Oxidation of rat insulin II, but not I, leads to anomalous elution profiles upon HPLC analysis of insulin‐related peptides

Abstract

Rat insulin II, unlike rat insulin I and other non-rodent insulins, contains a unique methionine residue at position B29. Reversed-phase HPLC allows for separation of the two rat insulins, with insulin I typically eluting faster than insulin II. An anomalous peak of insulin immunoreactive material was found eluting even faster than insulin I following acid extraction of rat insulin-producing cells. This early peak co-eluted with [Met-O ^B29]insulin II suggesting that during cell extraction and subsequent purification steps, rat insulin II is subject to selective oxidation at Met^B29. Such oxidation of rat proinsulin II affords improved separation from rat proinsulin I compared to the native form.