The Preparation of the A and B Chains from Reduced and S-Carboxymethylated Beef Insulin

Abstract

The A and B chains of reduced and S-carboxymethylated insulin have been prepared in a pure state by two procedures. The chains could be separated readily in almost pure form by chromatography on DEAE-cellulose in buffers containing 8[image] urea, or by pH fractionation in the presence of 0. 5[image] potassium chloride. These fractions were then freed from contaminating traces of each other and other impurities by gel-filtration on Sephadex G-50 in buffers containing 8[image] urea. The S-carboxymethylated B chain could also be purified on Sephadex G-50 using 50% formic acid. The retention of these polypeptides by Visking 18/32 cellulose dialysis tubing greatly facilitated their isola-tion. The rate of carbamylation of the B chain of S-carboxymethylated insulin by the cyanate formed in urea solutions has been studied. In agreement with Cole (1961) and Stark (1965) carbamylation at about pH 7. 5 was found to involve only the [alpha]-amino group. At pH 10 it was found that there was negligible carbamylation of either the a-amino or the e -amino groups. There was no evidence of peptide bond breakage when the S-carboxymethylated B chain was allowed to stand at room temperature in 50% formic acid for 24 hr.