ABSTRA 1. Green canthaxanthin-carotenoproteins were iso¬ lated and partially purified from three species 7 of marine idoteid isopods - lootea monter sis (Maloney), Idotea resecata (Stimpson), and Idotea kirchanskii (Miller and Lee). The proteins have similar absorption maxima at 2. 280 nm. and 683 nm., similar electrophoretic properties, and a molecular weight of greater than 480,000. 3. Although shown not to be lipoproteins, the pro- teins show both lipophilic and hydrophilic properties. 4. A comparison of canthaxanthin-carotenoproteins and astaxanthin-carotenoproteins is made. (2) INTRODUCTION Carotenoproteins, complexes in which a carotenoid and a protein are in stoichiometric combination, were first discovered in the invertebrates in 1883 (Merejkowsky, 1883) and since have been shown to have a very wide distribution (Cheesman et al., 1967). o Most study of the invertebrate carotenoproteli ns has centered on three proteins - crustacyanin, the blue protein of the lobster carapace (Wald, Nathanson, Jencks, & Tarr, 1948; Jencks & Buten, 1964; Cheesman, Zagalsky & Ceccaldi, 1966), ovoverdin, the green pigment of lobster eggs (Kuhn & Sorensen, 1938; Stern & Salomon, 1937, 1938), and ovorubin, the red protein of the eggs of the gastropod Pomacea inalicul ata (Cheesman, 1958; Norden, 1962). Of these, crustacyanin has been analyzed the most thoroughly, probably due to its availability and ease of pur ification. All of these carotenoproteins have astaxanthin as their prosthetic group and for quite some time it was believed that astaxanthin was the only carotenoid found as a caro¬ tenoid prosthetic group in invertebrate caroteno¬ proteins. In 1966, astaxanthin esters were found to be the prosthetic group in the egg carotenoproteins in (3) Eunagurus (Cheesman & Prebble, 1966) and in a sub- sequent publication in the same year canthaxanthin was found in the green carotenoprotein of the marine isopod Idotea montereyonsis (Maloney) (Lee, 1966a). Canthaxanthin was also shown to be involved in the ea resecata (Stimpson) (lee a carotenoprotein of Ido Gilchrist, 1972) and in a variety of Anostraca (Gil¬ christ, 1968). Other than very preliminary work little is known about these canthaxanthin-caroteno¬ proteins. Recently, studies conducted on the egg astaxanthin- carotenoproteins of a variety of aquatic invertebrates indicated these proteins to exhibit remarkable simi- larities (Zagalsky, 1971). This relatedness combined with wide distribution may imply that these proteins have similar functions, perhaps developmental in nature. The function of the canthaxanthin-caroteno¬ proteins is unknown, but at least in the isopods, they may be involved in color change (Lee, 1966a, 1972). There were therefore two basic interests in initiating this study of canthaxanthin-carotenoproteins: 1) To compare them with the astaxanthin-caroteno- proteins; and 2) To determine if there exists inter- (4) specific similarities between the canthaxanthin- rotenoproteins of differont isopod species. For this purpose, Idotea montereyensis (Maloney), ta (Stimpson), and Idotea kirchanskii Idotea rese (Miller & Lee) - three closely related species of ma ine isopods - were used. The present studies were undertaken to compare various properties of the reen carotenoproteins isolated from three species of marine idoteid isopods, including absorption spectra, electrophoretic patterns, composition, . minimum molecular weight, and stability to light and heat. AN THOD ATERIALS Samples of the green variety of I. montereyensi skii were collected from beds of the and I. kird eelgrass, Phy scouleri, both at Tomales adi Bay, Marin County, California and at Seventeen Mile Drive, Monterey County, California. Samples of the brown coloured ve riety of I. resecata were collected D7 ra off the Hopkins from the kelp! oysis Marine Station, Pacific Grove, California. c (5) ate The following materials were utilized: Sephadex G-200 (Tharmacia, Inc.); DEAE-Cellex 50 (Bio-Rad Laboratories); and Cyanogum-41 (E-C Apparatus Corp.). All other chemicals were of reagent grade. Phosphate buffers were prepared by mixing solutions I KHyPO, and NagHPO, to the appropriate pH. teins eno xtraction andp car iono From 100-200 animals, all of which had been maintained in the laboratory in cold aerated sea water, were starved at least seven hours to remove + gut contents. They were subsequently frozen unoll extracted. Green protein complexes were extracted in 0.01 M phosphate buffer (KH,PO, + NagHPO,), pH 7.0 ing the method of Lee (1966a); the crude extract was centrifuged at 12,000 RPM at 0°0. for one hour to remove nonaqueous residues (Gilchrist, 1968). Solid ammonium sulfate was added to the solution to igit to 30 percent saturation and the resulting bri precipitate spun down and discarded. The green itated protein, which remained in solution, was prec ith 70 percent ammonium sulfate, centrifuged and redissolved in 0.001 M phosphate buffer, pH 7.0. 0 (6) The crude protein extract was placed in concen¬ trated solution on columns of Sephadex G-200 and eluted with 0.1 M phosphate buffer, pH 7.0. Columns were approximately 2.5 x 60 cm. and were thoroughly equilibrated in the cold with 0.1 M phosphate buffer, pH 7.0 before the protein solutions were added. The partially purified green protein was resatur¬ ated with 70 percent ammonium sulfate, centrifuged, and redissolved in a small amount of 0.001 M phosphate buffer, pH 7.0. The green solution was placed on DEAE-Cellex 50 Anion Exchange columns, prepared by the method of Lee (1966a), and was eluted with a igth of the perfusing stepwise increase in ionic sti phosphate buffer. urification of the Sephadex G-200 treated protein complexes was also attempted by means of treatment with diethyl ether and dialysis against istilled water. Diethyl ether treatment involved lightly shaking the protein solution with two volumes of diethyl ether, centrifuging the mixture for five minutes at 0°0., and removing the upper ether layer with suction; the lower aqueous layer was saturated with 70 percent ammonium sulfate, centrifuged, and the resulting precipitate redissolved in 0.001 M phosphate (7) buffer, pH 7.0 and recentrifuged to remove denatured tein and lipid. Dialysis of the Sephadex G-200 purified protein was performed in distilled water with stirring and frequent changes of water for two days at 5°0. under conditions of minimal illumination. The resulting precipitated protein was centrifuged, redissolved in 0.2 M phosphate buffer, pH 7.0, and recentrifuged. Absorption The absorption spectra of the carotenoproteins of the three species of isopods were obtained on a Beckman DBG Recording Spectrophotometer at successive fication. The ratio of absorbance sages of pui at the major peaks in the ultraviolet range (280 nm.) and the visible portion of the spectra (683 nm.) were recorded and calculated as E00/683. ectrophoresis D Electrophoresis was performed using an E-O Vertical Gel electrophoresis unit. Five percent Cyanogum-41 gels were utilized with Tris-NagEDTA- ric Acid buf. er at pH 8.4. All material was run between 200 and 300 v., in order to maintain a current (8) of 60-120 amps., for three to five hours. Proteins were stained for with Amido Black 10B and a lipo- crimson prestain (Gurr Inc.) was used to determine the presence of lipoproteins. All other procedures were carried out as noted in E-C Technical Bulletin No. 140 (E-C Apparatus Corp.). Apoprotein Protein complexes which had been purified by passage through columns of Sephadex G-200 and re¬ precipitated with 70 percent ammonium sulfate were treated with 6 M urea for one, fifteen, and 30 minute intervals. Spectra were taken of the resulting solutions and electrophoresis of protein samples treated with 6 M urea for 30 minutes was performed, sing 6 M urea-Cyanogum-41 gels and Tr s-NaEDTA¬ Boric Acid-6 Murea buffer, pH 8.4. In a subsequent experiment, Sephadex G-200 ified carotenoproteins were treated with equal volumes of acetone; the white precipitate was centri¬ fuged, redissolved in 0.1 M phosphate buffer, pH 7.0, and run on electrophoresis using a normal Cyanogum-+ gel and Tris-NajEDTA-Boric Acid buffer, pH 8.4. (9) i Approximate moleculary ltration studie it - Estimates of molecular sizes of the purified carotenoproteins (purified by Sephadex G-200 fil¬ tration, DEAE-Cellex 50 Anion Exchange, and dialysis against water) were obtained from a gel filtration study. A column of Sephadex G-200 (2.5 x 60 cm.) was equilibrated with 0.1 M phosphate buffer, pH 7.0, (flow rate was 18-20 ml. per hour) and was calibrated with a series of standard proteins, as explained by Cheesmane t al. (1966) and Andrews (1964). Blue dextran was used to determine the elution volume of the column. A molecular size estimate was ob¬ plotting log molecular weight against elution tained by volume (Andrews, 1964). o RESUDIS Absorption spectra The absorption spectra of the carotenoproteins of all three species of isopods under study were very similar. The spectra of the carotenoproteins, at various stages of purification, are shown in Fig. 1, 2, and 3. All display absorption maxima at (10) 280 nm. and 683 nm. In crude protein preparations, absorption below 500 nm. was usually so great that the 280 nm. peak was occluded and corresponding E200/683 ratios were either high or unobtainable. With Sephadex G-200 purified proteins, character- istic free carotenoid peaks were visible between 400-500 nm. and the absorption peaks at 280 nm. and 683 nm. became more clearly defined. At higher levels of purity, for all the proteins, the E00/683 sios decreased to an even greater degree and the 400-500 nm. absorption was largely removed. It should be noted that typically as canthaxanthin- carotenoproteins reach higher stages of purity with — +-- loss of contaminating proteins, the characteristic 00/683 ratio - the ratio of optical densities at 280 nm. to 683 nm. - is decreased and stabilized and thus serves as one indicator of carotenoprotein tio purity. The observed reduction in the E therefore may be attributable to removal of other proteins, including possibly some lipoproteins, from the crude carotenoprotein sample and the reduction in free carotenoid absorbance to a removal of lipid and lipid-dissolved carotenoids. These possibilities are suggested by the offects of treatment of the (11) carotenoproteins with diethyl ether and dialysis against water. Both of these processes firs decreased the E/683 ratio and second removed lipid and dissolved carotenoid, as evidenced by detection of lipid on the dialysis bags and by the change in color of the protein solution from green to blue. fic tion The purity of the carotenoproteins was deter- 1280 mined by absorption spectra (E /683) and electro¬ phoresis, each of which was performed at successive stages of purification. The five major stages of purity obtained for each protein are shown in Table 7 with corresponding E 0/683 ratios. As the proteins were passed through the various purification pro¬ cesses, the E/683 ratios decreased but never completely stabilized. Noteworthy is the fact that tereye only the carotenoprotein of I. is could be passed through DEAE-Cellex 50 Anion Exchange columns; that of I. kirchanskii remained immobile on the column, while that of I. resecata denatured on the column. 0 (12) Eloctroph resi The carotenoprotein complexes isolated from eee a sis, I. kirchan rese i, and I I. mont were remarkably similar electrophoretically as shown in Fig. 4. At all stages of purification and in all species three distinct protein bands were present. These are represented as bands 1, 3, and 4 on Fig. 4. Band 2 and other intormediary bands probably repre¬ es, as evidenced by the fact that they sent impur were colorless and were at least removed upon puri¬ ntereyensis. fication of the carotenoprotein in I. m The band which remained at the origin (band 1, Fig. 4), though originally believed to represent the caroteno¬ ein band, appears, as a result of this investi¬ prot ation, to be denatured protein or protein held back by lipid contamination. Evidence indicates in fact the green carotenoprotein of all three that species is represented solely by the two lower bands (bands 3 and 4, Fig. 4); green color was observed to move to these positions during the course of several electrophoretic runs. Upon treatment of the protein samples with 6 M urea, though a loss of absorbance at 683 nm. was noted, indicating a change 0 (73) in carotenoid-protein linkage, there was no change in the number of bands present, suggesting that the isopod carotenoproteins are not polymers made up of different molecular weight subunits as commonly seen in crustacyanin (Cheesman, al., 1966). Similar results were obtained with acetone denatured protein as well. Lipocrimson prestain always remained at the origin and was never observed to migrate down the ing that the isopod caroteno- gel, againsug proteins are not lipoproteins, although they do ppear to have strong lipophilic properties. + eight estim G-200 liltration ation -S eph Molecu Preliminary evidence, based on an experiment in which the carotenoproteins of all three species were mixed together and observed to move homogene¬ sly through a column of Sephadex G-200 (1.5x 30 cm.), indicated that all have approximately the same molecular weight. Sephadex G-200 gel filtration of the purified nsis carotenoprotein against protein 1.T tere standards show it to have a molecular weight greater than 480,000, perhaps as high as 1,200,000 (Fig:5). (74) This work, however, can only be considered as reliminary and more careful determination must be made before a definitive statement of molecular size can be made. DT 10 D As a result of this brief investigation the green carotenoproteins of the three marine idoteid isopods - Idotea mo itereyensis (Maloney), Idotea resecata (Stimpson), and luotea kirchansk i (Miller Lee) - were all isolated (that of I. kirchansk time) in at least a partially purified for the firs form and were shown to display remarkable inter- specific similarities in absorption spectra, elec¬ trophoretic patterns, molecular weight, and stability to sunlight and heat; in addition, all were shown to exhibit similar and unique behavior in both lipid and aqueous environments. Purification Purification of the carotenoproteins represented a major part of the work of this investigation. As /683 ratios obtained at evidened by thel 0 (15) successive stages of purification (Table 1), the proteins were never completely purified. The major difficulty seemed to be lipid contamination, occurring in varying degrees with the respective species. Lipid and lipid-dissolved carotenoid con¬ tamination was evidonced by the behavior of the proteins after treatment with diethyl ether and after dialysis against water. As a result of both of these procedures, the E/683 ratios of all proteins decreased (Table 1), indicating a loss of contami¬ ating protein, perhaps lipoprotein in nature, and the protein solutions tended to change in color from green to blue or blue-green, the latter implying that lipid and dissolved carotenoid were being re¬ moved from association with the carotenoprotein. — Lipid was, in fact, detected on the dialysis bags after such treatment. The levels of lipid contamination varied with the species. Based on absorption spectra and mobil- ity on DEAE-Cellex 50 columns, it appeared that the e sis contained the carotenoprotein of 1. monter least lipid contamination, while those of 1. kircha ski resec a seemed to be more highly contaminated, and (76) as they were both immobile on DEAE-Cellex 50. In addition, following diethyl ether extraction and subsequent resaturation and centrifugation with ammonium sulfate, the carotenoprotein complex of is formed a solid blue-green precipi¬ I. monte tate at the bottom of the centrifuge tube, while har secata often those of I.I i and of I. formed a green paddy at the air-supernatant inter- phase as well as a precipitate, indicating a higher content of lipid. Although, as a result of the lipid contamination, completely purified proteins were never obtained, the finding that lipid contamination of the caroteno- proteins is present to a considerable degree in all species implies that these molecules have both lipophilic and hydrophilic ends. Perhaps this duality of properties has significance in color change and other possible functions. It is noteworthy that the the only species carotenoprotein of I. monter capable of undergoing color change (Lee, 1966a), has the least lipid associated with it. That this molecule can associate with both lipid and aqueous environments may have bearing on such a process. - Furthermore, such a molecule might even facilitate (17) movement of materials between lipid and water within a biological system. In fact, green caro¬ tenoproteins have been observed to occur in the prevensis (Lee, personal communica¬ eggs of I. tion, 1973) as well as in the carapace, suggesting possible roles in transport or storage of nutrients. sorptio bectra The characteristic 280 nm. and 683 nm. absorption anthaxanthin-carotenoprotein are paks of the likewise present in all species, but with varying amounts of free carotenoid contamination, depending on the species and degree of purification (Fig. 1, 2, and 3). This carotenoid-lipid contamination may in fact be an artifact of the isolation procedure; within the animal, as noted above, the environment of the carotenoprotein could be either lipid or aqueous in nature and therefore the degree of asso¬ ciation with lipid dissolved carotenoid in the animal night be entirely different than that obtained in ein extraction. pro Tlectroh sis Electrophoretic studies resulted in several (78) interesting findings. First, the electrophoretic patterns of the three green carotenoproteins inves¬ tigated are identical (Fig. 4), with the two lower dark-staining bands (bands 3 and 4 in Fig. 4) repre¬ senting the carotenoprotein complex. The observa¬ tion that green color migrated to the positions occupied by these two bands implies that this mole- cule is composed of two subunits. Treatment of the carotenoprotein with 6 M urea or acetone did not change the electrophoretic pattern, although some change in carotenoid-protein linkage was evidenced in absorption spectra as there was a complete loss of absorbance at 683 nm. Furthermore, when samples of protein were prestained with lipocrimson, the red stain never grated away from the origin, as did the green color. This finding suggests that the canthaxanthin-caro- rchanskii, e ansis, I.k tenoproteins of I. monter a are not lipoproteins and that the and I.1 resecat lipocrimson stain remains attached to lipid-contam¬ inated protein at the ori in. Perhaps the lipid found associated with the isopod carotenoproteins serves in some way to hold the subunits of the molecule together, which are then quite easily disso¬ ciated upon electrophoresis. (19) These findings are in contrast to the situation found in crustacyanin, an astaxanthin-protein of large molecular weight which appears as a homoge- neous band upon electrophoresis under normal condi¬ tions (Cheesman et al., 1966); however, when treated with a strong denaturing agent such as 6 Murea a number of bands appear in the electrophoretic pattern, indicating dissociation into subunits (Cheesman, e 1966). The fact that no such increase in electro¬ phoretic protein bands occurs upon treatment of isopod carotenoproteins with 6 M urea implies that erent the association of subunits is of a much dif nature than that seen in crustacyanin. i to othe rotenoproteing 1 - Rel The present studies therefore indicate quite close interspecific similarity between the green canthaxanthin-carotenoproteins of three species of marine idoteid isopods; these proteins are pre¬ dominantly cuticular in nature, although they occur in other body regions as well as in the eggs. In addition to perhaps indicating a similarity in function of these isopod carotenoproteins, this relationship is in contrast to that found in the vertebrate astaxanthin-carotenoproteins in which 0 (20) close interspecific similarity has been found only in the egg carotenoproteins (Zagalsky, 1974). No such relatedness has been observed in the carapace astaxanthin-carotenoproteins so far studied. A comparison of the invertebrate canthaxanthin¬ carotenoproteins and the astaxanthin-carotenoproteins shows there to be some similarity (both classes of proteins are carotenoid-proteins of high molecular weight which bleach after varying amounts of time in sunlight), but the two classes of proteins are by no means identical (Table 2). Differences are seen to exist in: 1) Absorption maxima; the typical isopod carotenoprotein absorbances at 280 nm. and 683 nm. (in the pure form) are in contrast to the wide variety of absorption maxima seen in the hin-proteins (for example at 278 nm., 476 nm., aal and 660 nm. for ovoverdin); in addition, the astaxan- thin-proteins appear to have no lipid-carotenoid ssociations as do the isopod carotenoproteins in the impure form as evidenced by a lack of wide absorbance in the 400-500 nm. range; 2) Color; the canthaxanthin¬ proteins tend to be green to blue-green in color, depending upon the amount of lipid-carotenoid association, while the astaxanthin-carotenoproteins (21) appear in a wide variety of colors - for example, crustacyanin is blue, ovoverdin is greon, and ovorubin red; and 3) Prosthetic group; in addition to differences in carotenoid prosthetic group, some astaxanthin-proteins such as ovoverdin are lipoproteins while present studies indicate the isopod carotenoproteins to have no lipid as a prosthetic group; both ovoverdin and ovorubin are ycoproteins while to date it is not known if the isopod canthaxanthin-proteins contain a carbohydrate moiety. Very preliminary evidence indicates the isopod carotenoproteins to be of a much greater molecular size than that of either crustacyanin (MW 380,000) al., 1966), ovoverdin (MW 300,000) (Cheesman (Ceccaldi et al., 1966), or ovorubin (MW 330,000) (Cheesman, 1958). The evidence suggests the isopod canthaxanthin-proteins to have a molecular weight in xoess of 480,000; however, this remains to be con¬ irmed on totally purified material. This brief investigation of the green caroteno¬ proteins ol luotea monterey s (Maloney), Idotea rii (Miller & ote ata (Stimpson), and I Lee) gave results which can only be regarded as 0 (22) preliminary and much work is yet needed to gain further insight into the structure and function of these canthaxanthin-carotenoproteins. However, these studies do indicate that the invertebrate canthaxanthin- and astaxanthin-carotenoproteins are quite different in a number of respects and that there exists striking interspecific similarities between the carotenoproteins of three closely related species of marine idoteid isopods. 0 (23) T CKNOL The author wishes to thank Dr. Welton L. Lee for initial introduction to the study of caroteno- sistance proteins and for providing stimulation and as throughout the course of this investigation. 0 (24) ERENGES ANDREWS P. (1964) Estimation of molecular weights of proteins by gel filtration. Biochem. J. 91, 222-233. CECCALDI H. J., CHEESMAN D. F., and ZAGALSKY P. F. (1966) Quelques proprietes et caracteristiques de l'ovoverdine. C.r. Seanc. Soc. Biol. 160, 587-590. CHEESMAN D. F. (1958) Ovorubin, a chromoprotein from the eggs of the gastropod mollusc Pomacea canaliculata. Proc. R. Soc. B. 1 9, 571-587. CHEESMAN D. F., LEE W. L., and ZAGALSKY P. F. (1967) Carotenoproteins in invertebrates. Biol. Rev. 42. 132-160. CHEESMAN D. F. and PREBBLE J. (1966) Astaxanthin ester as a prosthetic group: a carotenoprotein from the hermit crab. Comp. Biochem. P siol. 17, 929-936. CHEESMAN D. F., ZAGALSKY P. F., and CECCALDI H. J. (1966) Purification and properties of crusta¬ 16. cyanin. Proc. R. Soc. B. 4, 130-151. IIGHPT GILOHRIST B. M. (1968) Distribution and relative abundance of carotenoid pigments in Anostraca 0 (25) (Crustacea: Branchiopoda). Comp. Biochem. 4, 123-147. Physiol. JENCKS W. P. and BUTEN B. (1964) The denaturation of crustacyanin. Arcl hs. Biochem. phys. 107, 511-520. KUHN R. and SORENSEN N. A. (1938a), Über astaxanthin und ovoverdin. chem. Ges. 71, dtsch 1879-1888. LEE W. L. (1966a) Pigmentation of the marine isopod eeyel nsis (Maloney). Comp. Biochem. Idotea mont: Phye ol. 18, 17-36. LEE W. L. (1972) Chromatophores and their role in color tereye ensi change in the marine isopod Idotea mo col. 8, 930-947. exp.! (Maloney). J mar. Biol. LEE W. L. (1973) Personal communication. Hopkins Marine Station, Stanford University. A LEE W. L. and GILUARIST B. M. (1972) Pigmentation, color change and the ecology of the marine isopod ldotea sata (Stimpson). J. exp. mar. 9, 1-27. Biol. Ecol. 1 - MEREJKOWSKY C. DE (1883) Nouvelles recherches sur la zoonerythrine et autres pigments des animaux. Bull. Soc. zool. 8, 81-97. (26) NORDEN D. A. (1962) Some properties of ovorubin, a carotenoprotein from the eggs of macea it iculata. Ph.D. Thesi s, Univers ity of London. can STERN K. G. and SALOMON K. (1937) Ovoverdin, a pig- ment chemically related to visual purple. 86, 310-311. e, N Scier STERN K. G. and SALOMON K. (1938) On ovoverdin, the carotenoid-protein of the egg in the lobster. biol. Chem. 122, 461-72. WALD G., NATHANSON N., JENCKS W. P., and TARR E. (1948) Crustacyanin, the blue carotenoid-protein of the lobster shell. ol. Bull. mar. biol o 22, 249-250. Woo Hole ZAGALSKY P. F. (1971) Comparative studies on the amino acid compositions of some carotenoid¬ ning lipoglycoproteins and a glycoprotein conta s and ovaries of certain aquatic from te siol. 47 invertebrates. Comp. Biochem 85-395. IGURE GAPTIONS Figure 1. Absorption spectrum from 250-750 nm. of the groen carotenoprotein complex isolated from Idotea montereyensis (Maloney). (A) represents the crude extract, (B) extract after being passed through a column of Sephadex G-200, and (C) Sephadex G-200 purified ttract after passage through a DEAE-Cellex 50 column. 00/683 - ratio of optical densities at 280 nm. to 683 nm. All spectra were taken of solution in phos¬ phate buffer, pH 7.0. 2. Absorption spectrum from 250-750 nm. of Figur — — the green carotenoprotein complex isolated from idotea kirchanskii (Miller & Lee). (A) represents the crude extract, (B) extract after being passed through a column of Sephadex G-200, and (C) Sephadex G-200 purified extract after being shaken with diethyl ether. 5280, 83 - ratio of optical densities at 280 nm. to 683 nm. All spectra were taken of solution in phos¬ phate buffer, pH 7.0. e 7 5. Absorption spectrum from 250-750 nm. of the green carotenoprotein complex isolated from Idotea resecata (Stimpson). (A) represents the crude extract, d (B) extract after being passed through a column of Sephadex G-200, and (C) Sephadex G-200 purified extract 280/683 - after being shaken with diethyl ether. E 0 e Captions, cont'd. Figu ratio of optical densities at 280 nm. to 683 nm. All spectra were takon of solution in phosphate buffer, pH 7.0 Figure 4. Electrophoresis patterns on Cyanogum- 41 gel of the green carotenoprotein complexes of 79. e ansis (Maloney), Idotea Kircha mon liller & Lee), and Idotea resecata (Stimpson). (a) - crude extract (b) - extract after passage through a column of Sephadex G-200 (c-1) = Sephadex G-200 purified extract after passage through DEAE-Cellex 50 (c-2) = Sephadex G-200 purified extract after di¬ alysis against distilled water at 5°0. for two days band (1) - lipid-contaminated protein band (2) and other unlabelled bands = contaminating protein bands (3) and (4) - isopod carotenoprotein All runs were made on 5% gels w. ith Tris-NaEDTA-Boric Acid buller, pH 8.4 for 3-5 hours at 200-300 V. Prot ins were stained for with Amido Black 10B. Figure ligul 5. Molecular weight determination of the - no e carotenoprotein of lde tea mon ansis (Maloney) 0 Figure Japtions, cont'd. Pigure by gel filtration on Sephadex G-200. The column was of a length of 60 cm. x 2.5 cm. Flow rate - 18-20 ml./hr. Fraction volume =- 3.0 ml. Samples were applied in 3.0 ml. solution in a single experiment. (1) - blue dextran (MW 2,000,o00) (2) - isopod carotenoprotein (estimated MW 1,200,000) 3) - apoferritin (MW 480,000) (4) - albumin (bovine) (MW 67,000) 55 L 8 1 — 0 f2 — — tta ta tt Kiisuap jeaiide 3 — — - — 5 — E 5 0 2 . s a 1eu 0130 — L 19.7 IABLE CAPTION Table 1. The I /683 ratios of Idoteid caro- tenoproteins at various stages of purification. () crude extract (2) crude extract after being passed through a column of Sephadex G-200 (3) Sephadex G-200 purified protein after being dialysed against distilled water for two days at 500. (4) Sephadex G-200 purified protein after being shaken with diethyl ether (5) Sephadex G-200 purified protein after being passed through a DEAE-Cellex 50 Anion Exchange column 280 /683 - ratio of optical densities at 280 nm. to 683 nm. All spectra were taken of solution in phos- ohate buffer, pH 7.0. able 2. A comparison of canthaxanthin-caroteno¬ proteins and as thin-carotenoproteins on the bas is of absorption spectrum, color, prosthetic groups, stability to light and to heat, and molecular weight. 0 — kava- 8 — H O - Tale 8 50 aa S QO 8- 1 35 8 8 8 88 11 . 86 + s oo . O — 11 8 8 — . o ao ot s 5 OO o 55 0 3 8 9. o 8 0 O OO O O O — o a 9 9 5 5 § 8 38 880 Jare 2