COMMUNICATIONS TO THE EDITOR
4494
this change was reported r e ~ e n t l y . ~Hydrolysis of D I T P (pH 3, GO") results in the initial formation of P-NH? and P-OH fragments which then condense to an extent of up t o 30y0 of the total phosphorus present. T M P m is converted almost quantitatively into D I T M P (pH 3 ) . n
/N\poo
OP -0
1 "P,
I
*
'0
/o\o
O!
O0P/o\poo
io
I
--,*.+
I
"\ /"
1
ZP/ O \
one H per mole of DITP6HlO) and a vague endpoint a t PH 10,s. 9detailed paper on this work is in preparation. THE PROCTER & GAMBLE COMPASY .1. NARATH & DEVELOPMENT DEPARTMENT F. H LOHMAN RESEARCH 0. T . QUIMBY hfIAMI VALLEYLABORATORIES 31, OHIO CIVCINNATI RECEIVED JULY 10, 1050
A b
/"
0%
Vol. 78
opo.
ISOLATION AND STRUCTURE OF MELANOCYTESTIMULATING HORMONE FROM PORCINE PITUITARY GLANDS'
> p i
Sir: O ' H O" -0 P-N-P-N-P 0 0
,011
o-
- - + '0
00 . "
\
0'
0'
P+P-N-P 0 0
n 0'
0' --*-O 0 " .
0'
0'
P-0-P-0-PO' 0 0
00
J.
Y n-
-0 P 00
The ring imidophosphates have been isolated from ThSPm hydrolysates in good yields by ion exchange techniques. Although the chain imidophosphates occur in the acid hydrolysates of TMPm to only a small extent, D I T P , ITP, and I D P have been prepared in high yields by selectively cleaving the corresponding ring imidophosphates a t the oxygen bridges in 3oyONaOH (73" or higher). At high NaOH concentrations P-N-P bridges appear to be inert, whereas they are cleaved more readily than P-0-P bridges in the pH range 2-1 1. Thus, P-N-P linkages differ markedly from P-0-P in dependence of hydrolysis rate on PH; more quantitative data will be reported soon. The various compounds involved in this study were characterized by elemental analyses, acidbase titrations, paper chromatographic and ionexchange separations, and X-ray diffraction (powder patterns). Products obtained by the methods of Stokes and de Ficquelmont had the elemental analyses of the monohydrate of DITLSP (some ITRSP.HrO), an acid-base titration curve of a ring polyphosphate (all H ' s strong), an X-ray pattern nearly identical to that of T?vSPm.HsO, and yielded the corresponding DITP.6Hz0, ITP.6H20 mixture in 30y0 NaOH ( 7 5 ' ) . The monohydrates of all three ring imidophosphates are isomorphous and form a continuous series of solid solutions. The hexahydrates of D I T P , ITP, and TP are similarly isomorphous. A typical analysis of DITP.6H20 gave 24.3yo Na, 19.8yo P, 5.92yo N (theoretical, 24.2GC/', Na, l9.(\% P, 5.91yo N ) , a titration curve with pronounced endpoints a t pH 5 and S.0 (spread (3) R. Klement and G. Biberacher. 24(i-25ii (195G).
Z. a n o r g . allgem. r h e i n . , 283,
We wish to report herein the isolation in pure form of the melanocyte-stimulating hormone (-\ISH, intermedin) from the posterior lobes of porcine pituitary glands. The structure of this hormone, a peptide consisting of 18 amino acids, will also be presented in this communication. -4 crude fraction of MSH was prepared from porcine posterior pituitary powder by glacial acetic acid extraction and fractional precipitation with acetone and ether, followed by adsorption on oxycellulose (l5Y0 of the weight of the ether precipitate).' Following elution with 0.1 N HCl, a highly potent3 concentrate was obtained. The eluate was de-acidified with methyldioctylaminel and brought to pH 6.3-7.0 with dilute ammonia. The precipitate that formed was discarded, and the supernatant fraction, after lyophilization, was submitted to zone electrophoresis on starch5 with a pyridine-acetic acid buffer of pH 4.9. A peak containing the bulk of the -\ISH activity could be eluted from a very narrow region; this peak was then submitted to countercurrent distribution for 1100 transfers a t 20", in the system 0,576 trichloroacetic acid DS. secBuOH. One main skewed peak was observed. The peak was divided in half and each half was recovered separately. IVhen both halves were re-run in the same system for 2-18 transfers, the distribution curve of each was virtually identical to the theoretical, and both possessed K xralues of 0.60. They were also shown to possess practically identical biological activities. Quantitative amino acid analysis of the 21- and 48-hour acid hydrolysates by the paper-fluorodinitrobenzene method6gave the following composition, based on molar ratios of the constituent amino acids: Aspi.o,Glu2.a,Serl.a,Glyi.9,Pro~.~ ,RZeta.r,Phei.i.Tyri.o,Lysi.s,Hisi.o,~g~.o, rry1.o.
Tyrosine and tryptophan were determined by a (1) This work is supported in part by the U. S. Public Health Serv-
ice (G-2907) and the Albert and Mary Lasker Foundation. Original manuscript received July 3, 195G. (2) (a) R . W. Payne, h l . S . Raben and E. B . Astwood, J. B i d . Chern., 187,719 (1950); ( b ) E B. Astwood, M.S. Raben, R . W . Payne and A , B . Crady, THISj o w R N A L , 73, 2989 (1951); (c) M. s. Raben, I . N Rosenberg and E . B. Astwood, Federation P r o c . , 11, 126 (1952). (3) Potency of preparations has been determined by t h e method described by K. Shizume, A . B . Lerner and T . B . Fitzpatrick (Endocrinology, 6 4 , 5 5 3 (1954)). Preparations purified by zone electrophoresis possess activities of 1 X 10'0 MSH u./g. T h e unit is t h a t described by Shizume, et 01. Thaugh a chemical purification occurs diiring t h e counter-current distribution procedure, s
