Vol. 77
201 4
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COMMUNICATIONS T O T H E EDITOR _ _
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STUDIES ON POLYPEPTIDES. VI. SYNTHETIC CONFIRMATION OF N-TERMINAL, AMINO ACID SEQUENCE OF CORTICOTROPIN-A'
vealed identical behavior of both compounds. Their Rfvalue in the Partridge system was 0.51, and both peptides appeared between glutamic acid s 7 : and lysine in the latter system. Paper chromatogWhite and Landmann2 isolated from a 24-hour raphy of a 1 : 1 mixture of the peptides produced one peptic digest of hog corti~otropin-~4 a pentapeptide spot only. to which they assigned the structure of seryltyroThe behavior of the synthetic specimen on treatsylserylmethionylglutamic acid (I), The fact that ment with carboxypeptidase and aminopeptidasel0 seryltyrosine had been previously identified as the duplicated that observed with the natural mateAT-terminusof corticotropin-A,3 ~ 4characterized the riaL2 The four constituent amino acids were libpentapeptide (I) as the N-terminal sequence of this erated in the expected molar ratios by both enpituitary hormone. The same N-terminal amino zymes. The recovery of serine from an acid hyacid sequence is also present in sheep a-corticotropin6 drolyzate (18 hours a t 105" with 6 N HC1) was only and porcine @-corticotropin.6 Since the ultimate 90% of the theoretical, reflecting the well-!;nown proof of the structure of the pentapeptide (I) de- lability of this compound to acid. Excellent repended on a comparison of the natural material coveries of tyrosine, methionine, and glutamic acid with a synthetic specimen of established chemical were realized. structure, we have undertaken the synthesis of (I) Thesc results establish the structure and optical by methods known not to cause racemization. purity of the synthetic peptide, and substantiate Carbobenzoxymethionylglutamic acid, diethyl structure (I) for the pentapeptide resulting from ester, was prepared from carbobenzoxymethionine the peptic digestion of corticotropin-.k. A detailed and diethyl glutamate, by the mixed anhydride description of our experiments will be presented a t procedure, and this material was converted into a later date. methionylglutarnic acid (111, [ c ~ ] ~ ' D 16.6" (in D. H . Sgackman, E. L Smithand D. M. Brown, J. Bioi. C h o n . , i v HCl). Anal Calcd. for C10I3&~2S: c, 212,(10) 255 (19.55). 43.2; H,6.5; N, 10.1. Found: C,d3.5; H, 6.7; K , (11) We wish t o express our thanks t o Dr. W. F. White of t h e 10.0, by saponification and decarbobenzoxylation. Armour Laboratories for comparing these properties of the natural and The dipeptide (11) was then treated with carbo- synthetic peptides. benzoxyserine azide' to give carbobenzoxyseryl- BIOCHEMISTRY DEPARTMENT KLAUSHOFMANN methionylglutarnic acid. Serylmethionylglutamic UNIVERSITYOF PITTSBURGH ALBERTJOIIL acid (111),from ethanol, [a]*'D -26.1' (in H20). SCHOOL O F MEDICINE PENNSYLVAXIA Anal. Calcd. for C13H2307N$3,C ~ H S O H C, : 43.8; PITTSBURGH, RECEIVED APRIL 9, 1955 H, 7.1; N, 10.2. Found: C, 44.5; H, 6 7 ; N, 10.8, was prepared from the acylated tripeptide by reduction with sodium in liquid ammonia. The coupling of (111)with the azide of carbobenzoxyserOXYGEN TRANSFER AND ELECTRON TRANSPORT BY THE PHENOLASE COMPLEX' yltyrosine afforded carbobenzoxyseryltyrosylserylmethionylglutamic acid which was again decarbo- Sir : benzoxylated with sodium in liquid ammonia, to The metabolic role of the phenolase complex? is form (I). The crude pentapeptide was repeatedly controversial, particularly in respect to a n hypothprecipitated from water by ethanol, and was finally esis suggesting that this enzyme system catalyses obtained as its crystalline monohydrate by slowly terminal transfer of electrons to ~ x y g e n . ~ I n order cooling a concentrated aqueous solution : [ a I z 6 D to throw some light on the problem and to under-20.6" (in 2 N HC1). Anal. Calcd. for C25H37- stand the molecular events occurring a t the cataOIINsS,H 2 0 : C, 47.4: H, 6.2; N, 11.1. Found: lytic configuration of the enzyme complex, we have C, 47.0; H, 6.4; N, 11.6. The peptide gave a posi- studied its hydroxylative phase using Ol8? and tive ninhydrin reaction and produced a dark pur- Hz018as tracers. ple color with diazotized sulfanilic acid in sodium We find that all oxygen enzymically introduced carbon'ate solution. as hydroxyl into the benzene ring of the substrate The paper chromatographic comparison of the comes from molecular oxygen (Table I). None synthetic product with the natural material in the comes from solvent water, Hydroxylation mechPartridgeR and 2-butanol-ammonia s y ~ t e m s ,re~ anisms inconsistent with this observation4 must accordingly be incorrect. The phenolase complex is (1) The authors wish t o express their appreciation t o Armour and Company for their generous support of this investigation an oxygen transferase. (2) W. F. White and W . A. Landmann, THISJ O U R N A L , 7 7 , 771
+
(195.5). (3) \V.A . Landmann, M , P. Drake and W. F. White, ibid., 7 6 , 43iO
(1953). (4) W. F. White and W, A. Landmann, i b i d . , 76, 4193 (1954). ( 5 ) J. Harris and C. H. Li, ibid., 7 6 , 3607 (1954).
(6) (7) (8) (9)
P. H. Bell, ibid., 7 6 , 5565 (1954). J. S. Fruton, J . Biol. Chcm., 146,463 (1942). S. M . Partridge, Biochem. J . , 42,238 (1948). J. Roland and A. Gross, A n d . Chem., 26, 502 (1954).
(1) This study has been supported by a grant from the United States Public Health Service (C-2291). (2) We mean by "phenolase complex" t h a t pair of enzymic activities occurring together, associated with copper-protein, and responsible for phenol o-hydroxylation and for dehydrogenation of o-diphenols t o oquinones. (3) W. 0. James, "Plant Respiration," Oxford Press, New York,
N.Y.,1953. (4) D. Kertesz, Biochim. cl Biophys. Acto, 9, 170 (1952).
