Heterogeneity of insulin. II. Chromatography of insulin on carboxymethyl cellulose in urea containing buffers.

Abstract

Chromatography of insulin, on carboxymethyl (CM) cellulose columns in urea-containing buffers, revealed the presence of at least five components, one major and four minor, in commercial insulin. This fractionation is related to other chromatographic fractionations of insulin and the nature of the minor components is discussed.Insulin which had been incubated in HCl was chromatographed. The behavior of the incubated products on the column showed that two or more amide groups were hydrolyzed simultaneously but at different rates. Because of the anomalous behavior of one of the components, it is postulated that a group other than the amides is also transformed during acid incubation. This component is present in commercial insulin samples.The electrophoretic separation of insulin in urea–starch gels was shown to be due to charge differences on the molecules rather than size differences. Correlation of the components separated on the CM-cellulose column with those separated in the urea–starch gel electrophoresis showed that much smaller charge differences on the molecules are distinguished on the CM-cellulose column than in the gel.