May 20, 195s
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a maximum a t 2728 A. with inflections a t 2950 and 3080 A. The fluorescence and ultraviolet absorption were characteristic of hydroxyindoles. (7) Esso Standard Oil Company Fellow, 1955-57. The active material was rechromatographed and CHEMISTRY DEPARTMEST HASS B. JOXASSEN eluted in three successive solvent systems. The TULASE UNIVERSITY ROBERT I. STEARNS' biologic activity, characteristic fluorescence, and S E W ORLEANS, LOUISIAN.4 JOUKO KENTTXMAA E.S . NAVALORDXANCE blue color with Ehrlich reagent remained excluTESTSTATION DONALD \V. MOORE sively together as a spot on these chromatograms. CHISALAKE,CALIFORSIA A. GREESVILLE ~VHITTAKER The solvent systems were isopropyl alcohol, conRECEIVED MARCH 25, 1958 centrated ammonium hydroxide, water (16 : 1: 3 ) Rf= 0.S3; 1-butanol, acetic acid, water (4: 1:5 ) RE = 0.87; isopropyl alcohol, concentrated ammonium ISOLATION OF MELATONIN, T H E PINEAL GLAND FACTOR THAT LIGHTENS MELANOCYTES' E = 0.SB. hydroxide, water ( 1 O : l : l ) R Siu: In preventing darkening of frog skin by XSH, During the past forty years investigators have melatonin, the active pineal gland factor, was a t reported that injection of pineal gland extracts into least 100 times as active on a weight basis as adrentadpoles, frogs, toads and fish produces lightening aline or noradrenaline, 200 times as active as triof skin color.'-* Recently i t was found that such iodothyronine and 5,000 times as active as seroextracts, by causing aggregation of melanin gran- t0nin.O Melatonin had no adrenaline nor noradrenules within the melanocytes of isolated pieces of frog aline-like activity on rat uterus and no serotoninskin, reverse the darkening effect of the melanocyte like activity on clam heart. N o melatonin activity stimulating hormone (A1SH).5 We wish to report was detected in beef pituitary, hypothalamus, isolation from beef pineal glands of the active fac- thymus, thyroid, adrenal, ovary, testis or eye. tor that can lighten skin color and inhibit MSH. SECTION OF DERMATOLOGY AARONB.LERNER JAMES D. CASE O F MEDICISE I t is suggested that this substance be called rnela- DEPARTMENT SCHOOL TOSHIYATA TAKAHASHI YALEUNIVERSITY tonin. OF MEDICINE TEHH. LEE Fii'ty grams of powdered lyophilized beef pineal SEW iyATARC MOR1 HAVEN 11, COSNECTICUT glands6 was extracted with petroleum ether for two RECEIVED MARCH 2 8 , 1958 hours in a soxhlet extractor. The defatted powder was mixed with 900 ml. water in a JT-aring Blendor. T H E STRUCTURE OF BOVINE CORTICOTROPIN1~2 After centrifugation a t 16,000 X g for 30 minutes the supernatant was extracted with 900 ml. ethyl Sir: acetate. The ethyl acetate layer was concenThe isolation of bovine corticotropin, a 39 aminotrated in vacuo a t 50" and subjected to distribution acid polypeptide possessing ACTH activity, has in a 30-tube countercurrent apparatus with the been reported from this laboratory3; its amino acid solvent system ethyl acetate, heptane, water composition is identical with that found for ovine (1:1:2 v.1.r.). Tubes S-15 were combined. The a-corticotropin but different from that of the porwater layer was extracted twice with SO ml. portions cine hormone. I t was further demonstrated that of ethyl acetate. 411 the organic solvent extracts the porcine, ovine and bovine hormones possess were combined and evaporated to dryness in vacuo identical T\i- and C-terminal residues. We wish to a t 50". The residue was sublimed a t 80" in vacuo. report herein the complete amino acid sequence of The sublimate was transferred with ethanol to bovine corticotropin. It will be noted that there IVhatman KO. 1 filter paper and chromatographed is a difference in certain portion. of the amino acid by descending technique with solvent system ben- sequence among the hormones of all three species. zene, ethyl acetate, water (19: 1 : 20). A test strip By means of the paper-strip modification4 of on reaction with Ehrlich reagent (p-dimethylamino- the phenyl isothiocyanate method,j the E-terminal benzaldehyde) showed a blue spot a t R,0.38. The amino acid sequence Ser.Tyr.Ser.1let.Glu. , , . "as unreacted strip was cut into sections and eluted established for bovine corticotropin. The rate with ethanol. Bioassay was performed using iso- of release of amino acids from the carboxyl end of lated Rana pipiens skin darkened with caffeine. the peptide hormone by the carboxypeptidase proThe lightening effect of the test substance on the cedure6 indicated the sequence . , .Leu.Glu.Phe a t melanocytes was measurzd photometrically with the C-terminus. transmitted light. This revealed that 95yGof reChymotryptic digests of the hormone (subcoverable biologic activity was present a t the posi- stratelenzyme = 100/0.6 (w./w.), PH 9.0, 40", for tion of the blue spot. Spectrophotofluorometric 24 hours) were fractionated by zone electrophoresis analysis of the acti\;e eluate showed a single fluores- on paper for 7 hours a t 200 volts with a collidinecent peag a t 8380 A. which was excited maximally acetic acid buffer of pH 7 ; after elution of each a t 2930 A. Ultraviolet absorption analysis showed band, the peptide fragments were further purified by paper chromatography in either n-BuOHl (1) T h i s investigation was supported by grants from the American
berg for stimulating discussions, and to Esso Standard Oil Company for financial support.
Cancer Society and t h e United States Public Health Service. ( 2 ) C. P. McCord and F. P. Allen, J . E x f l . Zool.. 23, 207 (1917). (3) 0. Bors and W. C. Ralston, PYOC.Soc. E x p . B i d , 77, 807 (195 1). (1) J. 0 . Kitap and 31. D. Altschule, "The Pineal Gland," Harvard University Press, Cambridge, Sfass., In,? t , p . 5G. (.i) I'.Takahashi and A . H. I,erner, t o h e published. ( 0 ) We are grateful t o tlie A r m o u r Labi,ratc,rieq for supplying LIS with se\.rritl kilograms of beef piueal glands.
( I ) Paper XI\' of t h e corticotropins ( A C T H ) series; for Paper X I I I , see C. H. Li, R. D. Cole, D. Chung and J. Leonis, J. B i d . C h e m . , 227, 207 (1957). (2) This work is supported in part b y t h e U. S. Public Health Service (G-2907) and t h e Albert and X a r y Lasker Foundation. (3) C. H. Li and J. S. Dixon. Science, 124, 934 (1956). ( 4 ) H Fraenkel-Conrat, TrfIs J O U R N A L , 7 6 , 3iiOG ( 1 0 5 i ) . (.i) P. Bdman, A d a C h e m . S c n n d . , 4, 253 (1950). ( ( i ) J . 1. Harris a n d C . H. Li, J . Bioi. C h i i n , 213. I!)!, (19>.F5).
