HARVARD UNIVERSITY Go Si LIBRARY OF THE Museum of Comparative Zoology MUS. COMP. ZOOL LIBRARY Postilla FEB 14 tues HARVARD UNIVERSITY. PEABODY MUSEUM OF NATURAL HISTORY YALE UNIVERSITY NEW HAVEN, CONNECTICUT, U.S.A. Number 91 October 22, 1965 THE RED-YOLKED EGG OF THE TOURACO, LAURACO’ CORY THAIX* TIMOTHY H. GOLDSMITH DEPARTMENT OF BIOLOGY, YALE UNIVERSITY The touracos are of biochemical interest because of the strik- ing red and green colors imparted to their plumage by the copper porphyrins turacin and turacoverdin. It is therefore interesting to note that Tauraco corythaix lays an egg with a bright vermilion yolk. As might be expected, however, the color of the yolk is caused by carotenoids, a class of fat soluble pigments unrelated to turacin. Following is a brief description of the carotenoids of a touraco egg, in which it is shown that the principal member is either the red pigment astaxanthin (3, 3’-dihydroxy-4, 4’-diketo- 8-carotene) or astacene, the closely related tetraketo compound. MATERIALS AND METHODS A single egg of Tauraco corythaix was made available by Dr. S. D. Ripley. The bird had been reared in captivity and was about a year old when it layed. The egg which we received had been accidentally broken. Several days elapsed before it was extracted, but for most of this period it was kept frozen (—15° C). 1] am indebted to Lana Warner Palumbo for her skillful assistance. This work was supported in part by a grant (NB-03333) from the U.S. Public Health Service. ho Postilla Yale Peabody Museum No. 91 The bird had been fed fruit, but because it was kept in an open aviary, it couid possibly also have eaten occasional insects and other animal matter. The yolk was ground with anhydrous sodium sulfate until dry. The resulting powder was placed in a glass tube and extracted exhaustively by pouring acetone onto the top of the column. The red solution recovered from the bottom was diluted with water and the pigments transferred to petroleum ether in a separatory funnel. The petroleum ether solution was washed with water, dried over anhydrous sodium sulfate, filtered through a small plug of cotton, and evaporated to dryness under reduced pressure to remove the last traces of acetone. The colored material was redissolved in fresh petroleum ether and chromatographed on a column of aluminum oxide (Merck reagent, “suitable for chromatographic adsorption”) whose ad- sorptive strength had been weakened by the addition of 5 per cent (w/w) water. The column was developed with increasing concen- trations of acetone in petroleum ether. The final fraction would not migrate in acetone or ethanol. It was recovered by extruding the top of the column and extracting the alumina with glacial acetic acid. This fraction was then transferred again to petroleum ether in a separatory funnel. The eluted fractions were evaporated to dryness, dissolved in a known volume of fresh solvent, and their absorption spectra measured on a Cary recording spectrophotometer. Additional tests were performed in several cases; these will be described with the results. RESULTS The fractions recovered from the alumina column are listed in Table I. They are numbered in the order they were removed from the column but listed in order of abundance, which is simply the reverse of the sequence of elution. Each fraction will be de- scribed in turn, starting with No. 5. No. 5 accounts for nearly two-thirds of the total pigment and shares a number of properties with free, i.e. unesterified, astaxan- thin and astacene. (Astacene, tetraketo-S-carotene, is readily formed by the oxidation of astaxanthin and is spectrally similar. ) Like the red carotenoid of lobster shells, it was so tightly bound to the top of the column of aluminum oxide that it could not be et 22,1965 The red-yolked egg of the Touraco 3 removed with acetone or ethanol; glacial acetic acid was required. It possessed a single broad absorption maximum in the visible, (Fig. 1) which is similar in shape and position to astaxanthin and astacene (Table II). Based on the molar extinction coefficient of astaxanthin, the yolk contained about 2.4 x 10° moles of this pigment. MUS. COMP. ZOOL TABLE I LIBRARY Chromatographic fractions from the yolk of the ee elative amount present (percentage of total) is approximate, as it 1 ed“on the” ~ assumption that the molar extinction coefficients of all the carotenoids are the same, which is only roughly true. The 1.3 per cent offthe which does not appear in the fourth column was eluted between jhands.c jppy Absorption maxima (and Per- shoulders) in Band Color Eluant centage petroleum number of Band Required of total ether (mu) red glacial acetic acid 62 464 4 yellow 25-30% acetone in petroleum ether 20.5 471, 443-444, (420) 3 diffuse pink 20% acetone in petroleum ether 7.4 470-471, (448) 2b = pale yellow 10% acetone in petroleum ether 5.6 438, (472) 2a pale yellow 4% acetone in petroleum ether 2.1 439, (470) 1 pale yellow petroleum ether el 468, 426, 400 From the percentage of acetone required for elution, band No. 4 was clearly an unesterified xanthophyll with two hydroxyl groups. Precise identification, however, was not possible. The degree of fine structure in the absorption spectrum (Fig. 1) is intermediate between isomerates of lutein (3,3’-dihydroxy-a-ca- rotene) and zeaxanthin (3,3’-dihydroxy-S-carotene). In petroleum ether the pigment exhibited absorption maxima at 471 and 443-444 my and in ethanol at 473 and 447 my. These features suggest a mix- ture of zeaxanthin and lutein; however, the band appeared uniform in color cn the column, and aliquots from the front and trailing portions were spectrally indistinguishable. There was no significant a Postilla Yale Peabody Museum No. 91 400 500 600 my Fig. 1. Absorption spectra of touraco astaxanthin in carbon disulfide (filled circles, solid curve) and a xanthophyll (fraction No. 4) in ethanol (open circles, broken curve) from the yolk of Tauraco corythaix. TABLE II Comparison of the absorption properties of touraco pigment and lobster astaxanthin. -———— Absorption maximum (mp) ——————} petroleum carbon ether chloroform disulfide pyridine Touraco pigment 464 484 501 490 Astaxanthin Pa AGS — 502** 4917 (from lobster shells) * crystals, unpublished observations ** Goodwin (1952) 7 Karrer and Jucker (1950, pg. 354); also saponified material (astacene ) after adsorption to alumina, unpublished observations of the author. er. -22;. 1965 The red-yolked egg of the Touraco 5 spectral shift in the presence of traces of HCI in ethanol, indicat- ing the absence of 5:6 epoxide bridges. No. 3, a diffuse pink band eluted with 20 per cent acetone in petroleum ether, possessed a rather unusual absorption spectrum (Fig. 2) with its principal maximum at 470-471 my» in petroleum ether, 483 my in chloroform, and 484 my» in benzene. The fraction appeared homogeneous when rechromatographed. It showed no change in absorption properties in the presence of potassium borohydride in ethanol. On partition between petroleum ether and aqueous methanol, the pigment distributed 33:67 (epiphase: hypophase) with 95 per cent methanol and 65:33 with 85 per cent methanol. This corresponds to an Ms» coefficient (cf. Krinsky, 1963) of 90. The possibility that the pigment was present as an ester was not examined. With respect to the position of the principal peak and the 400 500 600 my Fig. 2. Absorption spectrum of pigment No. 3 from the egg of Tauraco corythaix in chloroform (open circles, dotted curve) and petroleum ether (filled circles, dashed curve). 6 Postilla Yale Peabody Museum No. 91 relative degree of fine structure in the absorption spectrum, this red pigment is suggestive of capxanthin; however, it differs in other spectral details and in its chromatographic properties. No. 2a and 2b were spectrally indistinguishable, with d,,,, at 438-439 mp and a shoulder at 470-472 my» in petroleum ether. They differed only in the extent to which they were adsorbed on aluminum oxide (Table I). No. 1 showed sharp maxima at about 468, 426 and 400 mp, but there was much end absorption which interfered with precise measurement. The small amounts of these three pigments precluded further work. DISCUSSION Birds tend to accumulate xanthophylls in preference to caro- tenes, and in this respect the egg of the touraco is no exception (for reviews see Fox, 1953; Goodwin, 1952). Astaxanthin, al- though not found in higher plants, is frequently encountered in animals. In birds, it has previously been reported from the eggs of a gull (Larus ridibundus) and a stork (Ciconia ciconia), as well as the wattles of pheasants (Brockmann and Volker, 1934), the cone oil drops of the chicken retina (Wald and Sussman, 1938), and occasionally in the feathers (VOlker, 1950). It seems to be made from plant carotenoids by the chicken (Wald and Zussman, 1938) and probably also the flamingo (Fox, 1960), but if the diet of the bird contains sources of astaxanthin—for example, when planktonic crustaceans occur in the food chain—the ability to synthesize astaxanthin is possibly not present. The present re- sults with the touraco are interesting, for it is doubtful that the parent bird received more than traces of astaxanthin in its food. The large amount of astaxanthin in the yolk therefore indicates that the touraco is able to form this pigment by oxidizing other carotenoids. There is evidence that one or both the red pigments of the egg occur elsewhere in the bird, for the red color of the bill and skin about the eye are reported to be carotenoid (L. Auber, cited in Moreau, 1958). SUMMARY The yolk of Tauraco corythaix is bright vermilion. The caro- tenoids responsible have been separated by chromatography and Get. 22, 1965 The red-yolked egg of the Touraco Z their absorption spectra recorded. About three-fifths of the color is astaxanthin (or astacene) and one-fifth a xanthophyll similar to lutein and zeaxanthin. Both pigments were found unesterified. Several other carotenoids are present in minor amounts, but these could not be identified from the sample available. REFERENCES CITED Brockman, H. and Volker, O. 1934 Der gelbe Federfarbstoff des Kanarien- vogels und das Vorkommen von Carotinoiden bei Vogeln. Hoppe- Seyler’s Z. f. Physiol. Chem., 224: 193-215. Fox, D. L. 1953. Animal Biochromes and Structural Colours. Cambridge, University Press. Fox, D. L. 1960. in Comparative Biochemistry of Photoreactive Systems, M. B. Allen, ed., New York, Academic Press. Goodwin, T. W. 1952. The Comparative Biochemistry of the Carotenoids. London, Chapman and Hall. Karrer, P. and Jucker, E. 1950. Carotenoids. Amsterdam, Elsevier. Krinsky, N. I. 1963. A relationship between partition coefficients of caroten- oids and their functional groups. Analyt. Biochem., 6: 293-302. Moreau, R. E. 1958. Some aspects of the Muscophagidae. Part 3. Some General Features. /bis, 100: 238-270. Volker, O. 1950. Astaxanthin als Federpigment. Die Naturwissenshaften, 37; 309 . Wald, G. and Zussman, H. 1938. Carotenoids of the chicken retina. J. Biol. Chem., 122: 449-460. WIKI Vivi lh 4 066 305 244 at ss Jone ST a ele sre » alee f—8 ae =e Sr SN a rie Speen anon nie at Pree ats ere ae I a ate i el IE =a eel Rr ms a earn ane A