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Carotenoid-protein complexes and their stability towards oxygen and radiation
Environmental and Experimental Botan_r, Vol. 20, pp. I to 6.
0098-8472/80/0101-0001 502.00/0
i(S Pergamon Press Ltd. 1980. Printed in Great Britain
C A R O T E N O I D - P R O T E I N COMPLEXES AND T H E I R STABILITY TOWARDS OXYGEN AND R A D I A T I O N T. V. R A M A K R I g I ' I N A N mad F. J. FRANCIS Depa.rtment of Food Science and Nutrition, University of Massachusetts, Amherst, MA 01003, U.S.A.
(Received 16 Ju~v 1975; accepted in revisedform 24 April 1979) RAMAKRISHNANT. V. and FRANCIS, F. J. Carotenoid-protein complexes and their stabilitr towards oxygen and radiation. ENVIRONMENTAL AND EXPERIMENTAL BOTANY '~[I, 1-6, 1980.Carotenoid-protein complexes isolated from fresh mangoes were found to be more stable to oxygen and radiation when dissolved in water as compared with fl-carotene in petroleum ether. Part of the pigment could be released from the complex by gamma irradiation. Observations on the stability of the carotenoid (98~o fl-carotene) in the complex indicated that the pigment is either associated with the lipid prosthetic group of the protein or loosely attached to the protein by weak hydrophobic bonds. INTRODUCTION protein complexes has a significant influence on A LARGE n u m b e r of scientific papers concerning the color characteristics of the fruit system. the fat soluble carotenoids, the the distribution of carotenoids in foods have Unlike been published and there are extensive mono- carotenoid-protein complexes are soluble in graphs in this fieldJ 3' 6, ~5) The overall carotenoid water and they might offer a suitable method for achieving proper clarity in water-based foods pattern may vary from relative simple mixtures to extremely complex ones. The simplest mix- where carotenoids are presently used for colortures are found in foods of animal origin be- ing. Despite the appreciation of the potential of cause of the limited ability of the animal to carotenoid-protein complexes, very- little is known about their presence and nature in fruit absorb, modify and deposit carotenoids. The opposite exteme is represented by the formidable systems. This investigation deals with the isolarray ofcarotenoids encountered in citrus products, ation of carotenoid-protein complexes from a dehydrated alfalfa meal or paprika. These pig- fruit source and a study of their nature and ments may exist in nature in a free form or be stability with special reference to oxygen and biochemicaltv, combined with fatty acids, <5~ pro~,-radiation. teins~ 7, 22, 26)orsugarsJl 1.23, 24)From thepoint of, physical distribution in biological systems, caroMATF.,R/A/~ AND IVIETHODS tenoids have been found dissolved in lipids, Isolation of carotenoid-proteins suspended as crystals or dispersed as proteincomplexes. <41 An attempt was made to isolate carotenoidThe physicochemical nature of the p i g m e n t - proteins from carrots, tomatoes, squash and
Paper No. 1032 from the University of Massachusetts Agricultural Experiment Station. This research was supported in part bv the Glass Packaging Institute, Washington, DC.
T. V. RAMAKRISHNAN and F. J. FRANCIS mangoes (grown in Florida, U.S.A.). The procedure adopted for the isolation and purification is essentially similar to the one described by BACON KE. ~2) The carotenoid-proteins were found in extremely small quantities in carrots, tomatoes and squash. Mangoes were found to have a relatively higher content of these pigment complexes and were used for further work.
Determination of molecular weight of the protein The approximate molecular weight of the protein was determined by the thin layer gelfiltration method of ANDRZWS~1~ using Sephadex G-100 superfine (Pharmacia). The gel particles were suspended in 0.05 M tris hydrochloric buffer, pH 7.5, containing KC1 (0.1M), for 3 days before use. A slurry of suitable consistency for making thin layers was obtained by allowing a suspension of the particles in buffer to settle for half an hour and then removing as much excess buffer as possible through a narrow tube with gentle suction. Thin layer plates of 0.5ram thickness were made. The flow of buffer through the layers was maintained for several hours before the application of samples, and the flow rate was regulated by altering the angle of tilt of glass plates, usually 10--20 ° from the horizontal. Good separations were obtained when the flow was sufficient to move cytochrome C, the reference protein, through 5--6 cm during an 8-10 hr period. Proteins (30-50/~g) in tris-hydrochloric acid buffer were applied to the plates. The separated proteins were detected as brown spots by placing the wet plates in a closed vessel containing iodine vapor. Carotenoid-proteins, because of their color, could be detected on the plates even without iodine treatment. Migration distance in mm was measured for the standard proteins and also for the carotenoid-protein.
Composition of the complex (a) .Nitrogen content. The acid digestion of protein samples, nesslerisation and measurement of the resultant color were done by the method of LANG. (I 5~ (b) Total lipids. The carotenoid-protein was extracted for 4 h r in a Soxhlet apparatus with
100ml of a chloroform-methanol (2: 1) mixture. The lipid extract was washed with 20°~ of its volume of water, taken to dryness on a rotary evaporator and finally extracted with chloroform-methanol-water (16: 8: 1) to decompose proteolipidsJ 9~ The residue was taken up in chloroform-methanol (2: 1), partially evaporated, transferred to a weighed specimen tube and the solvent removed under vacuum. The tube was transferred to a desiccator and evacuated to 0.1 mm Hg pressure over sulfuric acid, to remove traces of water, until constant weight was attained. (c) Phospholipids. The phospholipid content was indirectly determined by estimating the inorganic phosphorous content by the procedure of MARINETTI.(16) The phosphate was converted to phosphomolybdate, reduced to molybdenum blue and quantitated by colorimetric measurements. An appropriate percentage content of phospholipid, based on phosphatidylcholine, was obtained by multiplying the percentage of lipid phosphorous by 25. (d) Detection of sugars. For this qualitative analysis, lipids were removed from the pigment protein which was then hydrolysed "to release the sugars. The sugars were separated and identified by paper chromatography. This procedure was described by HOUGH and JONES.112~ (e) Carotenoids. Carotenoid pigments from the protein complex were extracted with methanol, transferred to petroleum ether, dried over anhydrous sodium sulphate and evaporated to dryness. The pigments were separated by thin layer chromatography using silica gel G as adsorbent and 109'o acetone in petroleum ether as solvent system. Identification of carotenoids was done by co-chromatography and spectrophotometry. (f) Ratio of carotenoid to protein. The pigment was extracted from the carotenoid protein using an aqueous methanol and petroleum ether system and quantitated on the basis of absorption of 450nm. The weight of the protein part was assumed to be equivalent to the weight of the pigment complex, for determining the ratio of carotenoid to protein on the basis of weight. The molar ratio was deduced from the knowledge of the molecular weights of the carotenoid and the protein.
CAROTENOID-PROTEIN COMPLEXES
Stability of the carotenoid in the complex (a) Autoxidation. The pigment protein complex (30.0 mg) was dissolved in 50ml of sterile double distilled water and poured into sterile glass vials. Sufficient headspace was left over the solution to initiate oxidation. The glass vials were stored in the dark at room temperature. A very. small portion of the sample was withdrawn periodically and the carotenoid content was determined by extraction and subsequent quantitation. (b) Effects of radiation, t-carotene was incorporated into three systems at a concentration of approximately 200 #g per 100 ml. The systems were: (1) t-carotene-protein complex in water; (2) a colloidal dispersion of t-carotene in water prepared by adsorbing t-carotene on sucrose and suspending the mixture in water; (3) a solution of t-carotene in petroleum ether. These were packed in glass vials and an equal amount of headspace was left over each system. They were irradiated with ?-radiation using Co 6° as the source. The dose rate was approximately 2500 rads per minute. After the appropriate dose was delivered, each system was analysed for the pigment content by solvent extraction and quantitation on the basis of absorbance at 450nm. The protein complex system was also analysed for the extent of release of fl-carotene from the complex. Pigment extracted in petroleum ether with no addition of a polar solvent was taken as the index of free carotenoid not attached to protein. RI~IJLTS AND DISCUSKION
Composition of the carotenoid-protein complex The pigment-protein complex was analysed for the presence of various components (Table 1). In addition to protein and carotenoid, it contained lipids and sugars, as well as phospholipids in relatively small amounts. Reducing and amino sugars were also detected, but could not be quantitated since they constituted such a small percentage of the protein complex. O f the total carotenoids in the complex, 98°o was composed of t-carotene. The other 2% could
3
Table 1. Compositionof the carotenoid-protoincomplex Component
Percentage
Nitrogen Total lipids Phospholipids Reducing and amino sugars
12.30 2.15 0.14 Detectable
not be identified samples.
due
to lack of sufficient
Molecular weight of the protein An approximate estimation of the molecular weight of the t-carotene-protein complex was done by measuring the migration distance under standard conditions and deducing the value from a standard curve. The migration distance for the pigment was 93 mm and the molecular weight was estimated to be 88,000. Under these experimental condition, the carotenoid proteins did not separate into more than one fraction.
Stability of t-carotene in the complex (a) Autoxidation. Data on the susceptibility of carotene to oxidation when the pigment-protein complex was dissolved in water are shown in Table 2. In 3 days, the loss of pigment was
Table 2. Stability of [3-carotene in the protein complex to oxygen Time (hr) 12 24 48 72
Loss of pigment* (%) 3.2 6.5 12.3 18.1
*Average of five observations.
found to be 18.1% Under similar conditions, t carotene in hexane underwent more than 60%b degradation, while the loss of pigment in colloidal dispersions of t-carotene in water was only 4.5%.
T. V. RAMAKRISHNAN and F. J. FRANCIS (b) Effects of radiation. On radiation treatment, some of the [J-carotene seemed to be released from the pigment-protein complex. In Table 3, data are presented on the extent of release of pigment for three different radiation doses. With an exposure to 0.75 Mrad radiation, almost ¼ of the pigment from the complex was released.
physical or chemical barrier. (2t) In the colloidal system, however, the free radicals formed from water are not readily accessible to the pigment molecules. Most of the damage to [J-carotene in the colloid is likely to be due to the direct effects of radiation. (2°) The solution of pigmentprotein complex has properties characteristic of both the other two systems. The protein mo-
Table 3. Release of the pigment from the protein complex on radiation treatment Radiation dose (Mrads)
Pigment release (% total)*
0.25 0.50 0.75
6 13 23
i
i
*Average of five observations. (c) Radiation sensitivity of [J-carotene in different physical states. In Table 4, data are presented on the extent of radiation damage to [J-carotene in three different systems. The pigment was extremely radiolabile when dissolved in petroleum ether, but relatively stable to radiation in aqueous collodial dispersions. In the protein complex, [J-carotene was found to have an intermediate radiation sensitivity. In petroleum ether, free radicals formed on irradiation of the solvent molecules can travel to the site of the pigment molecules without any
lecules help to solubilize the pigment, but a true solution cannot be achieved, due to the fat soluble nature of [j-carotene. The radiation damage to the pigment in the protein complex may probably be due to the orientation of the /3-carotene molecules in the system and accessibility of free radicals for attack. Further, the lipid prosthetic group of the protein complex may also promote the radiation sensitivity of/3carotene by ~olubilizing it and providing free radicals of lipid origin in the immediate vicinity of the pigment molecules.
Table 4. Effects of radiation on E-carotene in different physical states Radiation dose (Mrads) Nature of the system
0.25
0.50
0.75
Loss of pigment (%)* Protein complex of /J-carotene in water Colloidal dispersions of /~-carotene in water Solution of E-carotene in petroleum ether *Average of five observations.
9.6
19.8
30.6
2.0
4.2
6.5
56.0
82.0
97.0
C A R O T E N O I D - P R O T E I N COMPLEXES Combination of t-carotene and the protein T h e ratio of t-carotene to protein, on the basis of weight, was estimated to be 1: 380. The molecular weight of protein was estimated to be approximately 88,000. t-carotene corresponds to a molecular weight of 536. From these data, the relative distribution of the carotenoid and the protein, on a molar basis, was deduced to be 1:2. This ratio is only approximate and cannot be taken to represent the stoichiometry until the direct link between the protein and t carotene is clearly established. T h e carotenoid-protein complex isolated in this investigation could be easily extracted by acetone or methanol, T h e readiness with which the carotenoid was removed from the complex by water miscible organic solvents eliminates the possibility of any covalent linkage between the pigment and the protein, tv's'ls~ This also eliminates the possibility of glycosidic linkage between the carotenoid and sugar molecules. T h e absorption maxima of t-carotene in petroleum ether were at 480, 452 and 425 nm. The t - c a r o t e n e - p r o t e i n complex in water had absorption m a x i m a at 495 and 460 nm. There were bathochromic shifts of 8 and 15nm in the positions of the principal absorption maxima. This small red shift in the maxima of t carotene in the complex argues against the possibility of carotenoid-protein interaction which is characterized by a strong polarization of the chromophoric group. (7'19) Further, flcarotene, the only carotenoid part of the complex, does not have any carbonyl group for interaction with the amino acid residues of the protein molecule. The link between fl-carotene and the protein, in the present case, is probably quite different from the one found in ovoverdin~14,22) and ~-crustacyanin. t13) It is possible that fl-carotene is associated with the lipid prosthetic group of the lipoprotein. T h e small bathe.chromic shift found in the spectra of the carotenoid in the protein complex is consistent with the red shift expected of lipid-bound carotenoids/1°~ T h e sensitivity of fl-carotene in the complex to oxygen and radiation (Tables 2 and 4) supports the lipid association of the pigment in the protein complex. Further evidence in support of this conclusion is the complete recovery of the pigment
from the complex with even 50°'o acetone. Direct attachment of t-carotene to the protein by weak hydrophobic bonds which do not involve any strong or partial polarization, is another possibility. T h e gradual release of the pigment from the complex on irradiation is also compatible with the possibility.
~ C E S
I. ANDREWSP. (1964) Estimation of molecular weights of proteins by sephadex gel-filtration• Biochem. ft. 91~ 222-233. 2. BACON KE (1971) Carotenoproteins. Pages 624636 in A. S. PIETRO, ed. Methods in Enzynologr, vol. 23. Academic Press, New York. 3. BAUF_atFEZNDJ. C. (1972) Carotenoid vitamin A precursors and analogs in foods and feeds, ft. Agri. Food Chem. 20~ 456--473. 4. BAUERFEINDJ, C., BRUBACHERG. B., KLAUI H. N. and MARUSlCH W, L. (1971) Use of carotenoids. Pages 743-770 in O. ISLER, ed. Carotenoids. Birhauser, Basle. 5. BELL,'a~Y D. (1966) On the lipochromes in the skin of marine teleost fish with special reference to the painted comber (Serranus scriba L.). Comp. Biochem. Biophysiol. 17, 1137-1140. 6. BORENSTEIN B. and BUNNELL R. H. (1966) Carotenoids: properties, occurrence and util• ization in foods. Adv. Food Res. 15, 195-276. 7. CHEESMAND. F., LEE W. L. and ZAGALSKYP. F. (1967) Carotenoproteins in invertebrates. Biol. Rev. 42, 131-160. 8. FALUDI-DANIELA., NAGY A., GYURJA.N I. and FALUDI B. (1965). Characteristics of pigmentprotein complexes in normal and chloroplast mutant leaves. Photochem. Photobiol. 4, 359-367. 9. Fot~zn J. and LEES M. (1951) Proteolipids, a new type of tissue lipoproteins: their isolation from brain. J. Biol. Chem. 191, 807--817. 10. Fox D. L., HOPKINS I. S. and Z1LVERSMITD. B. (1965) Blood carotenoids of the Roseate Spoonbill. Comp. Biochem. Physiol. 1'1~641-649. ll. HERTZBERG S. and JENSEN S. L. (1967) The carotenoids of Mycobacterium phlei, strain \;era. 2, the structure of phlei-xanthophylls-two novel tertiary glucosides. Acta Chem. Scand. 21, 15--41. 12. HOUGH L. and JONES J. K. N. (1962) Chromatography on paper, pages 21-31 in R. L. WmS'rLER and M. L. WOLFROM, (ed.) Methods in Carbohydrate Chemistry, vol. 1. Academic Press, New York.
T. V. RAMAKRISHNAN and F. J. FRANCIS 13. JEscKs W. P. and BUTEN B. (1964) The denaturation of crustacyanin. Arch. Biochem. Biophys. 107, 511-520. 14. KuI-iN R. and S6R~-SF-N N. A. (1938) Uber Astaxanthin und Ovoverdin. Bet. Dtsch. Chem. Ges. 71, 1879-1888. 15. LArcO C. A. (1958) Simple micro determination of Kjeldahl nitrogen in biological materials. Anal. Chem. 30, 1692-1694. 16. M.ARXNETTIG. V. (1962) Chromatographic separation, identification and analysis of phosphatides. 07. Lipid Res. 3, 1-20. 17. NAGY A. H. and FALUDI-DANIEL A. (1967) O n the naturc of binding forces stabilizing carotenoid-protein chlorophyll-protein complexes in vivo. Photosynthetica 1, 69-75. 18. N m m M U R A M. and TAKAMATSU K. (1957) A carotcne-protcin complex isolated from green leaves. Nature (London) 180, 699-700. 19. PLAa'-r J. R. (1959) Carotene-donor-acceptor complexes in photosynthesis. Science 129, 372-374.
20. RAMAKRISHNANT. V. (1967) Radiation lability of carotenoids in natural and model systems. M.S. Thesis, University of Bombay, Bombay, India. 21. RAMAKRISHNANT. V., SAWA~rrP. L. and Ku~rrA U. S. (1970) In vitro stability of ~-carotene in aqueous dispersions. Radiat. Bot. 10, 395-399. 22. STERN K. G. and SALOMON K. (1938) On ovoverdin, the carotenoid-protein pigment of the egg of the lobster. 07. Biol. Chem. 122, 461--475. 23. TmRKELL D. and HUNTER M. I. S. (1969a) Carotenoid-glycoprotein of Sarcina tiara membrane..7. Gen. Microbiol. 58, 289-292. 24. TmRKELL D. and HUNTER M. I. S. (1969b) The polar carotenoid fraction from Sarcina tiara. 07. Gen. Microbiol. 58, 293-299. 25. WEEDON B. C. L. (1971) Occurrence. Pages 2959. in O. ISLER, ed. Carotenoids. Birhauser, Basle. 26. ZAGAtSKY P. F., CH~.ESMAND. F. and CECCALDX H. J. (1967) Studies on carotenoid-containing lipoproteins isolated from the eggs and ovaries of certain marine invertebrates. Comp. Biochem. Physiol. 22, 851-871.
0098-8472/80/0101-0001 502.00/0
i(S Pergamon Press Ltd. 1980. Printed in Great Britain
C A R O T E N O I D - P R O T E I N COMPLEXES AND T H E I R STABILITY TOWARDS OXYGEN AND R A D I A T I O N T. V. R A M A K R I g I ' I N A N mad F. J. FRANCIS Depa.rtment of Food Science and Nutrition, University of Massachusetts, Amherst, MA 01003, U.S.A.
(Received 16 Ju~v 1975; accepted in revisedform 24 April 1979) RAMAKRISHNANT. V. and FRANCIS, F. J. Carotenoid-protein complexes and their stabilitr towards oxygen and radiation. ENVIRONMENTAL AND EXPERIMENTAL BOTANY '~[I, 1-6, 1980.Carotenoid-protein complexes isolated from fresh mangoes were found to be more stable to oxygen and radiation when dissolved in water as compared with fl-carotene in petroleum ether. Part of the pigment could be released from the complex by gamma irradiation. Observations on the stability of the carotenoid (98~o fl-carotene) in the complex indicated that the pigment is either associated with the lipid prosthetic group of the protein or loosely attached to the protein by weak hydrophobic bonds. INTRODUCTION protein complexes has a significant influence on A LARGE n u m b e r of scientific papers concerning the color characteristics of the fruit system. the fat soluble carotenoids, the the distribution of carotenoids in foods have Unlike been published and there are extensive mono- carotenoid-protein complexes are soluble in graphs in this fieldJ 3' 6, ~5) The overall carotenoid water and they might offer a suitable method for achieving proper clarity in water-based foods pattern may vary from relative simple mixtures to extremely complex ones. The simplest mix- where carotenoids are presently used for colortures are found in foods of animal origin be- ing. Despite the appreciation of the potential of cause of the limited ability of the animal to carotenoid-protein complexes, very- little is known about their presence and nature in fruit absorb, modify and deposit carotenoids. The opposite exteme is represented by the formidable systems. This investigation deals with the isolarray ofcarotenoids encountered in citrus products, ation of carotenoid-protein complexes from a dehydrated alfalfa meal or paprika. These pig- fruit source and a study of their nature and ments may exist in nature in a free form or be stability with special reference to oxygen and biochemicaltv, combined with fatty acids, <5~ pro~,-radiation. teins~ 7, 22, 26)orsugarsJl 1.23, 24)From thepoint of, physical distribution in biological systems, caroMATF.,R/A/~ AND IVIETHODS tenoids have been found dissolved in lipids, Isolation of carotenoid-proteins suspended as crystals or dispersed as proteincomplexes. <41 An attempt was made to isolate carotenoidThe physicochemical nature of the p i g m e n t - proteins from carrots, tomatoes, squash and
Paper No. 1032 from the University of Massachusetts Agricultural Experiment Station. This research was supported in part bv the Glass Packaging Institute, Washington, DC.
T. V. RAMAKRISHNAN and F. J. FRANCIS mangoes (grown in Florida, U.S.A.). The procedure adopted for the isolation and purification is essentially similar to the one described by BACON KE. ~2) The carotenoid-proteins were found in extremely small quantities in carrots, tomatoes and squash. Mangoes were found to have a relatively higher content of these pigment complexes and were used for further work.
Determination of molecular weight of the protein The approximate molecular weight of the protein was determined by the thin layer gelfiltration method of ANDRZWS~1~ using Sephadex G-100 superfine (Pharmacia). The gel particles were suspended in 0.05 M tris hydrochloric buffer, pH 7.5, containing KC1 (0.1M), for 3 days before use. A slurry of suitable consistency for making thin layers was obtained by allowing a suspension of the particles in buffer to settle for half an hour and then removing as much excess buffer as possible through a narrow tube with gentle suction. Thin layer plates of 0.5ram thickness were made. The flow of buffer through the layers was maintained for several hours before the application of samples, and the flow rate was regulated by altering the angle of tilt of glass plates, usually 10--20 ° from the horizontal. Good separations were obtained when the flow was sufficient to move cytochrome C, the reference protein, through 5--6 cm during an 8-10 hr period. Proteins (30-50/~g) in tris-hydrochloric acid buffer were applied to the plates. The separated proteins were detected as brown spots by placing the wet plates in a closed vessel containing iodine vapor. Carotenoid-proteins, because of their color, could be detected on the plates even without iodine treatment. Migration distance in mm was measured for the standard proteins and also for the carotenoid-protein.
Composition of the complex (a) .Nitrogen content. The acid digestion of protein samples, nesslerisation and measurement of the resultant color were done by the method of LANG. (I 5~ (b) Total lipids. The carotenoid-protein was extracted for 4 h r in a Soxhlet apparatus with
100ml of a chloroform-methanol (2: 1) mixture. The lipid extract was washed with 20°~ of its volume of water, taken to dryness on a rotary evaporator and finally extracted with chloroform-methanol-water (16: 8: 1) to decompose proteolipidsJ 9~ The residue was taken up in chloroform-methanol (2: 1), partially evaporated, transferred to a weighed specimen tube and the solvent removed under vacuum. The tube was transferred to a desiccator and evacuated to 0.1 mm Hg pressure over sulfuric acid, to remove traces of water, until constant weight was attained. (c) Phospholipids. The phospholipid content was indirectly determined by estimating the inorganic phosphorous content by the procedure of MARINETTI.(16) The phosphate was converted to phosphomolybdate, reduced to molybdenum blue and quantitated by colorimetric measurements. An appropriate percentage content of phospholipid, based on phosphatidylcholine, was obtained by multiplying the percentage of lipid phosphorous by 25. (d) Detection of sugars. For this qualitative analysis, lipids were removed from the pigment protein which was then hydrolysed "to release the sugars. The sugars were separated and identified by paper chromatography. This procedure was described by HOUGH and JONES.112~ (e) Carotenoids. Carotenoid pigments from the protein complex were extracted with methanol, transferred to petroleum ether, dried over anhydrous sodium sulphate and evaporated to dryness. The pigments were separated by thin layer chromatography using silica gel G as adsorbent and 109'o acetone in petroleum ether as solvent system. Identification of carotenoids was done by co-chromatography and spectrophotometry. (f) Ratio of carotenoid to protein. The pigment was extracted from the carotenoid protein using an aqueous methanol and petroleum ether system and quantitated on the basis of absorption of 450nm. The weight of the protein part was assumed to be equivalent to the weight of the pigment complex, for determining the ratio of carotenoid to protein on the basis of weight. The molar ratio was deduced from the knowledge of the molecular weights of the carotenoid and the protein.
CAROTENOID-PROTEIN COMPLEXES
Stability of the carotenoid in the complex (a) Autoxidation. The pigment protein complex (30.0 mg) was dissolved in 50ml of sterile double distilled water and poured into sterile glass vials. Sufficient headspace was left over the solution to initiate oxidation. The glass vials were stored in the dark at room temperature. A very. small portion of the sample was withdrawn periodically and the carotenoid content was determined by extraction and subsequent quantitation. (b) Effects of radiation, t-carotene was incorporated into three systems at a concentration of approximately 200 #g per 100 ml. The systems were: (1) t-carotene-protein complex in water; (2) a colloidal dispersion of t-carotene in water prepared by adsorbing t-carotene on sucrose and suspending the mixture in water; (3) a solution of t-carotene in petroleum ether. These were packed in glass vials and an equal amount of headspace was left over each system. They were irradiated with ?-radiation using Co 6° as the source. The dose rate was approximately 2500 rads per minute. After the appropriate dose was delivered, each system was analysed for the pigment content by solvent extraction and quantitation on the basis of absorbance at 450nm. The protein complex system was also analysed for the extent of release of fl-carotene from the complex. Pigment extracted in petroleum ether with no addition of a polar solvent was taken as the index of free carotenoid not attached to protein. RI~IJLTS AND DISCUSKION
Composition of the carotenoid-protein complex The pigment-protein complex was analysed for the presence of various components (Table 1). In addition to protein and carotenoid, it contained lipids and sugars, as well as phospholipids in relatively small amounts. Reducing and amino sugars were also detected, but could not be quantitated since they constituted such a small percentage of the protein complex. O f the total carotenoids in the complex, 98°o was composed of t-carotene. The other 2% could
3
Table 1. Compositionof the carotenoid-protoincomplex Component
Percentage
Nitrogen Total lipids Phospholipids Reducing and amino sugars
12.30 2.15 0.14 Detectable
not be identified samples.
due
to lack of sufficient
Molecular weight of the protein An approximate estimation of the molecular weight of the t-carotene-protein complex was done by measuring the migration distance under standard conditions and deducing the value from a standard curve. The migration distance for the pigment was 93 mm and the molecular weight was estimated to be 88,000. Under these experimental condition, the carotenoid proteins did not separate into more than one fraction.
Stability of t-carotene in the complex (a) Autoxidation. Data on the susceptibility of carotene to oxidation when the pigment-protein complex was dissolved in water are shown in Table 2. In 3 days, the loss of pigment was
Table 2. Stability of [3-carotene in the protein complex to oxygen Time (hr) 12 24 48 72
Loss of pigment* (%) 3.2 6.5 12.3 18.1
*Average of five observations.
found to be 18.1% Under similar conditions, t carotene in hexane underwent more than 60%b degradation, while the loss of pigment in colloidal dispersions of t-carotene in water was only 4.5%.
T. V. RAMAKRISHNAN and F. J. FRANCIS (b) Effects of radiation. On radiation treatment, some of the [J-carotene seemed to be released from the pigment-protein complex. In Table 3, data are presented on the extent of release of pigment for three different radiation doses. With an exposure to 0.75 Mrad radiation, almost ¼ of the pigment from the complex was released.
physical or chemical barrier. (2t) In the colloidal system, however, the free radicals formed from water are not readily accessible to the pigment molecules. Most of the damage to [J-carotene in the colloid is likely to be due to the direct effects of radiation. (2°) The solution of pigmentprotein complex has properties characteristic of both the other two systems. The protein mo-
Table 3. Release of the pigment from the protein complex on radiation treatment Radiation dose (Mrads)
Pigment release (% total)*
0.25 0.50 0.75
6 13 23
i
i
*Average of five observations. (c) Radiation sensitivity of [J-carotene in different physical states. In Table 4, data are presented on the extent of radiation damage to [J-carotene in three different systems. The pigment was extremely radiolabile when dissolved in petroleum ether, but relatively stable to radiation in aqueous collodial dispersions. In the protein complex, [J-carotene was found to have an intermediate radiation sensitivity. In petroleum ether, free radicals formed on irradiation of the solvent molecules can travel to the site of the pigment molecules without any
lecules help to solubilize the pigment, but a true solution cannot be achieved, due to the fat soluble nature of [j-carotene. The radiation damage to the pigment in the protein complex may probably be due to the orientation of the /3-carotene molecules in the system and accessibility of free radicals for attack. Further, the lipid prosthetic group of the protein complex may also promote the radiation sensitivity of/3carotene by ~olubilizing it and providing free radicals of lipid origin in the immediate vicinity of the pigment molecules.
Table 4. Effects of radiation on E-carotene in different physical states Radiation dose (Mrads) Nature of the system
0.25
0.50
0.75
Loss of pigment (%)* Protein complex of /J-carotene in water Colloidal dispersions of /~-carotene in water Solution of E-carotene in petroleum ether *Average of five observations.
9.6
19.8
30.6
2.0
4.2
6.5
56.0
82.0
97.0
C A R O T E N O I D - P R O T E I N COMPLEXES Combination of t-carotene and the protein T h e ratio of t-carotene to protein, on the basis of weight, was estimated to be 1: 380. The molecular weight of protein was estimated to be approximately 88,000. t-carotene corresponds to a molecular weight of 536. From these data, the relative distribution of the carotenoid and the protein, on a molar basis, was deduced to be 1:2. This ratio is only approximate and cannot be taken to represent the stoichiometry until the direct link between the protein and t carotene is clearly established. T h e carotenoid-protein complex isolated in this investigation could be easily extracted by acetone or methanol, T h e readiness with which the carotenoid was removed from the complex by water miscible organic solvents eliminates the possibility of any covalent linkage between the pigment and the protein, tv's'ls~ This also eliminates the possibility of glycosidic linkage between the carotenoid and sugar molecules. T h e absorption maxima of t-carotene in petroleum ether were at 480, 452 and 425 nm. The t - c a r o t e n e - p r o t e i n complex in water had absorption m a x i m a at 495 and 460 nm. There were bathochromic shifts of 8 and 15nm in the positions of the principal absorption maxima. This small red shift in the maxima of t carotene in the complex argues against the possibility of carotenoid-protein interaction which is characterized by a strong polarization of the chromophoric group. (7'19) Further, flcarotene, the only carotenoid part of the complex, does not have any carbonyl group for interaction with the amino acid residues of the protein molecule. The link between fl-carotene and the protein, in the present case, is probably quite different from the one found in ovoverdin~14,22) and ~-crustacyanin. t13) It is possible that fl-carotene is associated with the lipid prosthetic group of the lipoprotein. T h e small bathe.chromic shift found in the spectra of the carotenoid in the protein complex is consistent with the red shift expected of lipid-bound carotenoids/1°~ T h e sensitivity of fl-carotene in the complex to oxygen and radiation (Tables 2 and 4) supports the lipid association of the pigment in the protein complex. Further evidence in support of this conclusion is the complete recovery of the pigment
from the complex with even 50°'o acetone. Direct attachment of t-carotene to the protein by weak hydrophobic bonds which do not involve any strong or partial polarization, is another possibility. T h e gradual release of the pigment from the complex on irradiation is also compatible with the possibility.
~ C E S
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