under conditions corresponding to
those of Figure 10.

photosynthesis followed by 5 min of darkness. The
slope in the total fixation curve in the light corres-
ponds to a 13 cm^ COj assimilation (N.T.P.) per hour.

Figure 11 shows the amount of radioactivity incor-
porated into sucrose and three phosphorus compounds
for the experiment of Figure 10.

Figure 12 gives the number of counts in sucrose,
glutamic, malic and citric acid, for a different experi-
ment of 15 min photosynthesis, followed by 10 min
dark, and again 5 min of photosynthesis.

Although the variation between experiments is quite
high, there are some striking features which are com-
mon to all :

(1) The curves of some of the compounds show a
marked decrease in slope after 5 min of photo-
synthesis. This quite clearly indicates the presence of
rapidly turning-over reservoirs in the photosynthesis
cycle which are then thoroughly labeled and reach the
specific activity of the fed carbon dioxide : Diphosphate
area (mainly ribulose diphosphate) ; hexose-monophos-
phate area (50% glucose-, 26% sedoheptulose-, some
fructose- and mannose-monophosphate) ; phospho-
glyceric acid. The leveling off of these curves permits
the calculation of the concentration of the reservoirs of
those compounds in the photosynthesis cycle, by divid-
ing the measured amount of radioactivity per carbon
atom by the specific activity of the fed carbon dioxide'.

Table II gives the steady state concentrations during
photosynthesis for some compounds determined by
this method.

(2) The fact that the activity vs. time curves show
a definite yet low slope for as long as 30 min can be
taken to indicate that the breakdown of carbohydrates

Substance

jumoles/cni' cells ^

Phosphoglyceric acid

Dihydroxyacetone phosphate . .

Fructose phosphate

Glucose phosphate

Mannose phosphate

Sedoheptulose phosphate ....
Ribulose diphosphate

1-4

0-17

0-12

0-4

0-05

0-18

0-5

0-2

continues throughout the illumination, i.e. their for-
mation from photosynthetic intermediates is revers-
ible. Thus, there are two sources of the intermediates:
{a) the carbon dioxide fed; the amount of compound
formed from this source reaches the maximum specific
activity in 5 to 10 min ; (6) the carbohydrate pool of the
ceUs; the amount formed from this source is labeled
only slowly since the specific activity of the carbo-
hydrate pool rises slowly due to the large size of the pool .
(3) Other compounds show almost constant rate of
labeling during the whole period of photosynthesis;
sucrose, malic and glutamic acid. For this and other
reasons it is clear that these compounds are not in the
photosynthesis cycle, but are formed during the photo-
sjmthesis at a constant rate. Their large reservoirs in
the cells are labeled only slowly.

Table 111

Phosphatase Treatment of H.M.P. Area after au min Photosynthesis
and 30 min Photosynthesis Followed by 5 min Dark

Substance

Number of counts/min
on paper

30 min P.S.

30 min P.S.
5 min D.

Glucose

Fructose

Sedoheptulose

Mannose

3140
910

1600
460

4280
1040

12102

(4) When illumination is interrupted there appears
a sudden great increase in the concentration of phos-
phoglyceric acid (followed by a slow decrease after 2
min), and an almost complete depletion of the diphos-
phate area. Analysis of the monophosphate area
showed that the amount of sedoheptulose phosphate
decreased also (cf. Table III). The concentration of

' The efficiency factor of the counting of spots on papers has been
determined by converting three cut out spots to barium carbonate
and measuring their activity in an ionization chamber. It is 19
disintegrations per count.

^ Volume measured as wet packed cells

- An appreciable fraction of this count is certainly hexosc so that
one may estimate a maximum value ot the heptose at around 800
counts/min.

88

115. XII. 19521

M. Calvin and P. Massini: The Path of Carbon in Photosynthesis

455

malic acid decreases as well. The rate of labeling of
glutarriic acid is increased greatly after a short induc-
tion period; citric acid, which contains little activity
during the whole light period, shows a sudden increase
in the dark, followed by a slow decrease. The labeling
of sucrose continues at the same rate as in light for
about 1 min, after which it is stopped almost com-
pletely.

Both experiments gave the same picture for most of
the compounds, with the two exceptions: In the second
experiment the diphosphate area, which in the first
contained almost the same number of counts as phos-
phoglyceric acid during the light, had only about 15%
of it in this second run. This value dropped to 5% in
the dark. The phosphoglyceric acid showed a hardly
significant rise in the dark during the first 2 min, but
again a slow decrease after 5 min. Although we do not
know why in this experiment the concentration of
ribulose diphosphate was so low in the light, the co-
incidence with the lack of increase of phosphoglyceric
acid points to a connection between both effects.

(5) In the light following the dark, the diphosphates,
phosphoglyceric and malic acid increase again.

^'^ "J^f GLUTAMIC

60sL-60sO^^HHH^M^^^^H 37

ACID

l20sL ^^Hi 10

60(L

126

CITRIC

60tL-60sO ^^^BH
I20SL ^^8 2

1169

■^^■1 '*3

ACID
ALANINE

eosi -eosD ^^^^m

^1 ^?7

I90O ^^^^m

^^H 37 3

60sL
60sL-60sO I
iZOsL

I 91

SUCROSE

1526

After one-half hour, the aeration bubbler was taken
out and a suitable amount of radioactive bicarbonate
(sodium) solution added. (The algae, which were grown
in slightly acid medium, had enough buffering capacit\-
to convert the bicarbonate to carbon dioxide). The
vessel was immediately stoppered and shaken in the
light. .After 1 min. the suspension was drained into

C*mOt4V0RATCS
NTS

I-'ig. 13.— Effect of light and dark on the labelingof glutamic and citric

acid. 0-1% suspension, light intensity 1-6 < 10' ergs/cm'-s (Numbers:

counts/min x 10~^ on paper per cui' cells).

The effect of dark on the labeling of glutamic and
citric acid was already reported in an earlier paper^
and studied more closely in the following experiments:
0-2 cm' wet packed algae (ChloreUa pyrenoidosa) were
suspended in 200 cm' distilled water, illuminated in a
flat circular vessel of 1 cm thickness by incandescent
lights through an infrared filter (intensity 1 -6 x lO^ergs/
cm^-s) and aerated with 008% carbon dioxide in air.
The low concentration of cells was chosen to avoid shad-
ing of cells in the suspension, so that during the light
period all the cells were illuminated continually.

• .\I. Calvin, J. Chem. Education SC, 639 (1949).

a darkened flask, and after another minute poured into
four times its volume of boiling alcohol. Control samples
were treated in the same way, but kept in the light, in
contact with radioactive carbon dioxide for 1 and 2
min, respectively. The analysis of the fixed radioacti-
vity was performed by paper chromatography and
radioautography with the technique already described.
The results are shown in Figure 13.

DiuHssioii

It has already been pointed out that photosynthesis
is not a mere reversal of respiration ; this was supported
by the observation that the carbon of newly formed
photosynthetic intermediates is not available for res-
piration while the light is on'. We may thus represent
the relationship between photosynthesis and respira-
tion by the following scheme (See Figure 14). The
labeling of the Krebs cycle intermediates through the
storage products (carbohydrates, fats, proteins) of the
cells is a slow process, due to the relatively large size
of the storage pools. The fact that the photosynthesis
intermediates find their way into the tricarboxylic acid
cycle very rapidly after the light is switched off means
that there is another connection between the two
cycles which is blocked as long as the light is on but
becomes accessible in the dark. This was interpreted in
earlier work^ in terms of the action of the light in
maintaining at low concentration the intermediate re-
quired for entry into the tricarboxylic acid cycle. A
closer specification of how this is accomplished is now
possible since the discovery that alpha-lipoic acid is a

' M. Calvin, J. Chem. Education J«, 639 (1949). - J. W. Weicl,
P. iM. Warrington, and M. Calvin, J. Am. Chcm. See. 73, .lO.^
(1951).

2 M. Calvin, J. Chom. Education J6, 630 (1949).

89

456

M. Calvin and P. Massini: The Path of Carbon in Photosynthesis

[ExpebientiaVol.VI 11/12)

cofactor for the oxidative decarboxylation of pyruvic
acid to an active acetyl group' which is the one reaction
known to feed the Krebs cycle'. The mechanism of the
reaction may be written this way:

CH,

CH, HC - CH,-CH,-CH,-CO-Thiamin+ CH,-CO-COOH

I I (Co-pyruvate oxidase) (Pyruvic acid)

CH,

/ \
CH, CH-

Cocnzymc A

/

CH,-CO

COOH

CH,

CH, CH-ff+ Acetyl CoA+ CO,

Tlie reduced lipoic acid complex would then be reoxi-
dized to the disulfide form by a suitable oxidant (e.g.
pyridine or flavin nucleotides). In order that the oxi-
dation of pyruvic acid can proceed, the enzyme has to
be present in its oxidized form. If it is kept in its reduced
form under the influence of the light-produced reducing
f)ower, the reaction cannot proceed and the pyruvic
acid formed during photosynthesis will not find its way
into the respiratory cycle. The reaction is inhibited
because only a small amount of the enzyme catalyzing
it exists in the required form, most of it being kept in
the other form under the "pressure" of the reducing
power generated by the light energy. This recalls a
similar phenomenon which has been known for a long
time, i.e. the suppression of the fermentation of carbo-
hydrates in favor of their oxidation under aerobic con-
ditions (Pastruk effect). This effect has been explained
in a manner similar to the one used here to account for
the inhibition of the respiration of photosynthetic in-
termediates'. The reduction of acetaldehyde to alcohol
requires a dehydrogenase in its reduced form; under
aerobic conditions the dehj'drogenase exists primarily
in its oxidized form, and the acetaldehyde instead of
beijig reduced is oxidized to acetic acid.

The sudden rise in phosphoglyceric acid and the
decrease in ribulose diphosjihate and sedoheptulose

' L. J. Rfed, I. C. Cunsalus, et at.. J. Am. Chem. Soc. ?J, 5920
(1951). -E. L. Patterson, rf a/., J. Am. Chem. Soc. 7J, 5919 (1951|.
- I. C. GuNSALUS, I.. Struclia, and U. I. O Kane, ,1. Biol. Cheni.
J9<, 859 (1952).- L. J. Reed and B. G. DeBusk, J. Am. Chem. Soc.
r<, 3457 (1952). -M. W. Bullock, <( a/., J. Am. Chem. Soc. 7<, 3455
(1952).

* S. OcpcoA, J. R. Stern, and M. C. Schm idfr. J. Biol. Chem.
/9J. 691 (1951). - S. KoRKEs, A.DelCamillo, I.C.Gvnsalus, and
S. OCHOA, J. Biol. Chem. /93, 721 (1951).

• O. Meverhof, Amer. Scientist iO, 483 (1952).

phosphate in the dark period, together with the obser-
vation that the dark rise in phosphoglyceric acid is
absent when the ribulose diphosphate concentration
was low during the light, confirms the earlier suggestion
that the phosphates of the C, and Cj sugars are pre-
cursors of the C, carbon dioxide acceptor*. ThLs, togeth-
er with evidence gathered in previous work* leads to
the following scheme for the photosynthetic cycle'
(Fig. 15).

Upon this basis an attempt might be made to relate
the two effects as follows ; when the light is turned off,
the reduction reactions requiring light are stopped,
whereas cleavage and carboxylation reactions continue
until their substrates are exhausted. Presumably, this
would lead to a depletion of the Cj and C, sugars, the
synthesis of which requires reduction steps (particu-
larly the six-equivalents leading to the tetrose which
itself is a very small reservoir), and a rise of phospho-
glyceric acid, the further fate of which is also dej)endent
upon reduction. However, a number of arguments seem
to contradict this view : (I) The observation that plants
fix radiocarbon in the dark immediately following a light
period at low carbon dioxide concentration, to form
a similar pattern of compounds as the one found in
photosynthesis shows that the sequence following phos-
phoglyceric acid is not blocked at once upon cessation
of illumination, but that the cells contain sufficient
reducing power to transform some phosphoglyceric
acid intocarbohydrates , (2) the cleavageof the pentoses
and heptoses into the Cj carbon dioxide acceptor and
a triose and pentose respectively is dependent on a
reduction step as well.

Fig. 15.

We are thus led to the suggestion that the rise in
phosphoglyceric acid is not be explained by a mere
interruption of the sequence, but that the rate of pro-
duction of phosphoglyceric acid at some time in the

' A. A. Benson, J. A. Bassham, M. Calvin, A. G. Hall, H. E.
HiRscH, S. Kawaguchi, V. H. Lvnch, and N. E. Tolbert, J. Biol.
Chem. ;9(i, 703 (1932).

' S. Kawaguchi, A. A. Benson, M. Calvin, and P. M. Mayes,
J. Am. Chem. Soc. 7/, 4477 (1952). - M.Calvin, The Harvey
Lectures 46, 213-251, 1951, in press.

' This scheme is intended to represent only changes in the carbon
skeletons. The reducing equivalents are indicated only to show redox
relationships between the known compounds. A number of the
isolated compounds are isoxiraers and have not been included.

90

[15. XII. 1952]

M. Calvin and P. Massini: The Path of Carbon in Photosynthesis

457

HO,P-OCH,-CHOH-CHOH-CO-CH,-0-PO,H

CO,. 2[H]

►■

CO,

HO,P-OCH,-CHOH— CHO
phosphoglyceraldehyde

-HOjP— OCHj— CHOH— COOH
phosphoglyceric acid

-> 2x-HOjP— OCHj-CHOH-COOH
^^^^ phosphoglyceric acid

first minute of darkness is actually higher than it is in
the steady state photosynthesis. This would be the case
if the C3-C2 cleavage of ribulose diphosphate, which in
photosynthesis presumably yields a triose phosphate
molecule beside the Q carbon dioxide acceptor, in the
dark yelds a molecule of phosphoglyceric acid instead
of the triose molecule. The overall reactions may be
represented above (not a mechanism).

This hypothesis is supported by the fact that the
triose phosphate also decreases in the dark.

The fact that the net result of the reaction sequence
in the light from ribulose diphosphate to phospho-
glyceric acid and triose phosphate is a reductive car-
boxylation and thus the reversal of the oxidative
decarboxylation which, in the case of pyruvic acid,
requires the presence of a cyclic disulfide compound
leads to the idea that the former sequence might be
catalyzed by a similar enzyme. This idea seems to be
supported by the results of an experiment performed
in this laboratory some time ago, which were difficult
to explain'.

In order to examine the relation between photosyn-
thesis and the glycolytic cycle, a series of experiments
similar to those described previously were performed
with added iodoacetamide which is known to inhibit
the action of triose phosphate dehydrogenase', pre-
sumably through a reaction with its sulfhydryl group'.
A 1% suspension of Chlorella in phosphate buffer was
allowed to photosynthesize in light of 2500 footcandles
and an atmosphere of 1% carbon dioxide, 5% oxygen
and 94% nitrogen. At various times before adding the
radioactive bicarbonate solution, iodoacetamide was
added to give a 1-5 x 10"* M solution. 1 min after
adding the radiocarbon, the cells were killed and
extracted.

After 8 min contact with iodoacetamide, the cells
were still able to fix 75% as much carbon dioxide as
non-poisoned cells otherwise treated the same way
(control). The amount of radioactivity in phospho-
glyceric acid was 50% of the control, and the amount
in sucrose had reached a sharp maximum of 3-5 times

' W. Stepka, Thesis University of California (June 1951).

» O. Meyerhof and W. Kiessling, Biochem. Z. 28/, 249 (1053).

' I.. Rapkins. C. r. See. Biol. (Paris) HJ, 1294 (1933),

that in the control. There was practically no radio-
activity in the ribulose diphosphate. After 90 min of
exposure to the poison the cells had practically lost
their ability of photosynthesis.

If, in the proposed photosynthetic cycle, the cleav-
age of the heptose and pentose phosphates is depen-
dent on an enzyme containing sulfhydryl groups, which
were more sensitive to iodoacetamide than the triose
phosphate dehydrogenase, a picture similar to the one
described would be expected : After short exposure to
the poison, in relatively low concentration, the lack of
Cj carbon dioxide acceptor would slow down the photo-
synthetic cycle. The synthesis of carbohydrates, how-
ever, would proceed almost without inhibition, thus
decreasing the concentrations of the intermediates in
the cycle. This would allow the compounds to reach a
higher specific activity during the period of exposure
to radiocarbon (cf. equation (2), change of specific
activity inversly proportional to concentration]. At
some time after administration of the poison, the su-
crose would be labeled faster than in the control due to
the higher specific activity of its precursors. After a
longer period, however, the rate of synthesis of sucrose
would decrease because the pool of its precursors would
be exhausted.

Zusammenjassung

Die Trennung des Phanomens der Photosynthese
griiner Pflanzen in eine Lichtreaktion und die vom Licht
unabhangige Reduktion der Kohlensaure warden di.s-
kutiert.

Die Reduktion der Kohlensaure und das Schicksal des
assimilierten Kohlenstoffs wurden untersucht mit Hilfe
der Spurenmethode (Markierung der assimilierten Koh-
lensaure mit C") und der Papierchromatographie. Ein
Reaktionszyklus wird vorgeschlagen, in dem Phosphogly-
zerinsaure das erste isolierbare Assimilationsprodukt ist.

Analysierung des Extraktes von Algen, die in einem
stationaren Zustand fiir langere Zeit radioaktive Kohlen-
saure assimilierten. lieferte weitere Auskunft iiber den
vorgeschlagenen Zyklus und gestattete, die am Zyklus
beteiligten Mengen einiger Substanzen ungefahr zu be-
stimmen. Die friihere Vermutung. dass Licht den Res-
pirationszyklus beeinflusst, wird bestatigt. Die Moglich-
keit der Mitwirkung von a-Liponsaure (a-lipoic acid) oder
einer verwandten Substanz, bei diesem Effekt und im
Photosynthesezyklus, wird erortert.

91

[Reprinted from the Journal of the American Chemical Society. 76, 1760 {1954).)
CopyriKht lfl54 by the American Chemical Society and reprinted by permission of the copyright owner.

[Contribution from Radiation Laboratory and Department of Chemistry, University of

California, Berkeley)

The Path of Carbon in Photosynthesis. XXI. The Cyclic Regeneration of Carbon

Dioxide Acceptor^

By J. A. Bassham, A. A. Benson, Lorel D. Kay, Anne Z. Harris, A. T. Wilson and M. Calvin

Received October 16, 1953

Photosynthesizing plants have been exposed to C'Oj for short periods of time (0.4 to 15 sec.) and the products of carbon
dioxide reduction analyzed by paper chromatography and radioautography. Methods have been developed for the degra-
dation of ribulose and sedoheptulose. These sugars, obtained as their phosphate esters from the above C'»Oj exposures and
from other experiments, have been degraded and their distribution of radiocarbon determined. The distribution of radiocar-
bon in these sugars, and other data, indicate that sedoheptulose phosphate and ribulose diphosphates are formed during
photosynthesis from triose and hexose phosphates, the latter being synthesized, in turn, by the reduction of 3 phosphoglyceric
acid. Further evidence has been found for the previously proposed carboxylation of ribulose diphosphate to phosphoglyceric
acid. Free energy calculations indicate this step would proceed spontaneously if enzymatically catalyzed. The efficiency
of this cycle for reduction of CO2 to hexose would be 0.9 if the reduction of each molecule of PGA requires the concurrent
conversion of one molecule of ATP and one of DPN (red) to ADP, inorganic phosphate and DPN (ox.).

Previously reported tracer studies of the path of action leading to phosphoglyceric acid (PGA)'
carbon in photosynthesis' led to the conclusion which is then reduced and condensed to fructose
that carbon is incorporated by a carboxylation re- (3, ^-he following abbreviations win be used throughout this paper:

(1) The work described in this paper was sponsored by the U. S. PGA, phosphoglyceric acid; DHAP, dihydroxyacetone phosphate;
Atomic Energy Commission. This paper was presented before the FMP, fructose monophosphate; GMP, glucose monophosphate;
Division of Biological Chemistry. American Chemical Society, at the SMP, sedoheptulose monophosphate; RDP ribulose diphosphate;
124th National Meeting. Chicago. Illinois. September. 19.53. ADP, adenosine diphosphate. ATP adenosine triphosphate; DPN,

(2) M Calvin. "The Harvey Lectures," Charles C Thomas Pub- diphosphopyridine nucleotide (Coenzyme I), oxidised form; DPNlHi],
lisbing Company. Spring&eld, 111., 1050-61, p. 218. diphosphopyridine nucleotide, reduced form.

92

April 5, 1954

Cyclic Regeneration of Carbon Dioxide Acceptor

1761

and glucose phosphates by a series of reactions simi-
lar to a reversal of glycolysis. These conclusions
were supported by the observations that when car-
bon-14 is administered to the photosynthesizing
plant as C'Oj, the first radioactive compound iso-
lated is carboxyl-labeled PGA, followed shortly by
dihydroxyacetone phosphate (DHAP), fructose
monophosphates (F.MP) and glucose monophos-
phate (GMP), both hexoses being 3,4-labeled. Af-
ter longer exposures of the plant to C'^Oj, radio-
carbon appears in other carbon atoms of PGA and
hexose and the distribution of activity is in agree-
ment with the above conclusions.

•C

I

*c + c*

•c

I

•c

I
•c

2[H|

•c

I
•c-

I
•c

PGA

••c

I

••c

I

•c

I

•c

hexose

Observations on the rate and distribution of la-
beling of malic acid*"' showed it to be the eventual
product of a second carboxylation reaction which
is accelerated during photosynthesis, and it was
proposed that this second carboxylation played a
part in the reduction of carbon in photosynthesis,
leading eventually to the formation of the two-car-
bon CO2 acceptor (A, above). Malic acid, itself,
apparently was precluded as an actual intermediate
by inhibition studies,' but was thought to be an in-
dicator of an unstable intermediate which was
actually the first product of the second carboxyla-
tion. The discovery' of rapidly labeled sedoheptu-
lose monophosphate (SMP) and ribulose diphos-
phate (RDP) led to their inclusion in the proposed
carbon reduction cycle leading to the two-carbon
CO2 acceptor.

The reciprocal changes in reservoir sizes of RDP
and PGA observed when algae were subjected to
light and dark periods' indicated a close relation-
ship, perhaps identity, between the RDP and the
two-carbon CO2 acceptor.

In order to test these conclusions, it was neces-
sary to design experiments involving very short ex-
posures of the plant to C'*02. In some of these ex-
periments, the C* was administered during "steady
state" photosynthesis, the environmental condi-
tions (hght, carbon dioxide pressure, etc.) being
kept as nearly constant as possible for the hour pre-
ceding and the time during the experiment. Deg-
radation methods have been developed for sedohep-
tulose and ribulose and complete distribution of
radioactivity within these sugars obtained.

The results of these experiments seem to obviate
the possibility that the second carboxylation reac-

(4) A. A. Benson, S. Kawaguchi, P. M. Hayea and M. Calvin. This
Journal, 74, 4477 (1952).

(5) A. A. Benson, et at., "Photosynthesis in Plants," Iowa State
College Press, Ames, Iowa, 1949, p 381.

(6) D. W. Racusea and S. Aronoff, Arch. Biochem. Biophys., 42, 25
(1953).

(7) J. A. Bassham. A. A. Benson and M. Calvin, J. Biol. Chem.,
18», 781 (1950).

(8) A. A. Benson, el at . ibid . 196, 703 (1952).

(9) M. Calvin and Peter Massini, Expcrienlia, 8, 445 (1952).

tion (leading to malic acid) is a step in carbon reduc-
tion during photosynthesis. Since no new evi-
dence has been found for the second "photosyn-
thetic" carboxylation, it would appear that a carbon
reduction cycle involving only one carboxylation
(leading to PGA) is more likely than the previously
proposed two-carboxylation cycle.

Experimental Procedure

Short "Steady State "Eiperiments. — Algae (Scenedesmus
obliquus) were grown under controlled conditions,' centri-
fuged from the growth medium, and resuspended in a 1% by
volume suspension in distilled water This suspension was
placed in a rectangular, water-jacketed illumination cham-
ber 6 mm. thick, through which was passed a continuous
stream of 4% COi-in-air (Fig. 1). From the bottom of the
chamber, a transparent tube led to a small transparent
pump constructed of appropriately placed glass valves and
two 5-cc. glass syringes mounted on a lever arm in such a
position that the syringe plungers moved in and out recipro-
cally about 5 mm. when the lever arm was moved back and
forth by a motor-driven eccentric. The output of the pump
was divided, the major portion being returned to the illu-
mination chamber and a smaller portion (20 ml. /minute)
forced to flow through a length of transparent "Transflex"
tubing of about 1 mm. diameter and thence into a beaker
containing boiling methanol. This solvent was found to
have an apparent killing time of less than 0.2 sec. as deter-
mined by the cessation of carbon fixation during photosyn-
thesis. The linear flow rate of algal suspension in the tube
was about 57 cm. /second. A solution of C'Oj in water
(0.0716 M, 110 MC./ml.) in a 30-cc. syringe was injected
through a fine hypodermic needle into the Transflex tubing
at a point a selected distance from the end of the tubing.
From the known flow rate of algal suspension in the Trans-
flex tubing and distance of flow from the point of injection
of C'*Oj to the killing solution, the time of exposure of the
algae to C'* was calculated. The flow of the C'Oj-contain-
ing solution was controlled by driving the syringe plunger
with a constant speed motor, and the flow rate was 0.5 ml./
minute. The resultant dilution of the algal suspension was
2.5% and the increment in total CO2 concentration less than
15%.

{hot pl>ti r

Fig. 1. — Schematic diagram of flow system for short exposure
of algae to C"Oi.

Since the flow of algal suspension in the tubing was not
turbulent, some difference in rates of flow at the center and
at the edge of the tubing was unavoidable. The extent of
this difference was approximately determined by injecting
a concentrated dye solution for about 0.5 sec. through the
hypodermic needle while the flow rate in the tubing was 20

93

1762

Bassham, Benson, Kay, Harris, Wilson and Calvin

Vol. 76

DEORAnATION OF SbDOHBPTULOSR

H

H

HC=N— N— CJI.

L

t=N— N— C^H,
I H

HOCH

HIO,

NaHCO,

Phenyl-
hyrirszine

Ha

CHjOH

I
HOCH

I
HCOH

\
HCOH

HCOH

CHjOH

H,
PtO,

CH,OH
HCOH
HOCH
HCOH

HCOH

I
HCOH

1
CHjOH

Dowex-50
100°

NalO.

HC=N— N— C«Hi

C==N— N— C,Hj + 3HC00H + HCHO
I H

CHO

1, 2, 3 4. 5, 6 7

CH,OH

I

-C 1

CHO I
b CHO ()

I i

•— CH

I
HjC

HCOOH
4

+

H-i

+

Acetobacter
suboxydans

H,C

CH.OH
HOCH
HOCH

HCOH

HCOH

HCOH

I
CH2OH

Acelokacter

CeCClO,),-

CO,

2

6HC00H

HIO,

suboxydans

W

Ce(CIO,),-

CHiOH

I
HCOH

I
HOCH

I
HCOH

HCOH

I

c=o

I

CHaOH
Guloheptulose

ml. /minute and observing the spreading of color during its
travel through the tubing. For the longest length of tubing
used, the dye was seen to reach the end of the tubing be-
tween 14 and 17 seconds, and at a shorter time between 9
and 11 seconds, so that the spread of flow in time appeared
to be about 20% of the flow time. The times given are
average times of exposure of the algae to C*. Use of the
dye also permitted observation of the mixing of C'Oj solu-
tkm with algal suspension and mixing time appeared to be
about 0.2 sec.

The entire apparatus was illuminated from each side by a
Bine- tube bank of 40-watt fluorescent lights (white) giving
a nniform intensity of abwit 2000 footcandles from each side.
During an experiment the algal suspension was illuminated
for an hour or more with 4% COj beifort the start of the flow
C* exposures. Exposures to C"Oj ranging from 1.0 to 16
sec. were then carried otit and the products of C'^ reduc-

2HCHO
1.7

4-

5HCOOH
2, 3, 4, 5. 6

Sedoheptulose

+

Mannoheptulose

CO,
6

v +

6HCOOH

tion analyzed in the usual way" by paper chromatography
and radioautography.

Short Soybean Experiments. — A single excised trifoUate
leaf from a soybean plant (var. Hawkeye) was placed in a
circular flat illumination chamber with a detachable lace.
The chamber was equipped with two tubes, the lower one
leading through a stopcock to an aspirator and the "Pper
one tlmMigh a two-way stopcock to a loop oonUining COj.
A loosely tied thread led from the leaf stem under the de-
tachable face gasket, thence through a boiling ethanol bath
and a glass tube to a weight. The illumination chamber was
partially evacuated, both stopcocks were closed, and clamps
removed from the chamber, the detachable face remaining
in position through atmospheric pressure. With the open-
ing of the upper stopcock, the C"Oi was swept into the cham-

(10) A. A. Benson, « •<., This Jootmal, W. 1710 (I960).

94

April 5, 1954

Cyclic Regeneration of Carbon Dioxide Acceptor

1763

Degradation of Ribulosb

H

Phenyl-
hydrazine

HC=N— N— CH.

C=N— N— CHi
I H

HC— OH

I
HC— OH

i
CHjOH

H

HIO,

NaHCO,

HC=N— N— CH,

i=N— N— CH, -f HCOOH 4- HCHO
I H

CHO

1, 2, 3 4 5

CHjOH

I

c=o

I

HCOH

I
HCOH

I
CHjOH

Ce(C10.)«-
H-^

COi

2

+ 4HCOOH

PtOa

HIO,

CHjOH

I
HCOH

I
HCOH

I
HCOH

CHjOH

ber by atmospheric pressure, the detachable face fell off
and the leaf was pulled into boiling ethanol. An estimated
exposure time of 0.4 sec. was obtained. The radioactive
products were extriicted and analyzed in the usual way.
In other experiments, longer exposure times were obtained
by holding the detachable face in position.

Degradation of Sugars. — The reactions used for the degra-
dation of the radioactive ribulose and scdohcptulosc are
shown in the accompany flow sheets

All radioactive material was purified on two-dimensional
paper chromatograms.'° Radioactive sedoheptulose was
converted to the anhydride by liealing at 11)0° with acid-
treated Dowex-.')0 for one hour, followed by chromatography
to separate the resulting equilibrium mixture.

Formation of the Osazones. — The hexosc and hcptose
osazones were made in the usual manner with phenylhydra-
zine hydrochloride, sodium acetate and acetic acid. Usu-
ally about 25 mg. of sugar carrier was used for the reaction.
Sedoheptulose osazone cocrystallized with glucosazone
sufficiently well for fructose to be used as carrier with sedo-
heptulose activity.

The radioactive arabinosazone was made by the method of
Haskins, Hann and Hudson" with 10 mg. of arabinose car
rier. The osazone was recrystallized once and diluted, as
desired for each degradation, with pure crystalline, non-
radioactive arabinosazone from a similar large-scale prepa-
ration .

Oxidation of Osazones. — The recrystallized osazones were
treated with periodate in bicarbonate buffer as described by
Topper and Hastings.'^ The reaction mixture was frac-
tionated to obtain all the products by centrifuging and
thoroughly washing the raesoxaldehyde osazone; distilling
the supernate plus washings to dryness in vacuo and treating
the distillate with dimedon to obtain the formaldehyde de-
rivative; and acidifying and vacuum distilling the residue
to obtain the formic acid, which was counted as barium for-
mate. All products were recrystallized before counting.

Cerate Oxidation of Ketoses. — The oxidation of the car-
bonyl carbon of a ketose to CO2 by cerate ion was performed
according to the method described by Smith." To a solu-
tion of an aliquot portion of radioactivity plus weighed
carrier (sedoheptulosan or fructose) was added a slight ex-
cess of 0.5 M cerate ion" in 6 iV perchloric acid, the final
concentration of acid being 4 N. The resultant COj was

(11) W. T Haskins. R. N. Hano and C. S Hudson. This Jodrnal,
U, 1766 (1946).

(12) Y. J. Topper and A B Hastings./. Biol C*«m, 1T9, 1255 (1949).

(13) G Frederick Smith. "Cerate Oiidimetry." G Frederick Smith
Chemical Company. Columbua, Ohio, 1942.

(14) We are indebted to Prof. John C. Speck, Jr , of Michigan State
College, East Lansing. Michigan, for valuable data and suggestions re-
garding the use of cerate in these oxidations.

2HCH0 4- 3HCOOH
1,5 2.3,4

swept with nitrogen into COj-free sodium hydroxide. The
reaction was allowed to proceed for one hour at room tem-
perature and then the COj was precipitated and counted as
barium carbonate. In all cases the theoretical amount of
carbon dioxide was evolved.

Formation and Oxidation of Sugar Alcohols. — The radio-
active sugars were hydrogenated with platinum oxide as de-
scribed previously' and chromatographed on paper for puri-
fication. Carrier ribitol or voleraitol was added to an ali-
quot of radioactive alcohol and a slight excess of paraperiodic
acid was added. The reaction was allowed to stand at room
temperature for 6-7 hours. Then the formic acid and form-
aldehyde were distilled off in vacuo. After the formic acid
was titrated with barium hydroxide, the fonnaldehyde was
redistilled and precipitated as formyldimedon. Both the
residue of barium formate and the formyldimedon were re-
crystallized before plating and counting.

Bacterial Oxidation of Hepitola from the Reduction of
Sedoheptulose. — The radioactive reduction products of
sedoheptulose gave only one spot on chromatography.
After elution these were oxidized by Acetobacter suboxydans
in a small-scale modification of the usual method." Two
mg. of volemitol and about 100 d- of solution of radioactive
heptitols were placed in a 7-mm. diameter vial. An amount
of yeast extract sufficient to make a 0.5% solution was
added. The vial was sterilized, then inoculated from a 24-
hour culture of Acetobacter and left for a week at room tem-
perature in a humid atmosphere.

When the bacteria were centrifuged from the incubation
mixture and the supernatant solution was chromatographed,
three radioactive spots were obtained. The two major
spots were mannoheptulose and sedoheptulose, the oxidation
products of volemitol. The third had R, values very simi-
lar to those of fructose and cochromatographed with au-
thentic guloheptulose'" ( if f in phenol = 0.47; Ri in butanol-
propionic acid-water = 0.24). After treatment with Do-
wex-50 in the acid form at 100° for one hour, this third com-
pound gave a new compound which cochromatographed
vrith guloheptulosan (Ri in phenol = 0.62; i?i in butanol-
propionic acid-water = 0.30). It thus appeared that the
radioactive heptitols are volemitol and 0-sedoheptitol which
cochromatograph in the solvents used.

Both mannoheptulose and guloheptulose have carbon
chains inverted from the original sedoheptulose. In the
small-scale fermentations, however, the oxidation appeared
to be incomplete. The original alcohol did not separate
chromatographically from mannoheptulose. Therefore,

(16) (a) L. C. Stewart, N. K. Richtmyer and C. S. Hndsoo, TBM
JoDRNiL, 74, 2206 (1952); (b) we wish to express oor •ppreci»tion to
Dr. R. Clinton Fuller for his development of the micro-fermentation.

(16) We wish to thank Dr. N. K. Richtmyer for his generons gift of
crystalline guloheptulosan.

95

1764

Bassham, Benson, Kay, Harris, Wilson and Calvin

Vol. 76

the easily purified guloheptulose was used for subsequent
degradations witli cerate ion, despite its much poorer yield.

Oxidation of Sedoheptulosan. — The radioactive sarnple
and carrier were treated with sodium periodate as described
by Pratt, Richtmyer and Hudson" and allowed to stand at
room temperature for 3-4 days to give time for most of the
formate to be released from the intermediate ester. Then
the mixture was acidified with iodic acid and the formic
acid was distilled in vacuo. This was then counted as
barium formate.

Results

In Fig. 2, the radiocarbon fixed in a "steady
state" photosynthesis with Scenedesmus is shown as
a function of time of exposure of the plant to C'^Oj.

glucose monophosphate and fructose monophos-
phate ciu-ves although individual points are more
erratic, probably due to the relative instability of
the ribulose diphosphate.' The appearance of
compounds other than PGA with a finite rate of
labeling at the shortest times is demonstrated in
Fig. 4 in which the percentage distributions of
PGA and of the total sugar phosphates are shown.

TIME (SECONDS}.

Scent

e e 10

TIME (SECONDS),

Fig. 2. — Radioactivity incorporated in "steady state" photo-
synthesis with Scenedesmus.

The rate of incorporation of C'*Os appears to be
reasonably constant over the period of the experi-
ment. The distribution of radioactivity among
various labeled compounds is shown in Fig. 3. The

a 10 12

TIME (SECONDS) ,

Fig. 3. — Distribution of radioactivity among compounds formed during "steady
state" photosynthesis with Scenedesmus.

curve for the sugar diphosphates, principally ribu-
lose diphosphate, is not shown but lies between the

(17) J W. Pratt, N. K. Richtmyer aod C. S. Hudson, Tais Joumnal,
T4, 2200 (ieS2).

Fig. 4. — Distribution of activity in "steady state"
desmus.

The extrapolations of the PGA and sugar phos-
phates to zero time would give about 75 and 17%,
respectively. The remaining 8% not shown is dis-
tributed among malic acid (3%), free glyceric acid
(2%) and phosphoenolpyruvic acid (3%).' The
percentage distribution among the sugar phos-
phates is shown in Fig. 5 where it is seen that no
single labeled sugar phosphate predominates at
the shortest times.

These data alone do not permit
assignment of an order of preced-
ence of the various labele(l com-
pounds in the path of carbon reduc-
tion. In order to make such an
assignment it would be necessary to
measure the relative rates of in-
crease in specific activity of the
various compounds. If the slopes
of the ciu^es shown in Fig. 3 are
measured between 2 and 10 sec,
rates of increase in total radioactiv-
ity are obtained. If these rates are
divided by the cellular concentra-
tion of the compounds involved,
rates of specific activity increase are
obtained. This has been done using
measurements of concentrations
made by two independent'" meth-
ods which agreed fairly well in rela-
tive Older {i.e., PGA concentration:
GMP concentration = 4:1). The
resulting values ranged from 0.3 for
GMP to 1.0 for PGA, with FMP,
DHAP, RDP and SMP falling be-
tween these values when the rates
for these compounds were divided by 2, 1, 2, 1, 1
and 3, respectively, to allow for the number of
carbon atoms which degradation data reported be-

(18) A. A. BeiuoD. Z. BUUrochtm. U, 848 (19&2).

96

April 5. 1954

Cyclic Regeneration of Carbon Dioxide Acceptor

1765

low show to be labeled significantly at these short
times. This calculation is quite approximate, the
concentration of compounds involved being meas-
ured in experiments with algae photosynthesizing
under somewhat different conditions {i.e., 1% CO2
instead of 4%). However, such a calculation does
show more clearly the rapidity with which radio-
carbon is distributed among the principally labeled
carbon atoms and the difficulty in assigning an
order of precedence of labeled compounds on the
basis of labeling rates alone.

The fact that compounds besides PGA have fi-
nite initial labeling slopes (which results in their
percentage activity not extrapolating to zero at
zero time) might be explained in several ways. One
possibihty is that during the killing time some of
the enzymatic reactions (in this case reduction of
PGA and rearrangement of the sugars) may not be
stopped as suddenly as others (the carboxylation to
give PGA) or may even be accelerated by the ris-
ing temperature prior to enzyme denaturation.

Another explanation is that some of the labeled
molecules may be passed from enzyme to enzyme
without completely equilibrating with the active
reservoirs which are actually being measured. This
sort of enzymatic transfer of radiocarbon could
invalidate precedence assignments based on rates of
increase in specific activities since the reservoirs
would no longer be completely in the line of carbon
transfer. That the equilibration between reser-
voirs and enzyme-substrate complexes is rapid com-
pared to the carbon reduction cycle as a whole is
indicated by the fact that all the reservoirs become
appreciably labeled before there is an appreciable
label in the a- and /3-carbons of PGA, the 1-, 2-, 5-
and 6-carbons of the hexoses, etc. In any event, it
would appear to be safer to establish the reaction
sequences from qualitative differences in labeling
within molecules (degradation data) and changes in
reservoir sizes due to controlled changes in one en-
vironmental variable rather than from quantita-
tive interpretations of labeling rate data.

Table I shows the results of degradations on sug-
ars obtained from the soybean series. The first
column shows the variation in labeling of carbon

Table I
Radioactivitv Distribution in Sugars SEDOHEPTin.osE

AND HeXOSE from SOYBEAN LEAVES

Time,
sec.

0.4
0.8
1..")
3.5
.■i.O
8.0
10.0
20.0

300

Sedum

^ .Sedoheptiilose

C-4 C-1.2.3 C-4.5,G C-7 C-2

. Hexose

C-1.7 C-6 C-1,2.3 C-4,.'j.6

8
IS
24
20
29
24
28
21
14
12

3."'

4.''

3fi

44
37

.^7
fiO

(i4

47
48

52
51

35 12

7

12.5
12,5

28 15

-o

TIMC (SECONDS),

Fig. 5. — Distribution of radioactivity incorporated in
"steady state" photosynthesis with Scenedesmus: ©, sedo-
heptulose phosphate; 9, glucose phosphate; ®, dihydroxy-
acetone phosphate; O, fructose phosphate.

since the carbon dioxide is depleted just prior to the
administration of C'Oa. Included in the table is
a complete degradation of a sedoheptulose sample
from Sedum speclabile grown in radioactive carbon
dioxide for two days (kindly supplied by N. E.
Tolbert, Oak Ridge National Laboratory). As-
suming this sample is uniformly labeled, its degra-
dation indicates the probable limits of accuracy of
the other degradations — about ± 10% of the ob-
tained value, mainly due to plating and counting
errors resulting from the low amount of radioactiv-
ity available for degradation. The five degrada-
tions on sedoheptulose make it possible to obtain
separate values for all the carbon atoms. Although
the carbon-fourteen labels of carbon atoms 1 and 0
were not determined in the case of the Scenedesmus
experiments, they were assumed small and approxi-
mated equal to carbon-fourteen labels found in
carbons 2 and 7, by analogy with the soybean leaf
experiments where the labels of all carbon atoms of
the sedoheptulose were determined. The label in
each carbon atom of the ribulose can be obtained
individually from the three degradations performed.
The distributions in Table II should be interpreted
as a clear qualitative picture of the position of the
radioactivity within the molecule rather than as a

Table II

Radioactivity Distribution in Compounds from Flow
Experiments (Algae)

Glyceric
acid

S2

6

6

-5.4 Seconds-

Fructose

3

3

43

42
3
3

Sedohep-
tulose

2

2
28
24
27

2

2

Ribu-

11

10

09

5

3

8 5 Seconds

Sedohep- Ribu-

tulose lose

3
22

11

11

04

8

5

number four of sedoheptulose obtained from soy-
bean leaves exposed to C'''02 for very short periods.
These soybean leaf experiments are, of course, not
intended to represent "steady state" photosynthesis

quantitative picture. Fewer points were taken in
this "steady state" flow experiment than in the
one described earlier in order to obtain more la-
beled sugar per point for degradation purposes.

97

1766

Bassham, Benson, Kay, Harris, Wilson and Calvin

Vol. 76

In other experiments" the Scenedesmus have been
kept at a steady state of light, temperature, CO2
pressure, etc., and constant C'K)2 specific activity
until successive samplings of the suspensions showed
uniform labeUng ("satiu-ation") of all the common
photosynthetic reservoirs (PGA, RDP, GMP, etc.).
The total CO2 pressure was then rapidly changed
from 1% C02-in-air to 0.003% in air, all other en-
vironmental conditions, including the specific ac-
tivity of C"02, being kept constant. The condi-
tions of this experiment were, therefore, similar to
those used previously' to study changing steady
state except that CO2 pressure was changed in-
stead of illumination. In the case where the CO2
pressure was lowered (Fig. 6), the initial effects on
the reservoir sizes of PGA and RDP were just the
opposite of those observed when the illumination
was stopped. Lowered COi pressure resulted in an

RDP

Triose phosphate

4'

B -«-

A -*-

20

>

a
Pi

10

1% CO,

PGA

RIBULOSE Old
AREA

SCENEDESMUS 6' C

-600

-200

-100

45 minutes C"0, at 6° C.
Fig. 6.

100

Time in seconds.

increase in the reservoir size of RDP and a decrease
in that of the PGA. After a time the reservoir of
RDP passed through a maximum and dropped to
a lower level but the new steady state RDP res-
ervoir was now greater relative to that of PGA.
The labeled glycolic acid present, though rather a
small percentage of total activity, increased many
fold when the COa pressure was lowered. The res-
ervoir of glycolic acid increased much more slowly
than that of the RDP and did not pass through a
corresponding maximum, thus eliminating the pos-
sibility that most of the labeled glycolic acid was
formed by thermal decomposition of RDP subse-
quent to killing of the cells.

Discussion
1. Origin of PGA. — It has been suggested that
RDP is the compound which supplies the two-
carbon atoms for the carboxylation reaction lead-
ing to PGA.' If the reactions of these compounds
are represented by

(19) A. T. Wilioo, Thesis, to be submitted as partial fulfillment ol
requirements for the degree of Doctor of Philosophy. UnlTenltjr of
California.

Sugar rearrangements

then the initial changes in reservoir sizes which
would accompany changes in light or COj pressure
can be predicted. When the light is tmned off,
reducing power [H] decreases, so the reservoir of
PGA would increase and that of RDP decrease. If
CO2 pressure decreases, then the reservoir of RDP
would increase and that of PGA would decrease.
Both effects, as well as those opposite effects which
would be expected to accompany a resumption of
light or increase in COj pressure,
have been observed. These re-
sults support the proposal of a
carboxylation of RDP to give
two molecules of PGA or the
reductive carboxylation to give
one molecule of PGA and one
of phosphoglyceraldehyde as
the first step in the path of
carbon dioxide reduction.

It is also possible that the
products of this carboxylation
may be phosphoglyceraldehyde
and 3-phosphohydroxypyru-
vate. In this case subsequent
reduction of the phosphohy-
droxypyruvate would give first
PGA and then phosphoglycer-
aldehyde. The reaction of phos-
phoglyceraldehyde with hy-
droxypyruvate to give ribulose
monophosphate and COj has
been demonstrated by Racker^°
to take place under the influ-
ence of the transketolase en-
zyme. However, the increase in PGA concentra-
tion which is observed on stopping the illumina-
tion of photosynthesizing algae,' would probably
not be seen if a reduction of hydroxypyruvate
were required to form PGA since the reducing agent
would presumably no longer be formed in the dark.
Moreover, paper chromatographic analysis should
detect either phosphohydroxypyruvate or its de-
carboxylation product, phosphoglycolaldehyde, and
neither have been found in our experiments. When
C'*-labeled hydroxypyruvate was administered to
algae in this Laboratory, the labeled acid was me-
tabolized to give a variety of compounds, similar to
those formed from labeled pyruvate or acetate, which
were related more closely to the tricarboxylic acid
cycle and fat synthesis than to the compounds usu-
ally associated with carbon reduction in photosyn-
thesis.

There remains the possibility that the RDP first
spUts to give a three-carbon molecule and a free
two-carbon fragment which is then carboxylated.

(20) B. Racker, G. de la Haba and I. G. Leder, This Joijknal, Ti.
lOlO (1068).

0003% COi

200

300

98

April 5, 1954

Cyclic Regeneration of Carbon Dioxide Acceptor

1767

However, if the glycolic acid is an indication of the
free two-carbon fragment, then the observation
that its increase in concentration (following reduc-
tion in COj pressure) is not as rapid as the increase
in RDP concentration suggests that the Cj com-
fjound is not as closely related to the carboxylation
reaction as the RDP.

2. Origin of Ribulose Diphosphate. — If one
considers the principal labeling at short times of
PGA,^ RDP, SMP and the two hexose monophos-
phates^ as, respectively

CH20©
CHOH
•**COOH

PGA

CHjOH

Lo

•CHOH

I
♦CHOH

I
CHjO© 'CHOH

CHOH

CH,0<g)
RDP SMP

•CHjO©

I

*c=o

I

"CHOH

I
CHOH

I

C

4

c

I

c

HMP

it apj>ears that the ribulose is not derived entirely
from a Ce ^- Ci -f- C6 split or a C7 — >• C2 + Ce split.
No five carbon fragment of the hexose or the hep-
tose molecules contains the same distribution of
radiocarbon as ribulose. The combination of C3
with a labeled C2 fragment could account for the
observed radioactivity. However, some mecha-
nism for the labeling of the C2 fragment would be re-
quired. One such mechanism would be the break-
down of hexose simultaneously into three Cj frag-
ments,^' and since carbon atoms 3 and 4 of hex-
ose are labeled, a labeled Cj fragment might thus
be obtained. To our knowledge there exists no
precedent as yet for this type of reaction.

Another way of accounting for the observed dis-
tribution of radioactivity which seems quite plaus-
ible in view of the rapidly accumulating enzymatic
evidence for the reverse reaction '''•^'"-■' is the forma-
tion of ribulose from sedoheptulose and triose. This
reaction could result in the observed labeling

CH,OH •♦CHO

I I

=0 + CHOH

I

I

*CHOH CH2O©

I
•CHOH

I
•CHOH

I
CHOH

CHjO© phospho-
SMP glyceraldehyde

CHjOH

I

c— o -t-

I

•CHOH

I
CHOH

I
CHzO©

ribulose

•CHO

I
•CHOH

I
•CHOH

CHOH

I
CH2O© J

ribose

•C

I
•C

I

•••c

I

c

I

c

monophos- monophos-
phate phate

If the ribose-5-phosphate and ribulose-5-phosphate
are then converted to RDP the resulting distribu-

(21) H. GaffroD, E. W. Fager and J. L. Rosenberg, "Carbon Dioxide
Fixation and Photosynthesis," Symposia of the Society for Experi-
mental Biology (Great Britain), Vol. V, Cambridge University Press,
19S1.

(22) B. Aadrod, R. S. Baudurslii, C. M. Greiner and R. Jang. J.
Biol. Chem.. SOI, 619 (1953).

(23) B. L. Horecker and P. Z. Smymiotis, This Journal, T4, 212S
(1952).

(24) B L Horecker and P Z. Smymiotis, itruf , It, 1009 (1963).

tion of label would be that observed (carbon skele-
ton at right of reaction).

3. Origin of Sedoheptulose.— The degradation
data appear to eliminate the possibility of formation
of sedoheptulose by a simple 6 + 1 or 6 -f 2 addi-
tion, if we assume that no special reservoirs of pen-
tose and hexose exist with distributions of radioac-
tivity different from those measured. A reverse
of the reactions proposed above for formation of
RDP would require segregation of ribose and ribu-
lose distributions as well as some other mechanism
for labeling the ribose in the manner shown. It
does seem likely that all the reactions involving
rearrangements of sugars and perhaps those in-
volving reduction of PGA as well are at least par-
tially reversible in the time of these experiments.
If all these compounds are intermediates in a cycle
of carbon reduction, then during steady state pho-
tosynthesis there will be a net "flow" of radiocarbon
in the "forward" direction, but the possibility that
the distribution of radiocarbon in later intermedi-
ates may reflect to some extent that of earlier inter-
mediates cannot be entirely ignored.

The condensation of a triose with a C4 fragment
would give the observed distribution if the C4 frag-
ment is labeled in the carbon atoms 1 and 2

CHjO©

c=o

♦CHji

OH

•CHO

I
•CHOH

I
CHOH

I
CHsO©

CHsO©
C=

•i:

-o

DHAP

HOH

1
•CHOH

I
•CHOH

I
CHOH

I
CH,0©

Enzymatic evidence for this reaction and its re-
verse has been reported. ^''^^

4. Origin of the Four-Carbon Fragm«it. — Two
possible modes of formation of the four-carbon
fragment with the above labeling are a Cj + C3
addition, and a Ce -+ [C2] -|- [C4] split. The C, +
C3 addition which leads to malic acid produces a
C4 fragment labeled in the two terminal positions."
Therefore, the reduction of the dicarboxylic acid
formed as a precursor to malic acid could not result
in a C4 fragment with the C'^ distribution required
for the formation of 3,4,.5-C'* labeled sedoheptulose.
The rapid introduction of radiocarbon into malic
acid in earlier experiments* can be accounted for if
it is assumed that the reservoir size of malic acid,
depleted during the air flushing prior to the addi-
tion of HC'HDa", was increasing after the addition
of radiocarbon due to the increase in total CO2
pressure. Also, after the carboxyl group of PGA
and phosphoenolpyruvic acid have become appre-
ciably labeled, the mahc acid is doubly labeled.

It is interesting to note that in the long term
"steady state" experiments in which the light was
turned off,' the mahc acid concentration dropped
when the light was turned off rather than increas-
ing as PGA concentration increased. If maUc acid
were an indicator of a four-carbon intermediate in
carbon reduction, the product of a second carboxyl-

(25) B L. Horecker and P Z Smymiotis, ifriJ, 76, 2021 (1853).

99

1768

Bassham, Benson, Kay, Harris, Wilson and Calvin

Vol. 76

ation, then one would expect its concentration to
increase in the dark for two reasons. First, there
no longer is reducing power which would reduce
the carboxylation product to sugar if this product
were an intermediate in CO2 reduction. Second,
the rate of formation of malic acid should increase
since this rate depends on the CO2 concentration
(which remains constant), and the concentration
of phosphoenolpyruvic acid (which increases paral-
leUng the PGA concentration). The decrease in
malic acid concentration could be easily explained
on the basis of the proposed light inhibition of py-
ruvic acid oxidation.' The cessation of illumina-
tion should permit increased pyruvic acid oxidation,
thus providing more acetyl-CoA, which can react
with oxaloacetic acid derived from malic acid.

It is possible that there is a different "second
carboxylation" (Cj + Ci) leading eventually to a
four-carbon fragment which can react with those
to give sedoheptulose, but there seems to be no
evidence whatever for such a reaction at present.
Moreover, such a reaction should lead in short
times to a four-carbon fragment somewhat more
labeled in the terminal carbon position than in the
second carbon position due to dilution of the carbon
introduced in the first carboxylation reaction by
the PGA and triose reservoirs. This is not the
case — in fact in the very shortest times the ter-
minal carbon position of the hypothetical d frag-
ment (carbon four of sedoheptulose) is actually less
labeled than the second position, at least in the soy-
bean experiments.

The most likely source of the C4 fragment seems
to be a Co -► [C*] -f [C2] split. Trioses could then
react with [C4] and [C2] to give sedoheptulose and
ribulose, respectively. One possible formulation of
these reactions would be

C

I
C=

4
4

•CHO
=0 -I- CHOH
CHsO©

•C"

I
•C

CH.O©

+ c=o —

•CHjOH

CH,OH

c=o -f

I

•CHOH

I
CHOH

CH.O©

CH,0©

I

c=o

I

•CHOH

I
•CHOH

:hoh

HOH

CHjO©

The first reaction as written above would be a
transketolase reaction of the type reported by
Racker, et al.,^" who found that this enzyme splits
ribulose-.")-phosphate, leaving glyceraldehyde-.3-
phosphate and transferring the remaining two
carbon atoms to an acceptor aldehyde phosphate of
2-, 3- or 5-carbon atoms. No mention was made of
the effect of transketolase on ribulose-5-phosphate
with erythrosc-4-phosphate which would result in

the formation of fructose phosphate by a reaction
which is just the reverse of the Ce-*- [C2] + [Ci] split
written above. ^'

The labeling of carbon number 4 in sedoheptulose
observed in the case of the very short periods of
photosynthesis with soybean leaves seems to cast
some doubt on the Cj -»■ [C2] -f [C4] split unless
one can assume that the Ce which splits is itself
not symmetrically labeled at the shortest times, due
to different specific activities of the two trioses
which react to give hexose

CH,0©

CHOH

I
•**COOH
PGA

CHjO©

2[H

CHjO©

, I
>CHOH

- incomplete -
-equilibration-

I

•*CHO

It

later, hence
more complete
equilibration

w

CHsO©

CHjO©

. I
C=0

" I

•CHjOH

F-l,6-DiP

\

CHjOH

I

c=o

CH,OH

I

c=o

•CHOH

I
••CHOH

I
CHOH

CHjO©

••CHO

I
CHOH

I

I
•CHOH

I

CHOH

CHjO© I

CH2O'"

c=o C=0

••CHOH "CHjOH

•CHOH

I
••CHOH <

CHOH

CH,0©

Degradation of fructose from the 0.4- and 0.3-
sec. experiments showed no significant difference
between the two halves of fructose. It is quite
possible, however, that the differences in denatura-
tion rates of various enzymes mentioned earlier
may influence the results in these short times.

Combining these reactions with others aheady
proposed we have the following cyclic path of car-
bon reduction during photosynthesis. The car-
bon fragments specified only by the number of car-
bon atoms in their chains are all at the sugar level
of reduction

3Ci -I- 3C0j —

121H
6PGA

2C, -

C, -I- 2C, -

C, + C,

6PGA

> 6C,

C,

Cs-h C,
■>2C.

The net reaction for each turn of the cycle is

12 (HI -f 3C0,-

C.H,0, -I- 3H,0

The operation of this cycle is illustrated in Fig. 7.

5. Energetics of the Carbon Reduction Cycle. —
That the enzymatic rearrangements of sugars re-
quires no additional supply of energy in the form
of ATP or other sources seems to be indicated by
the experiments with isolated and partially purified
enzyme preparations in which such rearrangements
have been carried out without the addition of
energy donors. The free energy change of the car-
boxylation reaction can be roughly estimated. Es-
timating the free energy difference between ribose-

(26) Since this was written, a private communiration from Dr.
Racker has informed us that he has observed this reaction with F-6-P

100

Cyclic Regeneration of Carbon Dioxide Acceptor
Chl* [0]

1769

polytoechoridet

0 (ll*IM<)

(ribulott)

Fig. 7. — Proposed cycle for carbon reduction in photosynthesis. Heavy lines indicate transformations of carbon com-
pounds, light lines the path of conversion of radiant energy to chemical energy and the subsequent use of this energy
stored momentarily in some compound (E), to form a reducing agent [H] and oxygen from water.

.5-phosphate and RDP equal to that between GMP
and fructose diphosphate, the free energy change
for the reaction below is about —7 kcal.^'-^

CH,0©

c=o

CHOH

CHOH + CO, + H,0

i

HOH

CH,0©

2CHOH + 2H-'

i
CO,

CH,0©

(5 X \()-' M) (10-«A/)

(1.4X10-»Af) (10-' W)

Af 2© -176 -95

-57— 2(© -158) 2(-95)

AF - -7 kcal.

In the above calculation the concentrations of
RDP and PGA measured with Scenedesmus during
photosynthesis with 1% COz' are used. The mech-
anism of the reaction may consist of the addition of
CO2 to the 2,3-enediol sugar formed by enolization
of the RDP. The intermediate compound would
be 2-carboxypentulose-3. The free energy for the
formation of the ion of this acid and H+ {pH 7)
from COi and RDP is estimated as zero when the
concentration of the intermediate acid is 10~' M.
Subsequent hydrolytic splitting of this compound
to two molecules of PGA and another hydrogen ion
would proceed with a free energy change of —7
kcal.

The energy required to maintain the operation of
the proposed carbon reduction cycle might be sup-
plied entirely in the reduction of PGA to triose
phosphate. If this reduction were accomplished
by a reversal of the enzymatic reaction usually writ-

(27) The intemal energy of the -PCHH" group, exclusive of the
energy of bondiog to the remainder of the molecule ia here denoted by
© and assumed constant throughout.

(28) J. A. Bassham, Thesis, submitted as partial fulfillment of re-
quirements for the degree of Doctor of Philosophy, University of Cali-
fornia, 194B.

ten, each "turn" of the cycle would be represented
by three times the reaction

2DPN[H,1 -I- 2ATP -|- CO, — *■ |CH,0| -f (A)
+ 2DPN -t- 2ADP -t- 2© -f H,0

This is the sum of the reactions

2[DPN1H,1 + V1O. — »-

DPN -I- H,0) AF - -101 lead. (B)

2[ATP — »-ADP-f©I A^ - -21 kcal. (C)

CO, -f H,0 — *- O, -f- |CH,0| HF " -1-116 kcal. (D)

The efficiency of the transfer of energy of reactions
B and C to reaction D is 116/(21 + 101) = 0.96.

However, additional energy might be supplied
to the operation of the cycle by phosphorylation
reactions in which additional molecules of ATP are
required. One such reaction may well be the phos-
phorylation of ribulose monophosphate to give ribu-
lose diphosphate. In this case, one additional
molecule of ATP would be required per molecule
of CO, reduced. The efficiency of the net reaction
(A') would then be 116/132.5 = 0.88.

2DPN[H,) -I- 3ATP + CO, >-

|CH,0| -I- 2DPN -f- 3ADP -f 3© -t- H,0 (A')

The over-all efficiency of photosynthesis would
be the product of 0.96 or 0.88 and the efficiency of
the process by which water is photolyzed to give
oxygen with the production of reducing power, fol-
lowed by the conversion of the energy of this re-
ducing power to DPN[H,] and ATP.

If the mechanism for photolysis of water in-
volves thioctic acid, as has been proposed,^ the
energetics of the photochemical and following steps
can be estimated

[Y + HOH -^ If

(E)

S — S

SH SOH

(29J J. A. Barltrop, P. M. Hayes and M. Calvin, to be pnblbhed.

101

1770

Bassham, Benson, Kay, Harris, Wilson and Calvin

Vol. 76

(where the symbol / represents the side chain:
— (CH2)4COjH).

+ 1 I + HjO + 'AO, (F)
2SH SOH SH SH S — S

In this process, two quanta are required for each
dithiol molecule formed. The stored energy is the
sum of the energies of the two half reactions

2H+ + 2e- + "AOj Af = +37.5 kcal. (G)

H,0

C(

+ 2H
S — S
which is

H,0 +

+ 2e--

HS

E =• -0.3 v.»
AF= +13.8 kcal.

SH (H)

S — S

(2A») {\^

- — ► I I +'AO,

i>.F

HS SH

51.3 kcal.

(I)

Since the energy available from two light quanta at
7000 A. is 2 X 40.7 or 81.4 kcal., the efficiency of
this process would be 51.3/81.4 = 0.63.

If Co-I is used in the reduction of PGA, the re-
duced coenzyme could be formed with high efficiency
from the dithiol

DPN + SH SH

DPNlH.l + S — S

LP = -0.8 kcal. (J)

The required ATP could be formed in some way

by oxidation of SH SH or DPN [Hi] by an ener-
getic coupling of the reactions

DPNIH,] + 'AO, — *■ DPN + HjO

AF = -50.5 kcal. (K)
ADP + © > ATP Af - +10.5 kcal. (L)

Since from one to four molecules of ATP might be
formed per DPNfHj] oxidized, a wide range of ef-
ficiencies would be possible. A value of three has
been suggested*' and if this is used, the resulting
coupling reaction could be written

DPN[H,] + VtO» + 3ADP + 3© *-

DPN + H,0 + 3ATP (M)

Multiplying reaction J by 3 and combining with
reaction M we have

(T

3SH SH + 2DPN + 3ADP + 3© + 'AO, — *■

(SO) I. C. GunsaJus, Bymposlnm oa "Mechaaism of Bazyme Ac-
tion," McCollum-Pratt Inatitute. Johiu Hopkins Univcnity, 1033, to
be published.

(31) A, L. Lehnioger, "Phosphorus Metabolism," Vol.1, Johns Hop-
kins University Press, 1961, page 344.

+ 2DPN[H,] + 3ATP

0

+ H,0 + 3S — S

(N)

in which the stored energy is 132.5 kcal. and the en-
ergry expended is three times reaction I = 154 kcal.
The efficiency of the energy transfers represented
by reaction N is then 132.5/154 = 0.86.

Combining the efficiencies of reactions A', I
and N results in a calcidated over-all efficiency for
photosynthesis of 0.88 X 0.63 X 0.86 = 0.48.
Since Uie mechanism outlined above would require
six quanta for each molecule of carbon dioxide re-
duced (two quanta for each molecule of dithiol used
in reaction N) this efficiency can be obtained di-
rectly from the energy of these quata (244 kcal.) and
the energy of reaction D: 116/244 = 0.48.

Higher apparent efficiencies would be obtained
at low light intensities where the dark internal con-
version of prior storage products (involving no net
uptake of oxygen or evolution of COj) would sup-
ply appreciable amounts of ATP, DPNH, reduced
thioctic acid and possibly intermediates of the Oj
evolution chain as well."

Since reaction I as written stores only 51.3 kcal.
of 81.4 kcal. available, it is posable that some
mechanism may exist for the storage of some of this
energy in the form of either additional reducing
power or high energy phosphate. In this case, the
over-all efficiency would be higher.

6. Other Biological Evidence. — The intercon-
versions of the five-, six- and seven-carbon sugars
are being investigated by several laboratories. The
postulated cychc reactions which our data suggest
are consistent with the observations of these various
groups. Both the work of Axelrod, et al.,'* with
spinach preparations and the results reported by
Dische and Pollaczek" with hemolysates demon-
strate the sequence

ribose phosphate — >■ heptulose phosphate +

triose phosphate — *■ hexose phosphate

Recently studies have been made of the distribu-
tion of C" in products resulting from conversion of
l-C* labeled pentoses. Neish" has studied the
products of bacterial metabolism of several pentoses
while Wolin, et al.,** investigated the products of
enzymatic conversion of ribose-5-phosphate. In
both cases, the distribution of radioactivity in the
products coidd be accounted for by a reversal of
the reactions herein suggested, although a limited
number of other interpretations of their data are
possible.

BCKKBLBV, Cal.

(32) Z. Dische and B. Pollaczek, paper presented at Second Inter-
national Congress of Biochemistry, Paris, France. 1952.

(33) A. C. Neish. paper presented at American Society of Bacteriolo-
guts Meeting. San Francisco, Calif., 1953.

(34) H. B. Wolin. B. L. Horecker, M. Gibbs and H. Klenow, paper
presented at Meeting of American Institute of Biological Sciences,
Madison, Wisconsin, 1963.

102

BIOCHIMICA ET BIOPHYSICA ACTA 447

DYNAMICS OF THE PHOTOSYNTHESIS OF CARBON COMPOUNDS
I. CARBOXYLATION REACTIONS

J. A. BASSHAM and MARTHA KIRK

Lawrence Radiation Laboratory, University of California, Berkeley, Calif. {U.S.A.)

(Received January 30th, i960)

SUMMARY

Kinetic studies have been made of the rates of appearance of ^*C in individual com-
pounds formed by Chlorella pyrenoidosa during steady state photosynthesis with
"COjj. These rates have been compared with rates of COj and ^*C disappearance from
the gas phase during the same experiments.
The following results were obtained :

1. After the first few seconds, the rate of appearance of ^*C in compounds stable
to drying on planchets at room temperature is 95 to 100 % of the rate of uptake of
carbon from the gas phase.

2. After the first few seconds, the rate of appearance of carbon in compounds
isolable by usual methods of paper chromatography constitutes at least 73 to 88 %
of the rate of uptake of carbon from the gas phase. Compounds formed from the
carbon reduction cycle via the carboxylation of ribulose diphosphate account for a
least 70 to 85 % of the uptake, while carboxylation of phosphoenolpyruvic acid
appears to account for at least another 3 %.

3. The induction period in the appearance of ^*C in stable compounds may be
due to a reservoir of intracellular COj and HCO3 or to some other volatile or unstable
compound. If so, this reservoir contains no more than 1.5 )umoles of carbon, corre-
sponding to about 7 sec carbon fixation in the experiment in which it was measured.

4. No other carboxylation reactions, such as the carboxylation of y-aminobutyric
acid, could be observed. The rate of labeling of glutamic acid after 5 min of exposure
of the algae to i*CO, reached a maximum rate of about 5 % of the total uptake rate,
but this labehng appears to be due to conversion of labeled intermediates formed
from the carbon reduction cycle or phosphoenolpyruvic acid carboxylation.

5. The in vivo carboxylation of ribulose diphosphate in the light appears to be
followed by conversion of the product to one molecule of phosphoglyceric acid,
containing the newly incorporated ^^COj and one molecule of some other (kinetically
distinguishable) three carbon compound. This reaction would be different from the
one reported for the isolated enzyme system and the in vivo reaction in the dark,
which produces two molecules of 3-phosphoglyceric acid.

Abbreviations: PGA or 3-PGA, 3-phosphoglyceric acid; PEPA, phosphoenolpyruvic acid;
RuDP, ribulose 1,5-diphosphate; ATP, adenosine triphosphate; TPNH, reduced triphospho-
pyridine nucleotide.

103

448 J- A. BASSHAM, M. KIRK

INTRODUCTION

Much of the biochemical pathway through which carbon dioxide is reduced during
photosynthesis in algae has been established^-^ A principal feature of this pathway
is the carbon reduction cycle. A simplified version of this cycle is given in Fig. i,
which shows the key steps.

To map these paths, Calvin et al.^^" gave radioactive compounds, such as
"CO2 and KHj^^po^, to photosynthesizing plants. The plants made various reduced
organic compounds from these labeled substrates. They were then killed and the
soluble compounds were extracted from the plant material and analyzed by two-
dimensional paper chromatography and radioautography. The compounds were
identified and their radioactive content determined. From the amount and location
of radioactive elements within compounds following exposures of the plants for
various lengths of time and under various environmental conditions, biochemical
pathways were followed.

Fig. I. Carbon reduction cycle (simplified version), (i) Ribulose diphosphate reacts with COj to
01-.OL1GO-. AND GLYCEROL PHOSPHATES givc an unstable six carbon compound which

poLrssccHABiDES GALACTOSE PHOSPHATES spUts to give two three carbon compounds. At

least one of these is 3-phosphoglyceric acid. The
other three carbon compounds might be either
' ^'^^ 3-PGA, as it is known to be in the isolated en-

PENTOSE-5-PHospHATES fHEPTOSE PHOSPHATES zyme System, or some other three carbon com-

4TP (4 <HExosE PHOSPHATES pound such as a triose phosphate (dashed arrow) .

\| JTRiosE PHOSPHATES ^^j pQ^ jg reduced to triose phosphate with

RIBULOSE DIPHOSPHATE _^ ^^/ / j^jp ^^^ TPNH derived from the light reaction

.^^TPNH 2^OTP^ ^^^ water. (3) Various condensations and re-

arrangements convert the triose phosphates to
pentose phosphates. (4) Pentose phosphate is

c .„„„j£ phosphorylated with.\TP to give ribulose di-

-ALANINE phosphate. Further carbon reduction occurs via

conversion of PGA to phosphoenolpyruvic acid,
(s) andcarboxylation, (61, to form a four carbon
compound (probably oxaloacetic acid). Keac-
-ASPARTicAcio tions leading to the formations of some of the

secondary intermediates in carbon reduction are shown by the arrows lettered a through g.

In the present study we have extended our information about these pathways
by more precise control of the environmental conditions during exposure of the
plants to tracers. At the same time we have made measurements of the rate of entry
of tracer into the plant and of the rate of appearance of the tracer in specific com-
pounds.

We sought answers to the following questions: (a) How much of the total carbon
taken up by the plants enters the metabolic network via carboxylation of ribulose
diphosphate (reaction i)? (b) How much of the total carbon taken up enters by
carboxylation of PEPA (reaction 6)? (c) Are any other carboxylation reactions, such
as the carboxylation of y-aminobutyric acid", of any importance in steady state
photosynthesis? (d) Does the carboxylation of ribulose diphosphate in vivo lead to
one product only (PGA) or does it lead to two products (PGA and some other 3-carbon
compound)?

"Steady state photosynthesis" as used in this paper, is defined as a condition
under which unicellular algae are carrying out the reaction of photosynthesis, are
synthesizing all of the normal cell constituents, and are growing and dividing at

aTP =■

104

PHOTOSYNTHESIS OF CARBON COMPOUNDS

449

constant rates during the course of the experiment. Moreover, the rates of photo-
synthesis in experiments which will be reported here were between 30 and 80 % of
the maximum rates at which these algae are capable of photosynthesizing at room
temperature.

EXPERIMENTAL

Plant material

The plants used in all experiments were the unicellular green algae, Chlorella
pyrenoidosa, raised in continuous automatic culture tubes as described previously*.
The algae were raised and harvested as a 0.5 °o (volume wet packed cells/volume)
suspension. The algae were centrifuged from the culture medium and then suspended
in a special nutrient solution (described later). This suspension (80 ml) was placed
in the illumination chamber of the steady state apparatus.

Fig. 2. Steady state apparatus. (1) algae chamber. (2) water or nutrient solution reservoii, (3) acid
or base reservoir, (4) pH electrodes, (5) solenoid operated pH control valve, (6) solenoid operated
sampling valve, (7) small lamp, (8) photovoltaic cell, (9) large gas reservoir, (10) four-way stopcock.

Steady state apparatus

In the steady state apparatus, shown schematically in Fig. 2, a stream of gas
(i to 2 % CO 2 in air) is cycled through a closed system. The gas is bubbled through
the 0.5 % or 1.0% suspension of algae (80 ml) at a rate of approximately 1 1/min.
Gas and liquid mix rapidly in the algae chamber, which is 3/8" thick and 4" in
diameter (inside dimensions). The algae chamber is illuminated from both sides
by G.E. RSP2 photospot incandescent lights through an infrared absorbing glass in
a water bath, or in some experiments from one side by an incandescent lamp and
from the other side by a bank of eight 8", 6 W fluorescent lamps (blue and cool white).
In either case, the voltage to the incandescent lamps is adjusted just to give hght
saturation of the oxygen evolution rate. The algae chamber is water jacketed, and

105

450 J. A. BASSHAM, M. KIRK

the water is circulated in a thermostated bath. The temperature of this bath is set so
that during steady state photosynthesis the temperature indicated by the thermo-
meter in the algae suspension reads 25°.

The algae chamber is connected to a side loop through which the algae suspension
is made to circulate by the flow of gas into the chamber. A beam from a small lamp
passes through a window in the side loop to a photovoltaic cell which measures the
light absorption and hence the density of the algae. Electrodes in the side loop
measure pH, which is recorded on a multipoint recorder. The pH meter output is also
connected to a control relay which, through the activation of a solenoid-operated
valve, can cause acid or base from a reservoir to be added in small volumes to the
algal suspension. Another reservoir within the closed system contains distilled water
or nutrient solution, which can be added to the algal suspension to dilute it to the
selected concentration as the algae grow.

A solenoid-operated sampling valve at the bottom of the chamber permits one
to take I -ml samples rapidly (every 2 sec if desired). The inside of the algal chamber is
maintained at slightly above atmospheric pressure to force the algal sample out of
the chamber. When samples of algae are taken, they are run into 4 ml of methanol
at room temperature. This gives a mixture which contains about 80 % methanol
by volume. No significant difference in the resulting labehng pattern is seen whether
the algae are killed this way, in boiling ethanol, or in ethanol kept at — 40°.

After the gas in the closed system bubbles through the algae, it passes through
instruments which measure COg, ^*C, and Og, and each measurement is automatically
recorded. From the known sensitivities of these instruments and the volume of the
system, one can calculate rates of exchange of these quantities and specific radio-
activity. A large reservoir and small reservoirs may be connected or disconnected
from the closed system to obtain closed systems of various sizes. The volume of
the largest system is 6400 ml, while the volume of the smallest system is 435 ml. The
system can be open during the pre-labeling period by means of a stopcock.

Nutrient solution

For steady state experiments it is necessary to supply the algae with all the in-
organic compounds required for them to photosynthesize and grow at a normal rate.
Unfortunately, the nutrient solution in which they are usually grown in the laboratory
contains quantities of salts which make impossible an adequate separation of labeled
compounds by two-dimensional paper chromatography. Therefore, the algae are
suspended in much more dilute nutrient solutions of which that in Table I is typical.

TABLE I

STARTING NUTRIENT SOLUTION FOR STEADY STATE EXPTS. l8 AND 28

(NH^JijHPO^ 40 mg/1

MgSOi-yHjO 2omg/l

NH4CI 20 mg/1

KNO3 20 mg/1

Arnon's A-4 solution of trace elements plus

CoClj-6HjO (40 mg/1) and M0O3 (15 mg/I)i2 i ml/1

Fe'''+-versenol solution to give 90 xaM Fe++ i ml/1

NH.VOj (23 mg/1) I ml/I

106

PHOTOSYNTHESIS OF CARBON COMPOUNDS 451

This medium was adequate to maintain nearly a constant rate of photos3aithesis
in experiment steady state No. i8. In other experiments, such as steady state 28, the
algae growing under steady state conditions would in time exhaust the supply of
ammonium ion contained in this medium. However, it has been observed that as the
algae take up ammonium ion, the pH of the medium tends to decrease, presumably
due to the exchange into the medium of hydrogen ions for ammonium ions. Therefore,
dilute NH4OH was added to the algae suspension automatically by the pH control
system, thereby maintaining constant pH. At the same time ammonium ion concen-
tration was maintained approximately constant. The nutrient solution for pH
control was diluted by trial and error until its addition kept the algae density constant.
To it were added other inorganic ions in a ratio to the ammonium ion which was
estimated to provide the algae with an adequate level of these ions for growth for a
limited period. The resulting pH control medium used in steady state experiment 28
is shown in Table II.

TABLE II

CONTROL MEDIUM USED IN STEADY STATE EXPERIMENT 28

(NH^jHPO^ 6.6 mg/1

(NHi)jS04 6.6 mg/1

NH4OH 0.55 mg/1

FeClj-6H20 50 mg/l

KCl 8.0 mg/1

Trace elements as in starting medium

Administration of^*C

During the first part of the experiment the algae are kept photosynthesizing in
the light with a constant supply of 1.5 to 2 % unlabeled CO^ in air for 0.5-1 h. Constant
pH, temperature, and light intensity are maintained during this time, and during
the subsequent exposure to "COg. In the experiments reported here the pH was kept
at 6. Rate measurements of CO2 uptake and O2 evolution are made by making the
closed system small, 435 ml for a few minutes, and observing the rate of change of
CO2 and O2 tensions as indicated on the recorder. The closed gas system is made
large again, and at zero time, ^^COg is added to the system by turning a stopcock.
At the same instant a solution of NaH'^COj is injected directly into the algal suspen-
sion. The amount and specific radioactivity of the injected bicarbonate solution is so
calculated that it will immediately bring the specific radioactivity of the dissolved
COg and bicarbonate already present in the algal suspension to its final value. This
is the specific radioactivity which will obtain for all the CO 2 and bicarbonate in the
gas and liquid phases of the closed system after complete equilibration has occurred.
An example of this calculation is given in Table III. Samples of the algae suspension
of uniform size are taken every 5 or 10 sec for the first few minutes, and then less
frequently for periods up to i h. Each sample is taken directly into 4.0 ml of methanol
(room temperature) in a centrifuge tube (preweighed) . Sample tubes are reweighed
to give the sample size (± i %). After an hour at room temperature, the samples are
centrifuged and the 80 % methanol extract removed, i ml of methanol is added to
the residue and stirred a few minutes, then 4 ml water is added and the mixture

107

452

J. A. BASSHAM, M. KIRK

TABLE III

CALCULATION OF ^^C + '*C FOR STEADY STATE EXPT. li

Volume

%co.

fitnotes

mC"C

Specific activity

A Gas phase at start
B "CO2 loop
C Dissolved CO^, HCO3
D NaH^COj injected

Total
C + D

895
125"

T092

1.6

585

156
81.6
40.8

863

122.4

o

3767

607.5

4375
607.5

5.07 /iiClfimole
4.95 fiClfimo\e

' Effective volume.

warmed at 60° for 10 min. After centrifugation and a further extraction with i ml of
water, the combined clear extracts are concentrated at reduced pressure at below
room temperature. The concentrated extract, or an aliquot portion thereof, is trans-
ferred quantitatively to the paper chromatogram and analyzed in two dimensions
(phenol-water, butanol-propionic acid-water) as in earlier work^. The location of
the radioactive compounds on the chromatograph is found by radioautography
with X-ray film. When necessary, overlapping phosphate esters are eluted, treated
with phosphatase and rechromatographed.

Determination of radioactivity in compounds

The amounts of radiocarbon in each compound of interest on the chromatograms
from each sample is measured with a Geiger-Mueller tube. The paper chromatogram
is placed on top of the radioautograph, which rests on a horizontal light table, so
that the darkened areas of the film may be seen through the paper. The Geiger-
Mueller tube has a Mylar window, gold-sputtered for conductivity, but transparent
and thin (less than i mg/cm^) to permit the passage of "C beta particles. This tube
has an effective counting area of uniform sensitivity of about 17 cm^. The top of the
tube is transparent plastic so that paper and radioautograph may be viewed through
the top of the tube. Thus the counting area of the tube may readily be placed in posi-
tion over the radioactive compound on the paper. If the radioactive area is more than
4 cm across, or if it contains more than 20,000 counts/min (as counted by this tube
on the paper), the radioactive area is divided into smaller areas which are counted
one at a time (with the remainder of the spot covered by cards). The counting gas
used is helium-isobutane (99: i). The counting voltage is about 1300 V. The sensitivity
of the counter for '^C beta particles in an infinitely thin layer on an aluminum planchet
is about one count/3.1 disintegrations. However, only about one-third of the beta
particles escape from the paper (Whatman No. 4) and the actual sensitivity of this
tube for ^*C in compounds on the paper is about 1/11.2. These sensitivities were
determined by comparison of counts from three aliquot portions of a known ^■'C
labeled solution : (a) chromatographed on paper, (b) dried on a planchet, and (c) placed
in a scintillation counter with an internal standard. The radioactivity of each com-
pound is counted on each side of the paper and an average is taken of the counts
from the two sides. Comparison with determinations of radioactivity of compounds
quantitatively eluted and placed on planchets indicates that this method of counting
gives an accuracy of ± 5 %•

108

PHOTOSYNTHESIS OF CARBON COMPOUNDS 453

Rate measurements

Gas exchange: Measurements of the rates of COj uptake, ^*C uptake and O^
evolution by the photosynthesizing algae are made by taking the slopes of the three
traces on the recorder. In order to obtain accurate readings in 10 min or less, the
total effective gas volume of the closed circulating system is made small, about
435 ml. With 80 ml of 0.5 % algal suspension in the system the resulting change in
Oj or COg pressure is about 0.5 % in 10 min in a typical experiment. This corresponds
to a rate of 22 /umoles of gas exchange/min/ml of wet packed algae. The response of
the Beckman Infrared Analyzer, model 15 A, used in these experiments is not
completely linear in the range used (0 to 2.0 °o CO,) so that a correction based on a
previously obtained calibration curve is applied to the COg uptake curve plotted
on the recorder. The response of the A. O. Beckman oxygen analyzer is essentially
linear in the range used (19 to 21 °o). The level of '■*€ is plotted on the recorder as
millivolts response of the Applied Physics Corpn.'s Vibrating Reed Electrometer to
the ionization chamber (volume 118 ml, R = 10' ohms). From the known calibration
of the ionization chamber this reading can be directly converted to /xC of "C. From
the ^^COg reading and the ^*C reading the specific radioactivity of the CO2 may be
calculated at all times during the experiment. This specific radioactivity is used to
convert the rates of change of radioactivity in the system to rates of change of what
we shall call ""C" throughout this paper. For convenience of expression and calcula-
tion, this '^C will be expressed in ;umoles and represents the amount of ^^C and "C
corresponding to a given measured amount of radioactivity in the CO 2 administered
to the algae at any time during the experiment.

Total fixation in algae: In some experiments, small aliquot portions of each
sample of algal material, taken and killed in alcohol during the course of the experi-
ment, are spread in a thin layer on planchets with acetic acid, dried, and counted.
The amount of '^C found at each time of exposure of the algae to "CO 2 is plotted
and the slope of the curve drawn through these points gives the rate of appearance
of "C in stable compounds in the plant.

Fixation of ^*C in compounds found on the paper chromatograph : After the '^C
in individual compounds found on the paper chromatogram has been measured, the
amounts are sometimes totaled for each sample up to one minute, and a rate of ap-
pearance of "C in these compounds is calculated.

RESULTS
Steady state Expt. 18

The rates of exchange of gases before, during and at the end of the experiment
are shown in Table IV. We shall take 15.5 ^moles/min as an average value for uptake
of carbon during the experiment.

Ahquot portions of the samples were dried on planchets and their radioactivity
was counted. When results of these counts were plotted versus time of sampling, the
rate of fixation of "C into compounds stable to drying on the planchets was found
to be about 15 /xmoles/min (Fig. 3).

After chromatographic separation of the compounds, radioautographs, of
which Fig. 4 is typical, were obtained. The radioactivity of each compound in each
sample was determined and the total radiocarbon found in the various compounds

109

454

J. A. BASSHAM, M. KIRK
TABLE IV

HATES OF GAS EXCHANGE IN STEADY STATE EXPT. 1 8

All rates are given in /tmoles/ml of wet packed algae.

Carbon dtoxide

Initial rate 17-9

During experiment 16.6 15.1

Final rate 141 i3-7

* See section Methods of measurements of rate of gas exchange for explanation of expression of
"C in //moles. In theory the value for "C and CO^ should be the same. The difference is a reflection
of inaccuracy in measurement of the slopes, especially CO^.

Fig. 3. Appearance of '^C in stable compounds

(dried on planchets) in Chlorella pyrenoidosa vs. time

of photosynthesis with "COj.

JO 40 50 60 m
Time in Seconds with"C02

Fig. 4. Radioautograph of chromatogram of Chlorella
pyrenoidosa after 2 min photosynthesis with "COj.

^^<

\, titim tnjt, *»■

Fig- 4-

110

PHOTOSYNTHESIS OF CARBON COMPOUNDS

455

was plotted against time (Fig. 5). The maximum slope of the curve in Fig. 5 is 13
^moles. This is a lower limit for the rate of appearance of i*C in soluble compounds
which are also stable to chromatography. It does not take into account other com-
pounds, too weakly radioactive to be counted, or "lost" from the front of our chroma-
tograms. (In order to obtain good separation of phosphate esters we customarily
allow the phenol-water solvent to drip from the ends of the chromatograms. Small
amounts of labeled fatty material are lost in this way.)

After 30 sec, appreciable amounts of radioactivity are passing through the
extractable precursor compounds seen on the chromatograms into non-extractable
substances, which are not seen on the chromatograms. Consequently the rate of
appearance of **C in compounds on the paper decrease.

During the first ten seconds, the rate of appearance of i*C in stable compounds
is less than the maximum rate during the subsequent time. This could be ascribed
to mixing time of the added H^CO^with the H^^CO^ present initially, or alternately
to the presence of an intermediate pool of either HCO3 or some other unstable or

10

20

30 40 50 60 70
Time in Seconds with ^^COg

Fig. 5. Appearance of '•'C in compounds on chromatograms prepared from Chlorella pyrenoidosa vs.

time of photosynthesis with ^^COj.

volatile compound. Such a compound would precede the stable soluble compounds
in the fixation pathway. The size of this "pool", if it exists, cannot be greater than
the difference between the fixation curve after 10 sec and a line of the maximum slope
drawn through the origin (see Figs. 3 and 5). This is no more than i.o to 1.5 /nmoles,
which is equal to the carbon fixed in 4 to 6 sec in this experiment. A calculation of
the amount of HCOJ which would be found inside algae cells in a volume of i ml with
an internal pH of 7 in equihbrium with 1.7 % CO^ gives a value of about i to 1.5
/j,moles, depending on the volume available inside the cells. It seems to us to be not
unreasonable to suppose that this "pool" is merely intracellular COg and HCO3
but it does not matter to the subsequent argument whether it is this or some other
unstable or volatile substance.

From the measured rates of uptake of COj and "C and from the rates of ap-
pearance of i*C in stable compounds these experimental findings may be listed:
(a) The appearance of ^*C in stable, nonvolatile compounds, after the first 10 sec
of exposure of the plant to ^*C02, is equal to the rate of total uptake of "COj within

111

456

J. A. BASSHAM, M. KIRK

experimental error, (b) During the period between lo and 30 sec exposure to "COj,
the appearance of ^^C in individual compounds which can be isolated by our methods
of paper chromatography, is equal to at least 85 % of the rate of total uptake, (c) If
there is a pool of COj, HCOJ or other unstable or volatile compound lying between
administered COj and stable compounds in the fixation pathway, its amount is not
more than i.o to 1.5 /xmoles (4 to 6 sec fixation) and it is essentially saturated after
10 sec.

Let us next consider the question of how much of this fixed ^*C must pass through
the PGA pool.

In Fig. 6 are shown the labeling curves of some of the more rapidly labeled
compounds and groups of compounds. By 3 min, compounds of the carbon reduction
cycle are essentially saturated with radiocarbon. Secondary intermediates such as
sucrose, malic acid, and several amino acids are not saturated until longer times
(5 to 30 min). In order to evaluate the importance of the fixation pathway leading
through PGA, we have tabulated the actual measurements of "C found in compounds

Fig. 6. Appearance of '*C in PGA and sugar phosphates in Chlorella pyrenoidosa vs. time of photo-
synthesis with '^COj.

during the first minute (Table V). The "C found in all those compounds derived
from PGA without further carboxylation (see Fig. i) is added together (Tj). Com-
pounds labeled by C3-C1 carboxylation are totaled separately (Tj). Since three of
the carbon atoms in these compounds are derived from PGA, their total radioactivity
is multiplied by a factor which is 3/4 times the degree of saturation of the PGA,
which is presumed to be the same as that of their immediate precursor, namely, PEPA.
(The saturation curves for PGA and PEPA are in fact very similar in this and other
experiments.) The sum of Tj and Tjf, representing measured '^C derived from the
primary reaction which forms PGA, is plotted in Fig. 7. Again the "pool" of HCO3
or other volatile or unstable compound is about i /^mole and in this case it must
precede PGA in the chain of reactions. Where one draws the curve of maximum
slope through these points is somewhat arbitrary, but the maximum rate of ap-
pearance of '^C in these compounds falls somewhere between 11 and 13 /xmoles/min.
Thus on the basis of the appearance of "C in these extractable, stable compounds
alone, at least 70 to 85 % of all carbon fixed during photosynthesis (measured ex-
ternally) is incorporated via the carbon reduction cycle. It must be emphasized

112

PHOTOSYNTHESIS OF CARBON COMPOUNDS

457

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113

458

J. A. BASSHAM, M. KIRK

that this percentage is a lower limit based only on absolute measurements of identified
compounds.

A lower limit for the amount of carbon incorporated via C^ plus Cj carboxylation
is obtained by plotting Tg — ^Tjf (Fig. 7). The minimum rate of this incorporation is
about 0.4 /imoles/min/ml algae, or about 3 % of the total. Note that this value is

I

to 20 30 ■W 50 60

Time in Seconds with ^^C02

Fig. 7. Appearance of "C in compounds derived from PGA and in compounds derived from C, + Cj
carboxylation in Chlorella pyrenoidosa vs. time of photosynthesis with "COj.

for the actual introduction of COj and does not include the carbon derived from PGA
(Tgf). The rate of incorporation of '*C into these three compounds thus accounts for
about 4 times 3, or 12 % of the total in this experiment. Other experiments indicate
that the relative contribution of C3-C1 carboxylation varies considerably and tends
to be higher (up to 3 times that reported in this case) when the rate of COg fixation is
greater and when amino acid synthesis is more rapid. In addition to the three com-
pounds listed here, other substances may be derived in part from C^-Cg carboxylation,
such as glutamic acid and citric acid, discussed below.

While at least 73 % of the total rate of fixation of carbon has thus been shown
to be due to the carbon reduction cycle and C1-C3 addition, there is no indication
of any other significant fixation pathway. In Fig. 8 the ^*C found in glutamic acid
and in citric acid is shown. Could this labeling of glutamic acid be the result of a
carboxylation of y-aminobutyric acid? The maximum rate of labeling of glutamic
acid and in citric acid is shown. The maximum rate of labeling of glutamic acid is
about 0.7 /^moles/min or 4.5 % of all ^*C fixed. Since this rate is found between 5 and
20 min, it probably represents labeling of all five carbon atoms of glutamic acid,
because the precursors are surely at least partially labeled after 5 min. The labeling
due to carboxylation reaction would be expected to begin during the first 30 sec, if
one is to judge by the other known carboxylation reactions which were discussed
earlier. Yet, after the first 31.5 sec, the glutamic acid contains only 0.02 fimoles of
i*C. Between 40 and 60 sec, its labeling rate is only 0.2 /xmoles/min. Moreover,
y-aminobutyric acid itself would have to be synthesized from CO2 (by some as yet

114

PHOTOSYNTHESIS OF CARBON COMPOUNDS

459

unknown route), if it were a precursor to glutamic acid, and would have to be ap-
preciably labeled by the time glutamic acid reaches its maximum labeling rate. Yet
ije can detect no radiocarbon in y-aminobutyric acid in this experiment or in others
fy this series, even after the algae have been exposed to ^^COj for lo min. Clearly,
wottle if any of the labeled glutamic acid formed in our experiments is made b

^ — i—i — i — * — > — * — ir-io

Tims in Minuttt

Fig. 8. Appearance of ^*C in PGA, glutamic acid and citric acid in Chlorella pyrenoidosa vs. time of

photosynthesis with "COj.

carboxylation of y-aminobutyric acid. Rather, it must arise from other intermediate
substances such as those formed by the two carboxylation mechanisms already
discussed.

Note, however, that the rate of labeling of citric acid is by far too small to permit
it to be the precursor of the labeled glutamic acid in any sequence such as :
oxaloacetic acid + acetyl coenzyme A — >■ citric acid ->■->■->■
a-oxoglutaric acid -|- COj — > glutamic acid

Steady state Expt. 28

All the results described thus far were obtained in an experiment (steady state 18)
in which the nutrient solution, though not automatically replenished, was sufficient
to maintain the rate of photosynthesis at a nearly constant level during the course
of the experiment. The results of steady state Expt. 28, in which the nutrient solution
was replenished during the course of the experiment led to the same conclusions.

TABLE VI

COMPARISON OF STEADY STATE EXPERIMENTS 1 8 AND 28

Experiment

COi uptake

fimolesfminlml

algae

Rale of appearance

cf "C in compounds on

chromatograms

(20-40 sec)

RuDP

saturation
at 40 sec

PGA residual' carbon
saturalion according to

Reaction D Reaction L

I»

28

15-5
195

13
17-if

0-53
0.38

0-57
0-43

0.46
0.28

* See subsequent discussion for explanation of the term "residual". The degree of saturation
at 40 sec is obtained by dividing the measured value of '*C in the compound at 40 sec by the
saturation level of **C in the compound (or residual atoms) after 10 min exposure of the algae
to "CO,.

115

460 J. A. BASSHAM, M. KIRK

These results are summarized and compared with steady state Expt. 18 in Table VI.
Though not shown in the table, the maximum rate of appearance of ^*C in observable
compounds derived from the carboxylation reaction leading to PGA (the carbon
reduction cycle) was 70 to 90 % of the externally measured rate of i*C uptake.

DISCUSSION

When Calvin and Massini" reported the formation of PGA in an overall reaction
requiring ribulose diphosphate and CO 2 they proposed that the reaction in the light
gave one molecule of PGA and one of triose phosphate but in the dark gave two
molecules of PGA. Wilson^* discussed this possibility further after it was realized
that the carboxylation did not involve an intermediate splitting of the ribulose to
triose and diose. The dark reaction in whole plants'^ and the reaction in isolated
enzyme systems^*-!' was found to give rise to two PGA molecules. Also, it is clear
from previous kinetic studies^- ^^ of carbon fixation during photosynthesis that the ^^C
entering the carbon reduction cycle via the ribulose carboxylation passes through
the carboxyl group of PGA initially. This is consistent with the fact, established for
the isolated enzyme system by Horecker'*, that the CO 2 is bonded to the number
two carbon atom of ribulose diphosphate. More recently Park^' has shown by means
of inhibition studies in broken spinach chloroplasts that "C entering that system
must pass through PGA. That is, PGA is a biochemical intermediate compound — not
merely a compound formed by thermal breakdown after the plant is killed.

We shall present here an argument, based on kinetic data, which indicates that
the carboxylation of RuDP in vivo during photosynthesis gives rise to only one
molecule of 3-PGA.

If the i*C which has just entered PGA from "CO2 is subtracted from the total "C
in PGA, the i*C in the remaining carbon atoms of the PGA must all be derived
from ribulose diphosphate.

Let us consider the two reactions :

H2COPO3H- D)

H2C-OPO3H-

'co-j

2 1

c = 0

1

HOC-H

1

+

4I

HC-OH

1

3I
HCOH + ♦COj -^

1

1

•co-j

H2C-OPO3H

4I
HCOH

bI or L)
HjCOPOjH

HjC-OPOgH-

2I
HOC-H

1

+

3

ll

— c—

1

RuDP

•CO-,

PGA

— c—

1

The position of the '*C which has just entered the cycle as "CO^ is indicated by the
asterisks. In reaction D, there are five remaining carbon atoms of PGA (numbers i
to 5) which must be derived from RuDP, while in reaction L there are two such
"residual" carbon atoms (numbers i and 2). The steady state concentration of PGA
in steady state Expt. 18 is 3.0 /xmoles of carbon/ml algae, hence the carboxyl carbon
concentration is i!o jumole of carbon. However, if reaction D is correct, only one-half

116

PHOTOSYNTHESIS OF CARBON COMPOUNDS 461

of this carboxyl carbon, or 0.5 /xmole, is derived immediately from CO^; the other
half (carbon atom 3) comes from RuDP. We shall subtract the "C due to newly
incorporated "COg from the total i^C found in PGA at each time and for each of these
two cases. The specific radioactivity of the remainder may then be compared with
the specific radioactivity of the RuDP from which it must be derived.

In order to make this subtraction it is necessary first to calculate the radiocarbon
in the carboxyl group of PGA as a function of the time of exposure of the algae to
"COg. This calculation requires in turn a calculation of the saturation curve of the
"CO 2 pool", although this could be assumed to be saturated from the beginning
without seriously affecting the results.

Consider the steady state system :

R R

CO2 — > Pool 1 — > Pool 2 — >■ etc.

Let Ci and C^ be the steady state concentrations of Pools i and 2 and let x and v
be the degrees of saturation with "C of these pools (respectively) as a function of
time of exposure of the algae to "COj. R is the rate of flow of carbon into the system
and through the two pools. It is also assumed in this case that the rates of the back
reactions are negligible compared to the rates of the forward reactions.

For a small increment of time, the change in degree of saturation is the difference
between the rate of flow of "C into the pool (R) and the rate of flow of carbon out of
the pool (Rx), divided by the size of the pool C^; dxidt = (R—Rx)IC^. Integration
and determination of the integration constant at / = 0 gives x = i — expt ( — R/Ci)0 .

During a small increment of time, the change in degree of saturation of the second
pool is the difference between the rate of flow of "C into the second pool {Rx) and the
rate of flow out [Ry) divided by the pool size C^;

Integration and determination of constants at i = o leads to two solutions, one for
the case Cj + C^-

and another for the case C^ = C^'.

y = I — (I — RtjC) exp (— RtjC)

In applying these equations to the data from steady state Expt. 18 we have assumed
a value of Cj = 1.2 /xmoles for the "COg pool" (Fig. i) and a value of 0.2 /^moles/sec
(= 12 /ixmoles/min) for R. The resulting values for x are shown by curve A, Fig. 9.

If reaction D is correct, the PGA carboxyl pool arising from newly incorporated
CO2 is 0.5 /Limoles and its degree of saturation jy is given by curve B, Fig. 9. If reaction
L is correct, this pool is i.o /xmole and the saturation curve y is that shown as curve C.
Curve B times 0.5 and curve C times i.o give, as a function of time, the respective
/xmoles of "C in the PGA carboxyl pool derived directly from COj.

The degree of saturation of the residual carbon atoms of PGA (those which are
derived from RuDP) may now be calculated by subtracting from the experimentally
determined ["C]PGA these values of the COa-derived carboxyl (0.5 S for reaction D,
1.0 C for reaction L) and dividing by the pool sizes of the residual carbons (2.5 and

117

462

J. A. BASSHAM, M. KIRK

2.0 respectively). The resulting saturation curves are shown in Fig. 10. In the same
figure, Curve R is the saturation curve for ribulose diphosphate, obtained by dividing
the experimentally determined i*C labeling of RuDP by its steady state concentration,
which was 0.36 ^moles/ml algae.

If the carboxylation of RuDP were to lead to the formation of two molecules
of PGA (reaction D), then all of the carbon atoms of RuDP must give rise to the
"residual" carbon atoms of PGA. The degree of saturation of these residual carbon
atoms at no time could exceed the degree of saturation of the carbon atoms of RuDP.
Since the calculated values for these residual atoms, (PGA-0.5 B)/2.5, do exceed
those of RuDP at all times after 12 sec, reaction D does not appear to be correct.
The curve for reaction L does not exceed the saturation of RuDP until about i min.
In this case, the residual carbon atoms of PGA are derived only from carbon atoms 2
and 3 of RuDP, and thus may exceed the saturation of the average of carbon atoms

Time in Seconds

"T5 'X 30 40 50 eo'
Time in Seconds

ao 90 100

Fig. 9. Degree of saturation (vs. time of photo-
synthesis with "CO2) of "CO2 pool" and of
PGA carboxyl derived immediately from "COj
according to two proposed carboxylation re-
actions. Curve A is for "COj pool", curve B is
for PGA carboxyl derived immediately from
^^COj according to reaction D, curve C is for
PGA carboxvl according to reaction L.

Fig. 10. Degree of saturation of ribulose di-
phosphate (R) vs. time of photosynthesis with
"CO2 compared with degrees of saturation of
residual carbon atoms of PGA according to two
proposed carboxylation reactions.

I, 2, 3, 4, and 5 of RuDP. In fact, this is not surprising, since earher degradation
studies on RuDP' showed that, during i*C incorporation in photosynthesis, carbon
atom 3 is first labeled, followed by carbon atoms i and 2, followed finally by carbon
atoms 4 and 5. The saturation curve for the residual PGA carbon atoms according
to reaction L is thus about as would be expected.

Note that after 30 sec the carboxyl carbon of PGA would be saturated and the
same conclusion could be reached by looking only at the curves from 30 to 90 sec,
which are not dependent on the foregoing calculations of CO^ pool and PGA carboxyl
saturation. At these longer times it is sufficient to plot simply the curves for (PGA-0.5)/
2.5, (PGA-i.o)/2.o, and RuDP/0.32 all as a function of time.

We conclude, therefore, that the labeling curves for PGA and RuDP in this
experiment can best be interpreted as resulting from the occurrence of reaction L.
That is, the in vivo carboxylation reaction of the carbon reduction cycle during

118

PHOTOSYNTHESIS OF CARBON COMPOUNDS 463

photosynthesis appears to produce one molecule of PGA and one molecule of some
other three carbon compound.

Steady state Expt. 28 gav^e very similar results, from 10 sec to saturation (see
Table VI for comparison at 40 sec).

From these experiments alone we cannot identify this three carbon compound.
It could be merely a small pool of PGA itself, tightly bound to an enzyme, or in some
other way kept apart from the principal PGA pool. Such a pool of PGA molecules,
if sufficiently small (> o.i /xmole), would not be distinguishable from the other
PGA pool by our methods.

Alternatively, the six carbon product of the carboxylation reaction may be
reductively split to one molecule of 3-PGA and one molecule of triose phosphate.
In either case, the requirement for the reaction leading to PGA and triose phosphate
must be light (or cofactors derived from the light reaction), and the intact chloroplast,
or some intact sub-unit of the chloroplast, as it occurs naturally in the living cell.

One cannot say at the present time whether or not any of the chloroplasts or
chloroplast fragments isolated from broken cells retain the capacity to carry out such
a reductive splitting of the six carbon intermediate of the carbon reduction cycle. In
such cell-free systems, the carbon reduction cycle may well operate only via the
carboxylation reaction leading to two molecules of free 3-PGA. Recently Park^"
has prepared electron micrographs of chloroplast and chloroplast fragments which
had been found by him to have about as high a rate of photosynthetic COg reduction
as any such rates reported for cell-free systems. When compared with electron micro-
graphs of chloroplasts in intact cells, these isolated fragments appear to have under-
gone considerable physical change, particularly in regard to the apparent density
of the stroma and spacing between lamellae. It is possible that the reductive carboxy-
lation pathway, if correct, operates only in the unaltered lamellar system by means of
some rather direct transfer of photochemically-produced reducing power from the
pigmented layer to the carbon reduction cycle.

If two different three carbon compounds are formed in vivo in the light by the
carboxylation of RuDP, and if these two products are kept separate until they have
been converted to triose phosphate, and react with each other to give hexose, then
the resulting hexose molecule might be dissimilarly labeled in its two halves, nameyl
carbon atoms i, 2, and 3, and carbon atoms 4, 5, and 6. Such asymmetry has been
reported by Gibbs and Kandler^^-^^. However, other explanations of the phenom-
enon are also consistent with the carbon reduction cycle^.

ACKNOWLEDGEMENT

The work described in this paper was sponsored by the United States Atomic Energy
Commission, University of California, Berkeley, Calif. (U.S.A.).

REFERENCES

1 J. A. Bassham, a. a. Benson, L. D. Kay, A. Z. Harris, A. T. Wilson and M. Calvin, /. Am.

Chetn. Soc, 76 (1954) 1760.
* M. Calvin, /. Chem. Soc, {1956) 1895.
' J. A. Bassham and M. Calvin, The Path of Carbon in Photosynthesis, Prentice-Hall, Englewood

Cliffs, New Jersey, 1957.

119

464 J- A. BASSHAM, M. KIRK

* M. Calvin and A. A. Benson, Science, 109 (1949) 140-

' A. A. Benson, J. A. Bassham, M. Calvin, T. C. Goodale, V. A. Haas and W. Stepka, /. Am.
Chem. Soc, 72 (1950) 1710.

• A. A. Benson, Arch. Biochem. Biophys.. 32 (1951) 223-
' A. A. Benson, /. Am. Chem. Soc, 73 {1951) 2971-

» A. A. Benson, J. A. Bassham, M. Calvin. A. G. Hall, H. E. Hirsh, S. Kawaguchi, V. Lynch
AND N. E. ToLBERT, /. Biol. Chem., 196 (1952) 7°i-

» M. Goodman, D. F. Bradley and M. Calvin, /. Am. Chem. Soc, 75 (i953) 1962.

"" M. Goodman, A. A. Benson and M. Calvin, /. Am. Chem. Soc, 77 (i955) 4257-

" O. Warburg, Science, 128 (1958) 68.

" R. W. Krauss, in J. S. Burlew, Algal Culture from Laboratory to Pilot Plant, Carnegie Institu-
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Biochim. Biophys. Acta, 43 (i960) 447-464

120

Ind

ex

Index

Acetic acid

as precursor to glutamic

acid, 35
fat formation from, 57
formation of, 39, 47
from malate?, 38
Acetoacetyl CoA, 57, 60
Acetyl CoA, 38, 57

formation from pyruvate, 47
Acetyl phosphate, 48

formation from sugar phos-
phates, 41
Adenosine triphosphate (ATP)
free energy of formation, 4
function, 5, 6, 17
Alanine

an early product of photo-
synthesis, 29
mechanism of formation, 33,

34
in preillumination experi-
ments, 17
pyruvate formation from, 47

rate of formation in Chlo-
rella pyrenoidosa, 31

two pools of, 30
Aldolase, 9, 16,51
Algae, 12
Amino acids, 25

synthesis of, 29
y-Aminobutyric acid, 34
8-Amino levulinic acid, 64
Aromatic compounds, 65
Aspartic acid, 17,29,30,31
ATP {see Adenosine triphos-
phate)

Carbohydrates, 25

Carbon reduction cycle of
photosynthesis
cofactor requirements, 9
description of, 8-12
evidence for, 12-16
figure, 11

123

2-Carboxy-3-keto- 1 ,5-diphos-

phoribitol, 1 1
Carboxydismutase, 23, 51
Carboxyl carbon of PGA, 39
Carboxylating enzyme, 51
Carboxylation reactions
of 8-aminobutyric acid, 34
balance among, 26
evidence for RuDP as react-
ant and PGA as product,
20
keto acid, product of, 8
leading to C4 compounds, 22
number of times per cycle, 9
of phosphoenolpyruvic acid,

22
other carboxylations than

cyclic, 21
PGA, first stable product of,

15
quantitative importance of,

22
reductive, 1 1, 23
Carboxylic acids

formation of, 37, 38, 39
Carotenoids, 60
Chlorella pyrenoidosa, 12, 25,

30
Chlorophylls, 60, 64
Chloroplasts

amino acid pools in, 30
as biosynthetic factory, 68
compounds excreted by, 28
site of synthetic reactions, 27
transport of reducing power
from, 40
Citramalate, 35
Citric acid, 48
Citrulline, 31

Degradation

of glycolic acid, 39

of hexoses, 16

of PGA, 15,39
Dihydroxyacetone phosphate

early product of CO2 reduc-
tion, 49

glycerol phosphate forma-
tion from, 59

intermediate in carbon re-
duction cycle, 1 1

reactions of, 51

Epimerase, 9

Erythrose-4-phosphate, 11,49,
51,65

Farnesyl pyrophosphate, 61

Fatty acids, 25, 56

Fatty acid synthesis, 38

Fats, 56, 67

Free energy change

carboxylation reaction, 38

formation of ATP, 4

formation of TPNH, 4

relation to reversibility, 38
Fructose- 1 ,6-diphosphate

in carbon reduction cycle, 11,
49

reactions of, 51
Fructose-1-phosphate, 55
Fructose-6-phosphate

in carbon reduction cycle, 1 1,
49

reactions of, 51, 53, 55

124

Fructose-6-phosphate ketolase,

41
Fumaric acid, 37

Galactose, 49

Geranylgeranyl pyrophosphate,

61
Glucose- 1,6-diphosphate, 52
Glucose- 1 -phosphate, 49, 54
Glucose-6-phosphate, 49, 52
Glutamic acid

formation of, 34, 35
light-dark labeling of, 48
rate of formation, 31
two pools of, 30
Glutamine, 31
GlyceraIdehyde-3-phosphate,

11,51
a-D-Glyceryl-1-phosphate, 59
Glycine

as porphyrin precursor, 62
origin of, 36, 38
rate of formation, 31
slow labeling of, 32
Glycolaldehyde-thiamine pyro-
phosphate, 41
Glycolic acid

direct formation from CO2,

44,45
effect of CO2 level on forma-
tion, 40
effect of Mn+ + deficiency

on, 46
formation in barley seed-
lings, 39
formation from sugar phos-
phates, 43

labeling by T and C^*, 45
labeling of carbon atoms, 39
role in hydrogen transport, 46

Glycolyl CoA, 43, 44

Glyoxylate, 44, 62

Glyoxylate cycle, 48

Glyoxylic acid, 38, 39

Hematin pigments, 60, 64
Hexoses, degradation of, 16
/3-Hydroxy-/3-methyl glutaryl,

60
•y-Hydroxyglutamic acid, 35

Inorganic phosphate, 51
Isomerase, 9

Isopentenyl pyrophosphate, 61
Isoprene unit, 61

2-Keto-3-deoxy-D-araboheptonic

acid-7-phosphate, 65
Ketoglutaric acid, 33

Malic acid

early fixation product, 17, 29

formation of, 37

reactions of, 38, 63
Malonyl CoA, 57
Mannose, 49

Mannose-6-phosphate, 53
Mesaconic acid, 35

125

^-Methylaspartate, 35
Mevalonic acid, 61
Monosaccharides, 49

Oxalacetic acid, 1 1

Paper chromatography, 12
PGA, 19, 20, 30

degradation of, 15, 39
Phenylalanine, 65
Phosphatase, 51
Phosphoenolpyruvic acid
(PEPA)
in amino acid synthesis, 30,

32
in aromatic synthesis, 65
carboxylation of, 26
formation from PGA, 17
formed from carbon reduc-
tion cycle, 1 1
Phosphoglucomutase, 52
3-Phosphoglyceraldehyde, 9, 49
2-Phosphoglyceric acid, 1 1
3-Phosphoglyceric acid (3-
PGA), 11, 15
product of carboxylation re-
action, 9, 11
PhosphoglyceryI-3-phosphate,

51
Phosphoglycerylkinase, 9, 51
Phosphohexose isomerase, 52
Phosphoketolase, 41, 48
Phosphoribulokinase, 51
Phosphoroclastic cleavage, 41
Phosphorylated sugars {see
Sugar phosphates)

Phosphoshikimic, 35

Phytoene, 62

Phytol, 60, 61

Pigments, 60, 67

Polyglycerol phosphates, 59

Polysaccharides, 67

Porphobilinogen, 64

Porphyrin compounds, 62

Protein synthesis, 29

Proteins, 29, 67

Protoporphyrin 9, 64

Pyrophosphate, 52

Pyruvic acid

from glutamic acid, 35
oxidative decarboxylation

of, 47
transamination of, 34

Radioautograph

of photosynthesis experi-
ments, 15, 18
preparation of, 14
use of, 12
Radiocarbon (C^^), 12, 13
Ribose-5-diphosphate

in carbon reduction cycle, 11,

49
reactions of, 51
Ribulose-l,5-diphosphate (Ri-
bulose diphosphate)
(RuDP)
in carbon reduction cycle, 11,

49
carboxylation of, 20, 51
decrease in light-dark experi-
ment, 19

126

Ribulose-5-phosphate, 9, 11, 49
RNA, 67

Sedoheptulose- 1 ,7-diphosphate,
11,49,51

Sedoheptulose-7-phosphate, 49,
51

Serine, 30, 31

Shikimic acid, 65

Squalene, 61

Steady-state growth, 21

Steroids, 61

Succinate, 62

Sucrose, 53

Sucrose phosphate, 52, 54

Sucrose phosphatase, 52

Sugar phosphates

conversion to free sugars, 50

occurrence as photosynthetic

intermediates, 15, 16, 49

Terpene compounds, 60
Tetrahydrophytoene, 62
Thiamine pyrophosphate, 41
Threonine, 31, 36
TPN, TPNH {see Triphospho-

pyridine nucleotide)
Transaldolase, 16
Transaminase, 34
Transketolase, 9, 16, 51
Triosephosphate dehydrogen-
ase, 9, 51

Triosephosphate isomerase, 51
Triphosphopyridine nucleotide
(TPN, TPNH)
free energy of formation, 4
function, 4-6, 17
requirement in cycle, 9, 17
requirement for RuDP for-
mation, 19
Two-dimensional paper chro-
matography, 13
Tyrosine, 65

UDPG-4-Epimerase (Galacto-

waldenase), 52
UDPG-fructose-6-phosphate

transglycosylase, 52
UDPG-pyrophosphorylase, 52,

54
Uridine diphosphogalactose, 52
Uridine diphosphoglucose, 52,

53
Uridine triphosphate, 52
Uroporphyrinogen, 64

X-ray film, 13
Xylulose-5-phosphate

in carbon reduction cycle, 11,
49

phosphoroclastic split of, 41

reactions of, 51
Xylulose isomerase, 51

127