that these two peptide hormones are also structurally identical, a t least with respect to amino acid sequence. I n addition, it can be said that the carboxyl group of the C-terminal amino acid residue, valine, is blocked, probably by an amide structure, since very inild x i d hydrulysis leaves the valine susceptible to rernoval by carboxypeptidase digestion I>oth tu,,-lISK’’Ih a r i d abZIS€I” ” 1 1 , l ~ IW*II e \ ~ I O L V I I to l~ l ~ l o c h ~ t:Itl t h e
[ C O Y T R I n l T I O S FROM THE
h‘-terminus by an acetyl residue The failure to react with ninhydrin on the part of the S terminal amino group of a,-MSH indicates similar blocking a t this terminus of the equine hormone as well, possibly also by an wet$ group. Acknowledgment -1Ye wish to thank the S a tional Institutes of Health of the United States Public Health Service for , I grant ft;?O07) whicli iii:icle this w \ r k pi)sGlilt..
BIOCHEMISTRY S E C T I O S , A R M O U R AND CO?,IPASY, CHICAGO 9, JLLISOIS]
CESTRAI. R E S E A R C H I , A D O R A T U R I E S ,
The Fractionation of Insulin on Diethylaminoethylcellulose 317 MARGUERITE VOLINI AND
MILTON
MITZ
RECEIVED JANUARY 29, 1960 Insulin was separated into two components on the anion exchanger diethylaminoethylcellulose, using stepwise elution with carbon dioxide-Tvater solution and 0.1 J1 ammonium phosphate. T h e fractions were compared as to biological proper ties, crystallizability, solubility, amino-acid content, optical rotation and electrophoretic mobility. They were identical in qualitative amino-acid content but displayed significant differences in physical configuration as well as in net charge. Their apparent interrelationship is t h a t of native and partially denatured protein forms. It is postulated t h a t the two insulins are composed of polymeric forms each containing a different monomeric species.
Introduction Heterogeneity in purified insulin preparations has been demonstrated by means of several criteria.’-’ In his sedimentation studies, Fredericq’ has obtained evidence that the component differences lie in the elementary sub-units of the insulin polymer. The classical work of Sanger8 shows that these differences are not concerned with the nature or sequence of the constituent amino acids; the fractionation studies of Fredericq,‘ Harfenist and Craig2, and Timasheff, Brown and Kirkwood3 collectively suggest that the differences can be attributed to variations in a few chemical groups, resulting in slight differences in net molecular charge. Indeed, Harfenist and Craig2 have actually showed that their A and B components, isolated by countercurrent distribution, differ by only a single amide group per 6000 niolecularweight unit. The purpose of the present paper is to report the separation of purified insulin into two components apparently unlike those obtained in prior investigations. Fractionation was accomplished on the anion exchanger diethylaminoethylcellulose (DEAIEcellulose) ‘3 by using stepwise elution with carbon dioxide-water solution’” and 0.1 -11 ammoniuin phosphate. The relation of the two fractions to ( I ) E. Fredericq, “Ciba Foundatiun Collaquia on Dndocrinology: Internal Secretions of the Pancreas,” 9, .Iand A. Churchill 1,td.. I,rindon, l H > ( i , p . SS-lO‘r. ( 2 ) E. J. I-Iarfenist a n d 1.. C Craig, THISJ O I T R N A L , 7 4 , 3083 (1932); ibid.,7 5 , 5,528 (l9>:3) (:i) S. S. Timasheff, I
