252 SNORT COMMUNICATIONS VOL. 36 (Z959)

peak. As digestion progressed, an active peak had begun to emerge just ahead of the original main component, and, by the end of 59 h, had completely replaced it. Fig. i f was obtained with a highly active fraction which was precipitated from the 59-h digest by the addition of (NH,)2SO 4 to o.3 saturation followed by dialysis and lyophilization. Subsequent studies reported separately have shown that the protein corresponding to the active peak in Fig. If is homogeneous in the ultracentrifuge, contains a single N-terminal phenylalanine residue, and has a mol. wt. of about 6ooo.

This work was supported by grants from the National Institutes of Health (Grant No. RG 4614) and the National Science Foundation (Grant No. G 5830).

Department o~ Agricultural Biochemistry, University o/ Minnesota, IRVlN E. LIENER St. Paul, Minn. (U.S.A.) T. VISWANATHA*

1 T. VISWANATHA, R. C. WONG AND I. E. LIENER, Biochim. Biophys. Acta, 29 (1958) 174. 2 H. FRAENKEL-CONRAT, R. S. BEAN AND H. LINEWEAVER, J. Biol. Chem., 177 (1949) 385 • 3 G. W. SCItWERT AND Y. TAKENAKA, Biochim. Biophys. Acta, 116 (1955) 57 o. 4 W. TROLL AND R. K. CANNAN, J. Biol. Chem., 20o (1953) 803. 5 H. FRAENKEL-CONRAT,. J. I. HARRIS AND A. L. LEVY in: D. GLICI~, Methods o/ Biochemical

Analysis, Vol. II , Interscience Publishers, Inc., N.Y., 1955. s F. LANNI, M. L. DILLON AND J. W. ]3CARD, Proc. Soc. Exptl. Biol. Med., 74 (195 o) 4. 7 N. M. GREEN AND H. NEURATH, in: H. NEURATH AND I~. BAILEY, The Proteins, Vol. I I , pt. ]3,

Academic Press, Inc., N.Y., 1954. s E. A. PETERSON AND H. A. SOBER, J. Am. Chem. Soc., 78 (1956) 751.

Received March 6th, 1959

* Present address: National Ins t i tu te of Arthri t is and Metabolic Diseases, National Ins t i tu tes of Health, ]3ethesda, Md. (U.S.A.).

Studies on polypeptides

XI I I . The synthesis of a derivative of a-MSH possessing a high degree of melanocyte-expanding activity in vitro

We have completed recently z a synthesis of the entire amino acid sequence of the pituitary hormone a-MSH 2 in the form of the blocked tridecapeptide N-benzyloxy- carbonylseryltyrosylserylmethionylglutaminylhistidylphenylalanylarginylt ryptophyl- glycyl-e-tosyllysylprolylvaline amide (12 L), and have found that this substance possesses melanocyte-stimulating activity in vitro (0.8. lO s MSH units/g).

We now find that N-acetylseryltyrosylserylmethionylglutaminylhistidylphenyl- alanylarginyltryptophylglycyl-e-tosyllysylprolylvaline amide (12 L) (I) exhibits 25 times the melanocyte-expanding activity of the benzyloxycarbonyl derivative, i.e., 2.0" 109 MSH units/g. It is indeed notable that a derivative of a-MSH in which the charges of two amino acid residues (glutamic acid and lysine) are altered markedly, in the latter instance by the introduction of the bulky tosyl group, should possess such a high order of biological activity.

The scheme which we employed for the preparation of the acetyltridecapeptide amide paralleled that used in the synthesis of the benzyloxycarbonyl derivative.

Abbreviat ion: MSH, melanocyte-s t imulat ing hormone.

VOL. 3 6 (1959) SHORT COMMUNICATIONS 253

N-acetylseryltyrosylserylmethionylglutamine (5 L) was prepared from seryltyrosyl- serylmethionylglutamine 1 and acetic anhydride in water containing NaHC0a. Well formed needles from water; m.p. 214-216 °; [a]~-2o.3 ° in dimethylformamide; log. e at 275 m~, 3.13o; single spot (Partridge system) Re, o.6o; ninhydrin-negative; Pauly- and methione-positive. (Found: C, 49.2; H, 6.7; N, 12.6; N-acetyl, 6. 7. C2~H4oOnNeS requires C, 49.4; H, 6.1; N, 12.8; N-acetyl, 6.6.) The N-acetyl peutapeptide was cleaved by carboxypeptidase into an equimolar mixture of serine, tyrosine and methionine (glutamine present on chromatograms could not be determined by the ninhydrin technique because of pyrrolidonecarboxylic acid formation) and formed acetylseryltyrosine and serylmethionylghitamine (RF, o.71 and 0.39, respectively) when digested by chymotrypsin; the recovery of tripeptide was 93 % of theory. The azide of the N-acetylpentapeptide was coupled with histidylphenylalanylarginyl- tryptophylglycyl-e-tosyUysylprolylvaline amide (7 L) 1 to give crude (I) (activity, 5.4" 1°8 MSH units/g). Counter-current distribution in the system IO % acetic acid- n-butanol gave material containing 8.8. lO 8 MSH units/g which was subjected to electrophoresis on powdered cellulose in a pyridinium acetate buffer, pH 5.I. The most active fraction from electrophoretic purification (1.5.1o 9 MSH units/g) was again redistributed in the acetic acid-butanol system to give a final product which assayed 2.0. lO 9 MSH units/g. Of the total biological activity present in the crude material 80 % was recovered in the final product, demonstrating a high degree of stability of the peptide to the conditions employed in its purification. The melanocyte-expanding activity of the final product and of the lower potency materials was not increased by incubation for 18 h at 37 ° with a mixture of IO % acetic and thioglycolic acids (IO : I, v/v). A sample of the final product which was homogeneous on paper (Re, 0.78 ) was subjected to acid hydrolysis (24 h at IiO ° with 6 N HC1) and the hydrolyzate analyzed for its amino acid composition by quantitative paper chromatography. The molar ratios were: Serl.6TyrlMet0.sGlulHislPhelArglGlyl-s-tosLysl.lValo. 9, (tryptophan destroyed, proline present but not determined). Partial destruction of serine and methionine by acid hydrolysis has also been observed 8 with corticotropin A.

• Since the final product was formed by an azide coupling step from two fragments of established stereochemical homogeneity 1 it seems reasonable to assume that it is of the 12 L variety.

We wish to express our sincere appreciation to Drs. M. R. WRIGHT and A. B. LERNER of the Department of Medicine, Yale University School of Medicine for the biological assays which were done by the method of SHIZUME et al. 4.

This work was supported by grants from the U.S. Public Health Service, the National Science Foundation, the American Cancer Society, Armour and Co. and Eli Lilly and Co.

KLAUS HOFMANN

Biochemistry Department, School o/Medicine, HARUAKI YAJIMA University o/Pittsburgh, Pittsburgh, Pa. (U.S.A.) ELEANORE T. SCHWARTZ

1 K. HOFMANN, M. E. WOOLNER, S . YAJIMA, G. SPUHLER, T. A. THOMPSON AND E. T. SCHWARTZ, J. Am. Chem. Soc., 8o (1958) 6458.

2 j . I. HARRIS AND h . B. LERNER, Science, 179 (1957) 1346. 3 E. E. HAYS AND W. F. WHITE, Recent Progress in Hormone Research, io (1954) 265. 4 K. SHIZUME, A. B. LERNER AND T. B. FITZPATRICK, Endocrinol., 54 (1954) 553.

Received January i9th, 1959