Nippon Suisan Gakkaishi 55(9), 1583-1589 (1989)

Astaxanthin and/or Canthaxanthin-actomyosin Complex in Salmon Muscle*1

Hikaru Henmi,*2 Masahiro Hata,*2 and Mitsuo Hata*3(Received March 17, 1989)

It was proposed that astaxanthin and/or canthaxanthin in salmon muscle binds with one ƒÀ-

ionone ring to a hydrophobic binding site which exists on the surface of actomyosin . The binding of carotenoids to actomyosin is not specific. The hydroxyl and keto groups of ƒÀ-end group in

tensify the binding strength of carotenoids to actomyosin .

In a previous paper,1) we reported that asta and zeaxanthin (physalis). xanthin in salmon muscle existed in the water insoluble fraction, associated with actomyosin pre Measurement of the Absorption Spectrum of the pared from myofibrils, and solublized easily Muscle by Opal Glass Method with Triton X-100, suggesting that astaxanthin The absorption spectrum of the muscle was binds to protein with a weak hydrophobic bond. measured using a Hitachi 150-20 spectrophoto Astaxanthin, canthaxanthin, zeaxanthin, cyn meter according to the method of Shibata.6) Thin thiaxanthin, ƒÀ-doradexanthin and diatoxanthin freshly sliced muscle was used. were identified as major carotenoids of salmonid

muscle.*4,2-5) Astaxanthin, canthaxanthin and Reduction of Astaxanthin and Canthaxanthin

other carotenoid(s) were extracted from the The muscle astaxanthin and canthaxanthin

actomyosin in cultured coho salmon muscle.1) were reduced by the method of Ako et al.7) The

These results suggest that salmonid actomyosin water-insoluble fraction of the muscle was sus

can combine with many carotenoids. pended in 2 volumes (v/v) of distilled water and In the present study, we investigated how carot solid NaBH4 was added to the suspension at

enoids combine with salmon actomyosin and the a concentration of 100mM, and incubated at nature of the astaxanthin and/or canthaxanthin 5•Ž in darkness. The suspension was taken at actomvosin complex. various time intervals and acetone was added to

stop the reaction. Authentic astaxanthin and

Materials and Methods canthaxanthin were dissolved in 95% ethanol and solid NaBH4 was added to the solution to a con

Materials centration of 100mM and incubated at 0•Ž in dark

Sockeye salmon Oncorhynchus nerka and cul ness. Carotenoids were extracted with acetone

tured coho salmon Oncorhynchus kisutch were petroleum ether (PE) or acetone-ethyl ether, and used. Coho salmon were cultured at Onagawa the reduction products were analyzed by thin layer

Bay and fed canthaxanthin as a pigment source. chromatography (TLC) and high performance

These specimens were kept at -80•Ž until use. liquid chromatography (HPLC). The following carotenoids were used: synthetic astaxanthin, echinenone, canthaxanthin (Hof Extraction of Carotenoids with Heptane fman-La Roche & Co.), ƒÀ-carotene (Wako Pure The method described by Goodman and Raz8 Chemical Industries LTD.), astaxanthin mono ) was used for the extraction of carotenoids with ester (shrimp), astaxanthin diester (Antarctic krill), heptane.

*1 Studies on the carotenoids in the muscle of salmon-II. *2 Department of Fisheries, Tohoku University, Sendai, Miyagi 981, Japan (逸 見 光,秦 正 弘:東 北

大 学 農 学部). *3 Department of Fisheries, Nihon University, Shimouma, Setagaya, Tokyo 154, Japan (秦 満 夫:日 本

大 学 農 獣 医 学 部), *4 W. Miki, T. Mori, K. Yamaguch, and S. Konosu: Abstract of Papers, the Autumn Meeting of the Japanese Society of Scientfic Fisheries(Sendai), 1984, p. 114.

1584 Henmi, Hata, and Hata

VIS detector (JASCO UVIDEC 100-VI) with the The water-insoluble fraction of the muscle was suspended in 20ml of distilled water and 20ml specially made columns (stainless steel, colum A; of n-heptane was added. The solution was LiChrosorb CN-5, Merck, 4.6•~250mm or col shaken at 5•Ž for 20h under nitrogen gas at a umn B; LiChrosorb SIL 100-5, Merck, 4.O •~ 250mm) were used. rate of 100 strokes/min in darkness. After shaking, Astaxanthin and its reduction products were the absorbance of the upper heptane phase was measured and carotenoid content was determined. analyzed on column A. The column was eluted with a solvent mixture of 29% n-hexane contain Combination of Actomyosin with Carotenoids ing 0.1%•@ N-ethyldiisopropylamine and 71% di Combination of actomyosin with carotenoid chloromethane containing 3% methanol at 2.0ml/ was carried out by the method originally de min. scribed by Ando and Yamamoto.9) Canthaxanthin and its reduction products were Carotenoid solution was prepared by the fol analyzed on column B. The column was eluted lowing method. Carotenoid (1.3mg) was dis with a solvent mixture of n-hexane: acetone: solved in chloroform. After addition of octa dichloromethane=90.08: 9.54: 0.38 at 2.0ml/min. ethyleneglycol mono n-dodecyl ether (50mg), The peaks were monitored by measuring the chloroform was removed under a stream of nitro absorption at 470nm and identified by comparing gen gas and one ml of distilled water was added, their retention times with those of the standard and then the solution was stirred. carotenoids.

The actomyosin (4.0g) was suspended in 5ml of 0.1M phosphate buffer (pH 6.0), and 0.2ml Results of the carotenoid solution was added. The solu tion was then shaken gently at 5•Ž for 12 h under The Absorption Spectrum of the Muscle nitrogen gas. After shaking, the actomyosin The maximum of the absorption spectrum of was washed with water to remove the uncombined the muscle of sockeye salmon was 486nm with a carotenoid. shoulder at 420nm. The carotenoid extracted from the muscle showed an absorption maximum Preparation of Actomyosin at 468nm in hexane and showed one band on Actomyosin in the muscle of coho salmon was TLC. This carotenoid was identified as asta xanthin. Muscle astaxanthin showed a weak prepared by the method of Arai.10) bathochromic 2max (from 468nm in hexane to 486nm in the muscle) (Fig. 1). Treatment of Actomyosin with Triton X-100 Purified actomyosin was treated with Triton X-100 by the method of Ando11) to remove lipids Extraction of Carotenoids with Heptane associated to actomyosin. In the muscle of sockeye salmon, the ratio of extracted astaxanthin to total astaxanthin was 3.6%. In the muscle of coho salmon, the ratio Analytical methods Carotenoids were identified by the previously reported methods.1) The carotenoid content was determined from the absorbance at absorption maximum, using E1%1cm=2200. Lipids were extracted by the method of Bligh and Dyer.12) Protein was determined by the method of Lowry et al.13) Egg albumin was used as a standard.

Thin Layer Chromatography (TLC) TLC was performed on silica gel plate (Wako gel B-5) using petroleum ether-acetone (7: 3) as solvent. Fig. 1. Absorption spectra of the muscle of sockeye salmon and astaxanthin. High Performance Liquid Chromatography (HPLC) -Muscle Nihon Bunko (JASCO BIP-1) pump and UV/ - - - Astaxanthin in hexane l. Astaxanthin, Canthaxanthin-actomyosin Complex 1585

Table 1. Combination of coho salmon actomyosin is similar to that of the naturally occurring asta with carotenoids xanthin-actomyosin complex.

Carotenoids Carotenoid contents (ƒÊg/mg protein) Combination of Triton X-100 Treated Actomyosin

with ketocarotenoids Astaxanthin 0.70-0.86 Astaxanthin monoester 0.20 After treatment with Triton X-100, the lipid Astaxanthin diester 0.00 content of the actomyosin changed from 0.35 to

Canthaxanthin 0.49 0.14mg/mg protein. The contents of astaxanthin

Echinenone 0.33 and canthaxanthin combined with Triton X-100 Zeaxanthin 0.47 treated actomyosin are given in Table 2. These ƒÀ-carotene 0.09 values were similar to those of untreated acto

myosin complexes. The absorption maximum of Table 2. Combination of the Triton X-100 treated the astaxanthin-actomyosin complex was 486nm, actomyosin with astaxanthin and canthaxanthin. showing a weak bathochromic ămex (18nm), similar

to that of the naturally occurring astaxanthin-acto Carotenoid Carotenoids myosin complex. The absorption maximum of contents (ƒÊg/mg protein) the canthaxanthin-actomyosin complex was 480 Astaxanthin 0.88 Canthaxanthin 0.41 nm, showing a weak bathochromic ƒÉmax (15nm), similar to that of the naturally occurring cantha

xanthin-actomyosin complex. canthaxanthin of extracted canthaxanthin to total These results indicate that lipids in actomyosin was 38%.

Combination of Actomyosin with Carotenoids

Actomyosin combined with free-form asta

xanthin, astaxanthin monoester, canthaxanthin,

echinenone, zeaxanthin, and ƒÀ-carotene. The

colors of astaxanthin-, astaxanthin monoester-,

canthaxanthin-, zeaxanthin-, and ƒÀ-carotene-acto myosin complexes were red, red, orange, yellow,

and yellow, respectively. However actomyosin did not combine with astaxanthin diester (Table 1).

The absorption maximum of the astaxanthin

actomyosin complex was 477nm, showing a

weak bathochromic ămax (9nm). About 5.5% of

astaxanthin was extracted with n-heptane from

the astaxanthin-actomyosin complex. This value Fig. 2. Reduction of muscle astaxanthin.

Fig. 3. Reduction of astaxanthin-actomyosin complex dissolved in 0.6 M KC 1586 Henmi, Hata, and Hata have no effect on carotenoid-protein binding.

Reduction of Muscle Astaxanthin and/or Can thaxanthin

After addition of NaBH4, the color of the muscle turned from orange to yellow. The reduction products of the carotenoids were extracted with acetone and separated by TLC and three bands was observed. These carotenoids were identified as astaxanthin, idoxanthin and crustaxanthin, respectively, from the absorption spectra and the behavior on TLC (data not shown).

The changes of the existing ratio of these re duction products are shown in Fig. 2.

Astaxanthin-actomyosin complex prepared from Fig. 4. Reduction of authentic astaxanthin. sockeye salmon muscle was dissolved in 0.6M

KCl, and incubated at 5•Ž in the presence of Addition of NaBH4 caused a rapid decrease of

0.1M NaBH4. The reduction products of asta astaxanthin at first, followed by a slow decrease xanthin were extracted with acetone-ethyl ether in the ratio. Idoxanthin increased rapidly at and analyzed by HPLC (Fig. 3). first, followed by a slow decrease in the ratio.

Fig. 5. Reduction of muscle canthaxanthin.

Fig. 6. Reduction of canthaxanthin-actomyosin complex dissolved in 0.6M KCl . Astaxanthin, Canthaxanthin-actomyosin Complex 1587

Fig. 7. Reduction of authentic canthaxanthin. Fig. 9-a. Reduction of canthaxanthin combined with coho salmon actomyosin.

of astaxanthin combined with Fig. 8-a. Reduction Fig. 9-b. Reduction of canthaxanthin combined with coho salmon actomyosin. coho salmon actomyosin treated with Triton X -100.

These results indicate that muscle astaxanthin

is reduced to crustaxanthin via idoxanthin.

In ethanol, astaxanthin was reduced very rapidly

to crustaxanthin and a small amount of idoxanthin

was observed (Fig. 4). This result shows that the

greater part of astaxanthin is reduced to crusta xanthin directly. Astaxanthin suspended in water

containing a small amount of ethanol was reduced

very slowly. In this condition, astaxanthin was

scarcely reduced over 1 hr.

The changes of the ratios of reduction products

of canthaxanthin are shown in Figs. 5, 6. These

Fig. 8-b. Reduction of astaxanthin combined with results indicate that canthaxanthin is reduced to

coho salmon actomyosin treated with Triton X isozeaxanthin via 4•Œ-hydroxyechinenone.

-100. In ethanol, canthaxanthin was reduced very

rapidly to isozeaxanthin and a small amount of Crustaxanthin increased linearly and the existing 4•Œ-hydroxyechinenone was observed (Fig. 7). ratio reached 80%. 1588 Henmi, Hata, and Hata

Reduction of recombined Astaxanthin-, and Can thaxanthin-Actomyosin Complexes The changes of ratios of reduction products of astaxanthin and canthaxanthin are shown in Figs. 8, 9. The reduction processes of these recombined carotenoids were identical with those of the nat urally occurring ones. These results suggest that recombined asta xanthin or canthaxanthin binds to the actomyosin binding site by the same mechanism as those of naturally occurring ones.

Discussion

The absorption spectrum of astaxanthin in the com muscle showed a weak bathochromic ămax (about Fig. 10. A proposed astaxanthin-actomyosin

18nm) compared with that in hexane. A weak plex model.

bathochromic shift and easy release of astaxanthin

and canthaxanthin from actomyosin with acetone, bottom of the hydrophobic hole of the hydro

Triton X-100, and sodium dodecyl sulfate1) sug phobic binding site on actomyosin. Therefore, gest that these carotenoids locate on the surface the keto group in the ƒÀ-ionone ring bound to

of the protein with a weak hydrophobic bond. actomyosin may be protected from an attack of Resonance Raman spectra of the salmon muscles NaBH4.

showed that the electronic ground states of asta Carotenoids were extracted with n-heptane to

xanthin and canthaxanthin in the muscle are learn the strength of astaxanthin- and cantha

similar to those of authentic standard dissolved in xanthin-protein interaction. Astaxanthin and

organic solvents.* This result indicates that the canthaxanthin were extracted with n-heptane from

carotenoid-protein interaction in the salmon mus the muscle, 3.6%. and 38%, respectively. Asta cle is weak. The circular dichroism spectrum of xanthin has one hydroxyl group at C3 and one

the canthaxanthin-actomyosin complex showed carbonyl group at C4 in one ƒÀ-ionone ring. Can

cotton effect, even though canthaxanthin is optical thaxanthin has one carbonyl group at C4. These

ly inactive (data not shown). This cotton effect is hydroxyl and carbonyl groups from hydrogen

caused by the induced asymmetry canthaxanthin bonds with a side chain of protein. The strength

which is due to asymmetrical rearrangement of of combination is proportional to the number of

binding sites. Therefore, it may be possible that hydrogen bonds. Therefore astaxanthin, which

the spectral shift is caused partly by distortion of forms two hydrogen bonds per one ƒÀ-ionone ring, ƒÀ-ionone ring in addition to the electron polariza combines more strongly to actomyosin than other

•@tion in the polyene chain.14-16) It was recognized carotenoids do. that muscle astaxanthin was reduced to idoxanthin Astaxanthin diester did not bind to actomyosin

and crustaxanthin. The changes of the reduction but astaxanthin monoester did bind to actomyosin.

products of muscle astaxanthin showed that asta Astaxanthin monoester from shrimp and asta xanthin is reduced to crustaxanthin via ido xanthin diester from antarctic krill contain long

xanthin. In 95% ethanol, however, NaBH4 at chain fatty acids (C16-C22) and therefore it may

tacked the keto groups in both ƒÀ-ionone rings be considered that a long chain fatty acid residue

simultaneously in a very short time to form crusta prevents the combination of astaxanthin diester xanthin. These results suggest that a keto group with actomyosin by steric hindrance. The com

in one ƒÀ-ionone ring of astaxanthin bound to bination of astaxanthin monoester with actomyosin

actomyosin is difficult to reduce. Muscle cantha indicates that one of the ƒÀ-ionone rings of asta

xanthin and authentic canthaxanthin showed xanthin binds to actomyosin and the other es

similar reduction processes to astaxanthin. It is terified ƒÀ-ionone ring is free from actomyosin.

proposed that one ƒÀ-ionone ring anchors to the This result supports the conclusion derived from

* M. Hata, H. Henmi and M. Takeuch i: Abstract of Papers, Annual Meeting of the Japanese Society of Scientific Fisheries (Tokyo), 1988, p. 96. Astaxanthin, Canthaxanthin-actomyosin Complex 1589

the reduction experiments. A proposed asta 4) T. Kitahara: Comp. Biochem. Physiol., 76B, xanthin-actomyosin complex model is shown in 97-101 (1983).

Fig. 10. This model may be adapted to other 5) T. Kitahara: Comp. Biochem. Physiol., 78B, carotenoids. A small amount of ƒÀ-carotene com 859-862 (1984). 6) K. Shibata: "Bunko Sokutei Nyumon", Kyori bined with actomyosin, indicating that a keto tsu-Syuppan, Tokyo, Japan, 1976, pp. 66-67. group in ƒÀ-ionone ring is not essential for com 7) H. Ako, J. Foster, and Clarence A. Ryan: bination with actomyosin. Biochemistry, 13, 132-139 (1974). The actomyosin of salmon muscle can associate 8) De Witt S. Goodman and A. Raz: J. Lipid. with many kinds of carotenoids and lipids. This Res. 13, 338-347 (1972). characteristic resembles that of lipoprotein. The 9) R. Ando and T. Yamamoto: J. Biochem., 97, association of carotenoids to apoprotein in the 877-882 (1985). carotenoproteins from invertebrates is more 10) K. Arai: 'Preparation of proteins in fish muscle' specific than that of salmon actomyosin.15,17) in "Suisan Seibutsu Kagaku Shokuhingaku Jik kensho," Koseisha-Koseikaku, Tokyo, Japan, 1974, pp. 179-188. Ac㎞owledgements 11) S. Ando: Nippon Suisan Gakkaishi, 49, 927- The authors thank Hoffman-La Roche & Co., 932 (1983). Ltd. (Basel) and Nippon Roche Co., Ltd. (Tokyo) 12) E. G. Bligh and W. J. Dyer: Can. J. Biochem. Physiol., 37, 911-917 (1959). for kindly supplying astaxanthin, Miyagi-ken 13) O. H. Lowry, N. Rowebrough, S. Farr, and R. Freshwater Fish. Exp. Stnt. for kindly supplying Randall: J. Biol. Chem., 193, 265-275 (1951). cultured coho salmon, and Taiyo Gyogyo Co., 14) V. R. Salares, N. M. Young, H. J. Bernstein, and Ltd. for kindly supplying sockeye salmon. P. R. Carey: Biochim. Biophys. Acta., 576, 176- 191 (1979). Refbfences 15) G. Britton, G. M. Armitt, S. Y. M. Lau, A. K. Patel, and C. C. Shone: 'Carotenoproteins' in "Carotenoid Chemistry and Biochemistry" 1) H. Hemni, T. Iwata, M. Hata, and M. Hata: , Per Tohoku J. Agr. Res., 37, 101-111 (1987). gamon Press, Oxford, U. K., 1982, pp. 237-251. 2) M. Hata and M. Hata: Tohoku J. Agr. Res., 16) J. C. Merlin: J. Raman Spectrosc., 18, 519-523 26, 35-40 (1975). (1987). 3) T. Matsuno, M. Katsuyama, and S. Nagata: 17) P. F. Zagalsky: Pure and Apll. Chem., 47, 103- Nippon Suisan Gakkaishi, 46, 879-884 (1980). 120 (1976).

Nippon Suisan Gakkaishi : Formerly Bull. Japan. Soc. Sci. Fish.