COMMUNICATION J . Biochem. 89,1975-1976 (1981)

Methionine Sulfoxide in the Resilium Protein of Surf Clams

Yasuo KIKUCHI and Nobuo TAMIYA

Department of Chemistry, Faculty of Science, Tohoku University , Aobayama, Sendai, Miyagi 980

Received for publication, February 24, 1981

A high content of methionine sulfoxide was observed in the resiliums (internal hinge- ligaments) of surf clams. As no isolation procedure which might cause the oxidation of methionine to methionine sulfoxide was involved and the hydrolysis was carried out in vacuo, it is the first solid evidence for the presence of methionine sulfoxide as a constituent of natural protein.

The hinge-ligaments of bivalves open the shells hydrolysis (4), the analysis was carried out after against the action of the adductor muscles. The alkaline hydrolysis. Methionine sulfoxide, but no amino acid composition of ligament protein was or only a small amount of methionine, was detected determined by Hare (1), Kelly and Rice (2), and (Table I, columns B). Kawahara and Maita (3) for a mussel (1), scallops Methionine sulfoxide was isolated from the (2, 3), and a surf clam (3). The second mentioned alkaline hydrolysate of the resilium of S. (Ps.) authors named the ligament protein of scallops sachalinensis as follows; the methionine sulfoxide (Placopecten magellanicus and Aequipecten irradi containing part of the eluate from the amino acid ance) "abductin" (2). In the present paper, we analyzer was collected from the alkaline hydro report the presence of methionine sulfoxide in the lysate of 30mg resilium powder seven times. The protein of resiliums (internal hinge-ligaments) of eluate was passed through an Amberlite IR-120 surf clams. (Hi-form) column (2.4 cm x 15 cm) and methionine Surf clams, Spisula (Pseudocardium) sacha sulfoxide eluted with 3M NH4OH. After concen linensis (Sakhalin surf clam) and Mactra chinensis, trating the eluate to 0.1ml, methionine sulfoxide were collected from the north-east coast of the was precipitated with acetone (1 ml). The yield main island of Japan and Spisula solidissima was 9.9 mg. The IR-spectrum of the methionine (Atlantic surf clam) at Woods Hole, Mass., U.S.A. sulfoxide preparation agreed with that of authentic The resiliums removed from the shells were dried DL-methionine sulfoxide (Wako Pure Chem. Ind. and crushed into powder. The amino acid com- Co., Ltd., Osaka, Japan). position of the resiliums after acid hydrolysis is Table I shows that 70-90 mol per cent of shown in Table I (columns A). About 35 per cent methionine in the resilium protein of surf clams is of the dry weight of the resiliums was ascribed to present in the sulfoxide form. The content of the amino acids released on acid hydrolysis. One methionine sulfoxide (12-15 mol per cent) is ex of the components eluted earlier than aspartic acid tremely high compared with the methionine con in amino acid analysis coincided with methionine tents of usual proteins. sulfoxide in its retention time. As menthionine The presence of methionine sulfoxide in the sulfoxide is reduced to methionine during acid glomerular basement membrane (5), in the lens

Vol. 89,. No. 6, 1981 1975 1976 COMMUNICATION

TABLE I. Amino acid composition of resiliums of surf clams. Results are expressed in mol per cent. No cor rection was made for hydrolytic losses.

a After acid hydrolysis: The resilium powder (about 2mg) was hydrolyzed with 6M HCl (0.5ml) at 105•Ž for 24h in an evacuated, sealed glass tube. b After alkaline hydrolysis: The resilium powder (about 2 mg) was hy drolyzed with 2.5M NaOH (0.3ml) at 105•Ž for 15 h in a polypropylene tube placed in an evacuated, sealed glass tube. c Unidentified components. d Gave a single peak with the retention time of cysteic acid. e Gave two peaks with retention times close to that of cysteic acid.

capsule (5), and in some other proteins (6, 7) has liums of a surf clam (3) and scallops (2, 3). The been reported, although the contents are low and "oxidation product" of methionine described by there is a possibility that it was formed during the Hare (1) in the ligament of Mytilus carfornianus isolation process of the proteins (8). In the may correspond to methionine sulfoxide. present study, no isolation procedure was involved. The hinge-ligaments of bivalves are practically Fresh resiliums of S. (Ps.) sachalinensis and M. insoluble as reported for scallops (2). No amino chinensis gave the same results as the dried prepa acid or protein was extracted from the resiliums rations. The observation that the resilium protein of surf clams with 70%. (v/v) aqueous trifluoro resisted cyanogen bromide treatment also sug acetic acid, 6M guanidine hydrochloride, pH 6, gested the presence of methionine in its oxidized containing 0.1M EDTA and 10 % (v/v) ƒÀ-mercapto form: Dried resilium powder (168mg) of S. (Ps.) ethanol or 5%. (w/v) sodium dodecylsulfate con sachalinensis was treated with 1.2 g of cyanogen taining 5mm EDTA and 10 % (v/v) ƒÀ-mercapto bromide in 70 % (w/w) formic acid (10 ml) for 4 ethanol at 37•Ž in 48h. days at room temperature. Only 4%. (w/w) of the material was solubilized and could pass through The authors are grateful to Mr. H. Abe for amino acid a column of Sephadex G-25. The content of analysis and to Mr. Louis Herland and Mr. David S. homoserine in the solubilized material was less Roos for sending the dried resiliums of S. solidissima. than 1 mol per cent of amino acids. On the other hand, ligament proteins of other REFERENCES

bivalves usually contain both methionine sulfoxide 1. Hare, P.E. (1963) Science 139, 216-217 and methionine, although the contents are low. 2. Kelly, R.T. & Rice, R.V. (1967) Science 155, 208-210 The contents of methionine sulfoxide and methio 3. Kawahara, H. & Maita, Y. (1968) Hokkaido Daigaku nine in the ligament proteins are as follows; Patino Suisan Gakubu Kenkyu Iho (in Japanese) 19, 52-61; Chemical Abstracts 69, 84460k (1968) pecten (Mizuhopecten) yessoensis (Giant ezo scal 4. Morihara, K. (1964) Bull. Chem. Soc. Jpn. 37,1781- lop), 2.6 and 7.6; Chlamys (Azumapecten) farreri, 1784 2.4 and 8.5; Meretrix lusoria (Common orient 5. Hudson, B.G. & Spiro, R.G. (1972) J. Biol. Chem. clam), 0.5 and 8.4 and Ruditapes philippinarum 247,4299-4238 (Japanese littleneck), 0.2 and 1.9 mol per cent, 6. Brewer, B.H., Jr., Keutmann, H.T., Potts, J.T., Jr., respectively. Reisfeld, R.A., Schleuter, R., & Munson, P.L. (1968) J. Biol. Chem. 243, 5739-5747 The presence of methionine sulfoxide was not 7. Adelstein, R.S. & Kuehl, W.M. (1970) Biochemistry mentioned by Kelly and Rice (2) or by Kawahara 9,1355-1364 and Maita (3) in their reports on the amino acid 8. Doscher, M.S. & Hirs, C.H.W. (1967) Biochemistry compositions of the acid hydrolysates of the resi- 6,304-312

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