Author: Tarr, Philip Regan Title: The catalase and oxidase activity of Pennsylvania cigar-leaf tobacco as modified by fertilizer treatment Place of Publication: Copyright Date: 1933 Master Negative Storage Number: MNS# PSt SNPaAg031 .7 <102960>*OCLC* Form:manuscriptitem2 lnput:CHF EditFMD 008 ENT:980111 TYP: s DT1: 1933 DT2: LAN: eng 035 (OCoLC)38092085 037 PSt SNPaAg031 .7 $bPreservation Office, The Pennsylvania State University, Pattee Library. University Park, PA 16802-1805 090 20 Thesis 1933m $bTarr,PR $cst*7338724 $cax+(Archival) 090 20 Microfilm D344 reel 31.7 $cmc+(service copy, print master, archival master) 100 1 Tarr, Philip Regan. 245 14 The catalase and oxidase activity of Pennsylvania cigar-leaf tobacco as modified by fertilizer treatment $ca thesis by Philip Regan Tarr. $c1933. 32 leaves $bill. $c29 cm. Thesis (M.S.)~Pennsylvania State College. Bibliography: leaves 30-31. 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The slips are available in the Rare Books Room, in the Microforms Room, and at the Circulation desk. 650 0 Tobacco $zPennsylvania. 650 0 Fertilizers $zPennsylvania. 830 0 USAIN state and local literature preservation project. $pPennsylvania. 830 0 Pennsylvania agricultural literature on microfilm. 260 300 502 504 533 590 590 !'m TBE iXZISILYAIIIA STATE COLLEGE The fcadixate School Department of Aericaltural a:icl "Biolv^jical Ghenistry The Catalaae suid ^idase Activit;/ of PennsylT>aia Ci^ar-" eaf ToDacao As Modified bv_ Fertiliser Treatment A Thesis ^y Philip Regan Tarr Salxnitted in partial falfillment for the dec^^'^e wx Ha ate r of Science Jiaie 1933 Approve 1: mc^^ i^ ^""""^ ?rofes:;or tjf ^ofl mI ^h^tooliexu L-astr;/ tVNn. ^ ^ n ^.^ Head, Departasnt of^crl^uita: aral and Biclogicial Chernistry TABLE OF CX)liTaiTS ^—^—^^^—^—^^^—^^——^—^^■^—^^^^^—^^-—^^^^——-^^-—~^^-^— -^ page IlITBODUCTIOIT 1 REVIEW OF LITKR^ITUPE S OBJECT OF TIIE IinraSTiaATIOH 6 PLAN 0? Tx^E EXP3RIII2ITT 6 A. Materials Used 6 B. Fertilizer Treatment "Received 5 EXPERBIEirr/lL T.TSTIIOLS 8 A. Sotiaatiori of Catalase Activity B. Estiriitition of Oxidase Acstivity PRES2NTATI01T OF DATA DISOUS3I01T 0? BBSUI/PS BIBLIOGRAPHY ACK!I0'MiEDC.32n! e 9 10 24 30 38 i4G4aU lUTHODUCTION ^ i The inost difficult kind of to'oacco to prodace in perfect iori. is the leaf used in the nicuiafactare of csigars of the finest quality. Fertiliser experiiiients have acjcomplished a gre^t deal in the efforts to produce a hetter cigar-leaf toliaoco, tut attention must also Toe given to the curing and sweating processes. Curing of to^bacco involves far more turn, u^erely drying tlie leaf. A m&rked loss of dry matter as well as a loss of water occurs during the process. Tt is pro'ba'oly a life process, due chiefly (if not wholly) to the activity of the colls of the leaf. The sweating or fermentation of tobacco is the last process be- fore the tobacco is made i;-to cirars. It is usually packed tight and as the weather grows wander, the sweating hegins and contin'oes for a long time. The sweating process is to tohacco what ferm«lta~ tion is to sine; it ripens and prepares it for use, perfecting its color and Improving its flp.'xr. The acrid or pungent taste is sub- dued while the Tsuming qualities are increased. It also gives the tohacco a shin^-, oily surface, which is called "satin face". The loss of wei^t often amounts to 10 or 15 per cent. Since fermentation is a measure of tohacco quality, it is al- tPgtther lo-ical that the enzymes concerned with respiration should prove tu t>e an index of tohacco quality. With that in mind, this investigation «M initiated. Cvo-ed tob^.cco will he ui:ed wid its oatalase and oxidase activity will, if possihle, he correlated .vith the fertilizer treatment received. This work will he fciio-^ed hy •iallar deteirainations on the fenr.'inted tohacco. HEVISV OF T1!E LITERATURE Loew (1) in 1901 diocovered catalase in to^bacco and soijijested that it acts in a protective capacity, destroying the accumulation of hydrogen peroxide fonned in the cell as a result of intsi^e oxi- dation processes, 'x'his theory is generally accepte.: todsy althoush Reed (2) has contrastias vie.vs. Heed points out that some TDacteria axe wi thoat catalase which would indicate its protective capacity is ateall, if any really' exists. The terei "oxidizing ferr.ent" was first introduced in 1877 "by Traube in his later v.ritinG3 ci. fernentations. ..-cording to him, the ozidiaing ferments ] m aromatic sub- stance contai:-ing an ortho-dihydroxy grouping as in catechol, wid (3) a peroxidase. The oxygenase catalyzes the oxidation of the cate- chol substance with the formation of a peroxidase. The universally present pc-oxidase decomposes the peroxide with the formation of atomic oxygen. The atomic oxygen which iiie system fonns from moleea- Ur oxygen brings about oxidation which otherwise wouldn't occur. This vie -point is now generally accepted. Appier; ar. '5) found the oxidase content of potato juice gives no 9 indication of the intensity of respiration in the talkers. However, he found the aatalase activity in the potato juice showed a very striking correlation with respiratory activity in the tuhers* Reed (G) "believed oxidases must be universally distributed if they are related to ' lolo^ical oxidation* He pointed oat that most plants free from oxidases are of an acid or alkaline nature, aiid that the oxidates may "be present in a protected form which masks the Ptaotion in vitro • Indeed he proved the presence of oxidases in citrus fraits, which were supposed to he free from the en^^r.e. A year later Reed (7) proved that oxidases played the chief role in biological oxidation, repudirting Lillie^s (8) theory that the chief role is assigned to intracellular surfaces or phase boundar- ies* In working with germinating corn, Lantz (9) failed to show a close correlation between catalase activity and respiration. He con- cluded by stating there is no evidence for believing that catalase is the enzyme chiefly concerned in physiological oxidation. The evidence more nearly supports the theory that cata^-ase prevents ex- cessive oxidation. Some interesting work by Barge (10) at Illinois showed the cat- alase activity of pine needles was greater in the summer than in the winter. Catalase measurements were made on the blood of rabbits and the activity c^as greater in the winter than in the sixmner. It ia knomi that a Tail in temperature decreases the oxidative processes in plains and i*^rc-^e3 than in blooded piAm^li, and that the reverse is true for a rise in temperature. By comparing Barge's (10) results, It ma^ ^^ seei: that a fall and rise in temperature Iia^ the same effect on catalase that it lias on the oxidative processes, VoTj^c^Ls (11) has four.: the optimum reaction of oatalaae to "be from pH 6 to pH 8, although cetalai;e prepared from heef kidney showed a strong activity "between pH 5 and pH 9* c The optimum teinperatiire, accordintj to T.lorgulis (11), is between 0^ and lO^C. The loss of activity "between 10^ and Ew 0. is much anallcr thar. letv/een 20^ and 30^C^ , \7hile between ZO^ and 40^0. the loss is so rapid that the catalase reaction is very quickly ^chaast- ed* Knott (12) found that phosphorus and nitrogen, v4ien supplied to the plant through the roots, resulted in increased catalase activity-. As early as 1901, Loew (1) noticed that potassium salts retard the catalase reaction In vitro, and that the nitrates of the alkali group retard more than the other aalts# Working with the cridase of apple tissue, Cruess and Overholser (13) found the organic peroxide to be mujch more susceptible to heat Vkff\ the peruxidase. Tlie Tomtr was Inactivated at 73.5'^ to 76 C, while the latter was inactivated at 90*^ to lOO^G. Craess and Fong (14) found the effect of pE on the inactivation tetTiperat^re of oxidase to "he vsry aiyiificant. The resistance to heat was greatest in the range of pH 5 tc J.I 7. Sheae same workers (15) found that IlgOg greatly affects the intensity of the oxid^-se reaction, The optiman is naturally affected by the relative activ- ity of the catalase which tends to rapidly dastroy the n-Cg. B0»e and his co-rorkera flG) fcunl that T'/lC solution of all 9 A chlorides tested decreased oxidase activity v/hile IT/ 10 solution of all sulfates tested sliglitlj Increased the activity^ Potassiur., sodium, and masnesi'M nitrates had no effect on oxidation, v/Mxe nitrates of "barium, calcium, man^^anese, and iron decreased it# They concluded that the chlorides which retard the caritustion of tohacco at hi^h tem- peratures also retard the oxidase action at low temperatures. OBJECT 0? TI^S IirraCTlGATIC:: The ohject of these inTestitjatior^s .ere to make a study of the catalase ^.d o:dd?.se activities of ^Pennsylvania ci^ar-leaf to'bacco as related to different fertilizer treatenents. PLAIT OP THE EX?SBi:3IIf Km Materials Used The tolacco oerae from the new tohacco e-perimental field, one mile northeast of Lancaster, :>-. , and was of the Swarr-Hil^sliwm strain. It was grown on the three-year rotation fiei: on plots that are 1/21 of an acre in area* The tobacco was harvested, air-cured, and sent to The Pennsylvania State College where it was dried, stripped, and the Uaves jround in a ^^iley mill for analysis. B. Fertilizer Treatment Keoeived The fertilizers used include: 6 1. Varying anoarrti; of nitrogen, 1/3 in nitrate of soda and 2/3 in cotton seed rieal» 2» Varvln^ ari.ounts of phosphoric acid in 16% superphot^phate. ^ S. Var^^in^ amounts of potash in salphate of potash* 4* Three rates of application of a 6-8-12 (IT-P-K) fertilizer j namely, 500, 1000, and 1500 po*ands per acre, hoth iDroadcast- ed and in the row. 5. The position of iLaiiure, whether applied alone or as a sopple- ment application* Ihe different treatments are listed in the following talile* Tal)le 1 Tlie Plot Treatineiit, the Yield, the Fire-hoidin^ Capacity aiid Elasticity of the Cored ToLaccQ Plot Treatment Application Time of Barn Elasticity Yield L'bs.per Acre Minutes Lis. per acre 1 6-8-4 1000 5 fair 1783 2 G-3-8 1000 5 good 1911 6-B-12 1000 5 good 1925 4 e-8-15 1000 9 good 1627 5* O-B-12 1000 6 fair 1261 6 3-8-12 1000 7 good 159e 7 9-8-12 1000 « good 1596 8 ft-0-i2 1000 6k poor 1554 11 6-8-12 500 5 good 17S4 12 6-8-12 1500 6 good 1806 16 6-G-iS 500 in row 5 fair 1869 18 8-8-12 1500 in row 7 fine 2585 19 none noii.e 8 mm 1050 5>0* 0-8-12 1000 A fair 1680 23 3-6-8 1000 plus man lire 4 good S05B 27 10 Tons Manure 5 fair 1869 ♦Check Plots 8 EXPERBTEIITAI liETHODS A. Estimation of Catalase Activity .■■iiirw»ii liBh The method for determining catalase activity is the one ased "by Knott !12). One gram of the ground tohacco sample was mixed v;ith an eqaal weight of calcium carbonate ^to regulate the acidity!, transferred to a 50 cc» volun-etric fxask and filled to the mark v/ith water* The c^talaiie activity v;as determined "by withdrawing 2 co. in a pipette immediately after thorou<:iily shaking the flask* The sample was placed in one arm of a dry refaction tuhe, and in the other arm was placei Z cc. of fresh **Dioxogen'' (= EG cc» Og) which had heen previously neatrali::8cL with a slight excess of O-Coj. The apparatus was of the same type of shaking device as used by Knott (12). By adjusting the rheostat, the number of swings per min- ute could be controlled. For this work, a rate of 60 complete swings was found to be the minimum at which the liquids would be thrown from one arm to the other sufficiently to give good mixing. The reaction tube was placed in a bath of water at 15 C. for three minutes before mixing took place, and remained submerged in this bath during the reaction. Since the supernatant liquid in the catalase preparation is low in activity, there is always the possibility of error in drawing the 5 cc. sample if one :.as failed to mix the preparation well or does not mear;.re "he 5 co. quickly before the heavier material begins to settle out of the pipette* IWlare to get close checks between duj- 9 licatea can "be largely laid to error in thii^ operation. Since the catalac^e activity as determined l.cre is onlj used as ei relative measure, it was not deemed nece^'sary to carry the reaction to completion* Northrop [IB] aay- that the rate of decom^i^^ition of the peroxide is proportional at mvj time to the concentration of the catalase present* '-Phis viev; is now ^enerali^ accepted* In this v;ork. we shall express catalase activity aa the cahic oeiitiir.eters of oxygai liberated in thirty minutes. A "blank determination ^:as ran, using 5 cc# of the juiied i^rei/- aration* As expe-ted, it was ver^ lov;. The liberated oxygen .vas corrected for the olank and changed to standard conditions. Ttie obtained values were multiplied by ten to give the c- tale 3a 'activity of one gram of the tobacco* I B. Estimation of Oxidase Activity The iodimetric method of Guthrie (17) v/as used for determining oxidase activity, uluccce reduces iodine in acid solution and is a suitable substrate for the oidLirsf enzyme. This is the foundation of this me-Hiod which answered our purpose v.c:.^. Preparation of substrate; Fcr^ graT.3 of glucose were added to 400 cc. of H* sodium hydroxide, placed in a 500 cc. Fxorei.ce fla«k and inmersed in a water-lat.. at So^G. Tor fifteen minates; removed and neutralized at once with 10 cc. of 65; phosphoric acid. Twenty- five grama of decolorizing charcoal (iTorit A was iwed) .vere added and allowed to stax^ over night. A small portion of the filtrate ,;a3 filtered and diluted one to five and the p!T value deten.ined 10 E' (Lamotte method was usedU If not close to pH 6.5, it .vas adjusted to this pH with N sodiwon hydroxide or N hydrochloric acid. The additlOii of 2 CO. either tc 100 cc. of the filtrate shifts the acidity ahout 0.1 pF. The iodine value for 25 cc. should he equal to about 60 cc. of ^/dO. Before using, the filtrate diould he diluted with an equ-.l volume of vvater. The method: Pipette 10 cc. of the diluted substrate into Van Sl^C-Cullen aeration tubes. Add 5 cc. of the tooaceo preparation (as used in oatalaae) containing the enzyme. Add fire drops of paraf- fin oil to each tube and aerate for one hour. V/ash into ?0C vc* Srlenmiyer flasks containing 10 cc. of 10> trichloracetic acid, add- ing in ail atout 25 cc. of water. Add 10 cc* of !t/50 iodine in n/iO ^tasoium iodide nd allow to stand for 50 minutes. Titrate with k/iOO sodiur. thlosulfate, using 1 cc. of ifo starch paste as an indic- ator. The difference between this and a blank titration is a measure of the oxidase activity of the sample^ The blank ^as relatively hi^h as v,e would ordinarily expect. As in catalase, the results are multiplied by 10 to give us the values for a 1 grar.; sainple. 0::idase activity will be expressed as the '^io^ilne value"" of the preparation. PRSS^TTATION OF DATA All cataia3€ and oxidase results are expressed for a 1 gram sam- ple of curel toLacco. Ty.e cntalase values are the cubic centimeters of oxygen liberat- 11 ed from hydrogen peroxide "by the above .:e.nple at 15 0. 'Tlie jas vo-l- umes were corrected for the "blank, and changed to staindaxd condi- tions The oxidase values correspond to the iodine value (ir/50] of the tol)acco preparation, i*e*, if the sample has an oxidase activity of 25«0, it is equivalent to 25.0 cc* of n/sO iodine as an oxiIi2.er* t: '•€«! 12 Tatle 2 Total liesults of G'atalase and Oxidase iictivitj of Cured Totsoco ka llodifiei ty Fertili::er Treatment Plot Treatnent Application Gatalase** Oxidase'** Its. per ri-cre cc. og Liberated To dine Value (so. Tt/sO) 1 &-e-4 1000 38,0 24. S 2 0-8— c 1000 35.9 25,5 3 6- 8- IE 1000 28.8 13.8 4 6-8-16 1000 31.1 23.9 5* o-e-12 1000 24.5 4S.3 6 5-e-i£ 1000 43.4 15.4 7 9-8-12 1000 14, 8 15.5 8 6-0-12 1000 31.8 16.3 11 6-6-12 500 44.5 57.5 12 6-8-12 1500 56.8 94.1 16 6-6-12 500 in row 17.3 25.3 18 8-8-12 1500 in TOir 54.2 37.9 19 iione none 41.2 3 £.7 20* 0-8-lf? 1000 19.9 43.3 23 3-8-8 1000 plus manai^" 22.4 40.1 27 10 Tons lianure 27.1 46.4 ., — *CLeCz: plots ^ **i ^vanx sample used at 15 G» ***i gram ^f^rr.ple used .y'-Y^ Table 3 Catalase Activity of Cured Tobacco as filtered "bj Mode of Application of 6-8-12 Fertiliser Plot Application Gatalase* cc» Og Liberated 16 500 lbs* in ro\T 17.3 S IQQC lbs. broadcasted 28»B 12 1500 lbs* broadcasted 36«8 11 500 lbs. broadcasted 44.3 *1 gram sample used at 15 G. Table 4 Oxidase Activity of Cured Tobacco as filtered by Mode of Application of 6-8-12 Fertilizer Plot 16 12 11 Application 1000 lbs» broadcasted 600 lb3# in row 1500 lbs. broadcasted 500 lb3» broadcasted Oxidase* Iodine Value (cc* 1^50 ] 13* 6 25*3 34*1 37* 5 *1 gram sample osed 14 Table 5 Catalase Activity of Cured TolDacco as Altered by Varying the nitrogen Content of the Fertilizer Application* plot Treatment Catalase** cc. Og Lilierated 20 0-8-12 19.9 5 0-6-12 24.3 o >6-12 43.4 6-8-12 28.8 7 S-6-i2 24.6 ♦The fertiliser application was **1 gra^ii sf^aple ase^i at 15^C. IQQO 11. 3* "broed^aste fis/^ Figure 1 Cata- lase Activ- ity CatalMe .a3 Altered by Varying the Ilitrogea 15 10 t-^. ? 6 10 KitrOKon Application 15 Table 6 Oxidase Activity of Cured Tobacco as Altered by Varying the ITitrogen Content of the Fertiliser Application* Plot Treatineaat 20 0-3-12 5 0-fa-i2 6 2-e-ia 3 6-8-12 7 9-8-12 Oxidase** Iodine Vaiae (cc. Tl/50) 43.5 16.4 13.8 13t5 •fhe fertilizer application was 1000 lbs. "broadcax'ted **1 gran sample used Figure 2 Oxidase as Altered "by Varying the nitrogen 45 40 36 30 Oxidase Activity 2S 20 15 10 5 Nitrogen Application . '-■H - ..--: »« ;«./■ 16 Table 7 Gataiase Activity of Cured Toliacco as Altered "by Varying the HiOsphorus Content of the Fertilizer Application* Plot • Treati3ient 5-0-12 6-8-12 Catslase** cc« Ug Lil3erated 31.8 So« 8 ♦The fertilizer application ms ICQO Its ♦♦l -r^r. sample used at 15^C» ■broadcasted Figure 3 Catalase ns Altered l)y Varying the Phosphorus ONiktalase Activity 20 15 10 4 r 8 1^ Phosphorus Application 17 Tstle 8 Oxidase Activity of Cured TolDacco as Altered "by Varying the Phosfiiorus Cojitent of tiie Fertilizer Appliootlon* Plot Treatment 0::idase** 8 8 G-0-i2 6-8-12 Iodine Valufi (CG. ,!:/50) 16.3 12.8 *The fertilizer aHpiication was 1000 I'bs. "broadcasted **1 gra\'i sample u^ed Figure 4 Cxi las e ps Altered "by Varying the Phosphorus Oxidase Activity 15 10 r 10 Phosphorus Application 18 Tal)le 9 Catalaae Activity of Cured To'ba.cco as Altered "bj- Yaryinc the Potassiui:! Content of the Fertiliser Applic^.tion* riot Treatment Gataiaae** cc. Og Liberated 1 6-8-4 38.0 t e-ti-8 SS.9 s e-8-i2 26.3 4 G-8-16 Sl.l •The fertiiiaer application was 1000 lbs. 'broadcssted **1 gram saiiiple used at 15^0, Figure 5 Cataiase M Altered by Varying the Potassium 40 35 30 25 Catalatc Activity 20 15 10 5 n 3 — 4~5 6 7 8 9~l0 11 lF™l5 14 i5 IS* lota^sium Application IS Ta'ble 10 Oxidase Activit^r of Cared Tobacco as Altered "by Varying the Potaaaiuni Contei± of the Fertiliser Application* Plot Treatment & 3 4 6-8-lg 6-8-16 Oxidase** T A. odine Value (cc* IV^Q) 24:. B ^*^. ml *The fertilizer ai5)lication was 1000 lbs* broadcasted **1 gram sample ^. ed Figure 6 Oxidase as Altered by Varying the Potassium 50 Oxidase SO Aotivity 15 10 1234£67£S10 12 T^ iC'taaaium Application 80 Talkie 11 Catalase Activity of Cured Tol:)accc ^"2 Altered "by Varjlii^ the nitrogen- Pot as si on: Content of tL-- Fertilizer Plus the Additicii of Manare Plot Treatment Application Catalese* 23 3 3-8-8 &-e-12 lOoo l"bs# plus mancire 1000 I'bs^ "broadcasted cc« Og liberated 28*8 *1 gram sample used at 15^G» l^a'ble 18 Oxiinse Activity of Cured To'bacco as Altered "by Varying the Nitrogen-Potassium Cont: t of the Fertilizer Plus the Addition of Manure L Plwt 25 Treatmait 3-8-8 G-8-12 Application 1000 ITds. plus manure 1000 llDS^ "broadcasted Oxidase* "lodiliC Vali^ (cc* li/50) 40.1 13*8 *1 gram sanple used Zl Table 18 Oataiase A:5tivity of Cured TolDac^o «3 Altered "by Varyin^^ the Hitro^jen Content of the Fertiliser as well a:- trie Mode of Application riot Treatment Application GataiE:.-e* oc« ^2 Libei-ated le 8*8-lt 1600 llos. in ro^ S4«2 3 e-e-is 1000 lbs. hroadcasted 28* 8 7 9-8-12 lOuu lhs» "broadcaf^ted E4.8 *1 gram srisnple used at 15^G^ TalDle 14 Oxidase Activity of Cured ToT^acco as Altered hy Varying the nitrogen Content of the Fertilizer as well as the Mode of xipplication Plot Treatment Application Oxilase* 18 8-e-12 1500 11)3. in row :o:u..; Value (oo. K/50) 37.S 3 6-8-12 1000 ITds, 'broadcasted 13,8 7 9-6-12 1000 Its* 'broadcasted 13.5 *1 gram sarr-ple used 22 Tatie 15 Gatalase .stivit^- of Gored Tolasoo as Altered "by Using A Manure Application Only Plot 27 9 rn rreatment 10 Tons of "^'^niire 6-e-l£ 0, ♦1 gram sample used at i5 C. ApplicB.tion 10 Tons of -H^LU-e 1000 I'bs* l)roadcast- ed Gatalaae* ilierated 27.1 ^o# 8 Tatle 16 Oxidase Activity of Ci^r^d Tucecco as Altered "by Using A llaaore A^-pii^^rtion Only Plot 27 Treatment 10 Tons of ^^oi:,ire G-8-18 App 11 c ati on Ten Tons of Manure 1000 ll)s» "broadcasted Oxidase ,i/i^^ Value (co* n/501 46^4 13* 8 *1 gram sample used 23 Tal)ie 17 Catalase Activity of Cured Tooacco as Altered "by TTsing Ho Treatment at All Plot Treatment . . . Application Catalase* IS Ho treatment 6-8-12 Bfo treatment 1000 11)S* oroal-jasted ca# Og Liberated 41*2 E6.8 *1 £Tnr^ sample osed at 15^C, Ta^^le 18 Odcldase Activity of Cured Tobaooo as Altered "by Using Fo Treatment at Ail Plot Treo-tment Application Oxidase* 19 3 ISO tr9«tMllt 6-8-12 To treatment 1000 lbs. Iodine Value (cc. VSO} 36,7 13.8 *1 gram sample used 24 Table 1 shows a great variation in "both catalase and oxidase activity iDetvveen the samples aaalyzed. In this discussion, the var- iation will "be correlated with the IT-P~K ratio of the fertilizer applied, as well as the cmount and mode of application of the spec- ific fertiliser. Plot Z was "broadcast with a 6-8-12 fertilizer to the extent of 1000 I'bs. This plot is used as a standard for the comparison of the other plots regardless of the treatment. It mil l)e noted that l)Oth catalase and oxidase are lor, especially the oxidase. Usin^ the standard 6-8-12 fertilizer h\xt varying the applica- tiOtttt we find extreme changes in the catalase activity of the to- "bacco (Pit^ure 1). Using 500 pounds of fertilizer applied in the row, the lowest catalase results* By ^broadcasting the same amount, the highest catalase is found. By raising the application to 1500 pounds, the catalase is raised just as if it were lowered 500 pounds. The oxidaae activity varies with the catalase with one exception. The lo.vest oxidase value is for the standard application of luOO pounds. 'Ihe 500 pounds in row treatment is again much lower than the 1500 and 500 poonds "broadcast applications (Figure 2). By raising the nitrogen from 0 to 3 units, the catalase activity is douhled. However, by raising the nitrogen more in this same pro- portion we find the catalase iecreasing (Figure D- The decrease from raising the nitrogen S to 6 units is moh greater than the de- crease resulting from an increase of nitrogen from 6 to 9 unit.. It PR is seen that tte oatalaae with O-nitrogeia aid. S-riitrosei are ooth sligjitly lower than the staiidard 6-nitro.::ei, "but the 3-nitrttgen makes for an enormous increase in the activity. P^egarding oxidase activity, 0-nitrogen shows a great value* An increase to 3-0-12 depres^^ies the activity nearly three times, the activity sio;/ly drops (Figure 2) when increasing the nitrogen to ^' and 9 units respect ively^ The latter increase, however, is very slight showing the oxidase activity to "be at a minimoii* In fact the 9-8-12 fonuula shcv/ed the lov/est oxidase activity recorded* Phosphoric acid content was varied in only one plot, hut the change from 6-0-12 to 6-8-12 should tell us in which way the oxi- dase and catalese should change, if at all. The catalase activity shows a slight inverse variation (Figure 3) with the amount of phc:^horic acid in the fertili2.er applied to the plot* It is so slight, however, that we may phosphorus has litt:.c or no effect on catalaae activity. Oxidase, sii.ilr.rl,r to catalaae, is changed very Uttle hy var- iations of phosphoric acid in the fertilizer. Just as catala:3e, it is very slightly lowered hy raising the P in U-P-K from 0 to 8 (Figure 4]. Slight changes in the catalase activity of the tohacco are caused hy chaiioing the potash content of the Tertilizer. An in- verse variation is seen vvi.ai the potash is varied from 6-8-4 to 6-8-12 (Figure 5). However, ic^y raising the potash to 6-8-16, the catala.e is proportionally raised. This indicates ihe lowest cata- lase is when the potash has a ralae of IE. £6 By alterin^: the pota^sh, the cxldase activity varies in an un- orthodox wa^ as shown "by Figore 6. Very little change occurs on application of potash values of 4, 8, or 15# But on a 6-8-12 fertil- iser, the oxidase shows a decided dro^. Just as catalase, the 6-8-18 fertilizer has the lov/est oxidase with the activity increasing when the potash is increased or decreased. However, hy decreasiii^ the val- Oi from 6 to 4 we find a corresponding oxidase decree Jie. By using a 3-8-8 fertilizer, we are lowering the nitrogen aid potassicttn proportionally from the standard 6-8-12 application. Both of these treatL.ents should tend for higher catalase as was shown "by Figures 1 and 5. But here the cataia:i6 is lowered(Figure 3). This lowering in catalase must "be due to the addition of manure to the fertiliser tre^ittnent. The lowering of hoth nitrogen and potash from 6-8-12 to 3-8-8 should make for higher oxidase, hut the enonnous increase, as shown hy Figure 4, must he partly due to the addition of manure to the ahove treatment. The 8-8-12 fertiliser should produce a lowered catalase from the 6-8-12 treatment, according to Figure U However, the 1500 pounds in row application is responsible for a higher catalase activity (Figure 5). The enonrious increase In oxidase oao^ed hy the 1500 pounds in row application zan he seen in Figure 6. Just as in catalase, the 8-8-12 treatment should lower the oxidase theoretically. (Figure 2). This verifies the results showfi in Figure 1, that 1500 pounds applic- ation greatly raises catalase and oxidase activity. 27 By sulstltatirif; ten tons of manure for 1000 pounds o£ o-C-i2 fer- tilixer, scarcely any aifferance in catalase activity is noted (Figure 5)* However, the /alae for the manure application is slightly lowsr. This verifies Figure 3, in that manure lowers catalase. The great inc-*o^.oe in oxidase ca^^ei hy manure was shown in Fig- ure 4. Fijure 6 substantiates this, in that plot 27 had the highest oxidase activity recorded* This plot receiving no treatment at all showed "oth oxidase and cataiose to have a high value. By comparing Tables 1 and 2, we see little correlation "between catalase and time of bum. However, as a general rule, it can be noted that the average catalase activity makes for the longeit burn- ing time, while low and high catalase tend to cause low values for the bum# Regarding oxidase, with few exceptions, the longest burn goes hand in hand with the lowest oxidase, and vice versa. The elasticity varies with the time of burn except in plots 5 and 8 where nitrogen aM phosphorus were respectively omitted. In these plots, the bums were good but the elasticities were bad. Comparing cata.ase with yield, it is seen that the mediocre catalase shows the higher yields. Higher or lower activities go along with the lower yields. With several exceptions, a high yield of tobacco varies direct- ly with a high oxidase. 28 smn/AFi a,::d coiicltjsioiis !• The purpose of this investigation was to study the catalase and Ou.idr-^3 activities of Pennsylvania cigar-leaf tol:acco as modified "by different fertilizer treatments Z. Kie ina^uier of applying the aexne ^aontity and quality of fer- tilizer great xy affects ttie cataxase ^^d oxidase content. A 500 pounds in row apijlication shows a BOOh lower catalase and oxide :e t;.a^ a 500 pounds brtaicast application does# 3» The amount of fertilizer applied also shov/s a great variation. Both catala.se and oxidase are greatly increased when an application 1000 pounds of 6-6-12 fertilizer is either lor/ered or raised to the extent of 500 pounds* 4. By varying the nitrogen from 0-8-12 to 9-8-12, loth catalase and oxidase are decreased with one exception, viz., the catalase is great 1;,- raised v/hen the nitrogen is increased from 0 to 3 units* 5. Increasing the phosphoric acid content from 6-0-12 to 6-8-12 finds both catalase and oxidase decreasing slightly* 6. Catalase decreases slightly from 6-8-4 to 6-8-1^ increase in potash, hut when it is increased to 6-8-16 the catalase raises pro- portionally. 30 "behaves in an unorthodox manner ty modifying the pot- ash. Tncreasing the potash from 6-6-4 to 6-8-8 causea a slight in- cree-e in oxidase aotiviti'. But n raise from 6-8-8 to 6-8-12 shows m enormous decrense, while a raise from 6-8-12 to 6-8-16 cause;; a E9 great increase at>aiiu ?• /hei manure is ouJ stituted for fertilizer, we find the cata- lase slightly lowered ^.liile the oxidaoe reaches its maximum value of this investigation. /* t "f" o -• Ct The adC^ltion of manure to a 3-8-8 fertilizer cMffM the cata lase to "be decre-^^ed. '//ithout the manure it v/ould "be increased. Qxi- da^je is greatly increased hy this addition of manure, aithouah 3-8-8 should ^hQv/ a 3li^t increase over 6-8-12# 4- «- 9. By increasing the nitrogen from 6-8-12 to 9-8-12, l:>oth cata iMe and oxidase should T)e slightly lowered. Bat if 500 pounds addi- tional fertilizer arc uaed and it is a,.plied directly in the row, we find "both catalase and oxide se oonsideratly increased. 10. TTo tre-tnent at all on the plot produces tobacco with a high cataiase and oxidase activity^ 11, Severs! very general ffc»tements were discussed regarding barn, elasticity, and yield of tobacco as compared with its catalaa© and oxidase activities. BIBLIOCxRAPHI 30 !• Ioe\v, Oscar 2. Heed, '^. B. 3. Bert rand 4« 0ns iuu, M» "^Z, Gatalase, a x^e'^i en«y» of geiieral occurreiice, vdth special reference to the tot^acco plant. IT. S. Dept. of Agr. , Uept* 68 s 1-47, 1901 The relation betv/een oxidase and catalase in pl8Jit tii's^aes, Bot. ^asette, 62: 303-310, iei6 Siir les i^apports qui e:ci stent eiitre ia con- stitation ch^nique des composes orgai^iques et xear oxjda-ilite soas 1* influence de la lassase. Bull* 3oc« Chim* par«15:763, 1696 Practical Plant Biochoaistry (text], OBmhrid^e, 1923 5. x^ppleman, Charles C* Relation of catalaare and oxidases to y^spir- ation in plants^ lid# Agr. Bxp. Sta. BllI.ICI, 6. Heed, a. B. ?• Heed, (If. B. 1915 The oxidases of acid tissues. lTo.6, 1914 Bot* ^az» , 57s 8. Illlie, B. 3. 9« lantz, G» V/, The role of oxidases in respiration. Jour. Biol. Ghm. Vol.XXII, L'o.i, 1215 Jour. Biol, Che:^., XV, 277, 1213 Hesplration in com with special reference to c^^talase. ^:^. Jour. Bet., Vol. XIV, TTo.2, 105, 1927 10* Barge, ^^'. S^ Physiolo epical La'co rate ry , U. of Til., 193C 11. Morgulis, Sergius J. Biol. Chan., Vol.UCVIII, ITo.3, 132G 12* Knott, J. £. Gatalase in relation to growth and to other changes in plant tissue. O^omell r-orioir IOC, ang 1926 13. Overhoiser, ^. L. , ai.u orue^^, **• ^» ^,/^ .7 ^ ., ^ ..^^ of ap-lie tissue. Calif. Agr. i^xp. ota. rech.^Bul. 7, 1923 14. Fong, W. T., and Cr^^ss, ^/. T. The effect of pH value on the in- ^ ^' activation tauperature of fruit oxidase. Plaiit ?hysiolo£:y, 4; 537-541, 1929 31 15. iJ'ong, V^* Y., and Gruess, W. V. The effect of pH value and hy- drogen peroxide concentration on froit oxidase activity. Plant ih^^'-siolcQ^, 4:365-366, 1929 16. Kose, ^' H., Kra^rhill, H. ii. , and iiose, d. c;. iiiffect of salts apon oxidase activity of apple "bark. Bot. ^aa*, Vol.IXIX, No*3, 1920 17. authrie, John B. An iodimetric method for determining oxidase activity. Journ. Am. Ghem. Soc. , 52, 3614^ 1930 IB. Forthrop, John H# The kinetics of the decomposition of peroxide hy catalase. Journ. Qen. Physiol* 7s373-387, 1925 SB ACKNOQTL&DGUIEXrr hi The writer wishes to express his apprecia- tion for the many helpfai saggestions of Dr* D. E. Haley, Professor of Soils exi^ Plant Chemistry, under wtiose direction this investiga- tion was condacted, also to Joseph J. Thomas, Instructor in Agrlcultaral and Biological Chem- istry for his valuahle assistance.