The degradation of [125I]iodoinsulin (A14) by insulin protease (EC 126.96.36.199) was studied using HPLC. A reverse phase HPLC method is presented which allows the separation and quantitation of insulin degradation products. After incubation of [125I]iodoinsulin (A14) with insulin protease, there was an initial rapid loss of radioactivity from the [125I] iodoinsulin (A14) peak, which was quantitatively accounted for by the appearance of radioactivity in 11 different peaks, but was not accompanied by a proportional increase in the solubility of the sample in trichloroacetic acid. Two of the peaks showed appreciable accumulation before the others, and all but the firsteluted peak plateaued by 20 min. After 20 min of incubation, the amount of radioactivity present as the first-eluted peak, solubility in trichloroacetic acid, and insulin loss continued to increase at a steady, but slowed, rate. The order of appearance suggests that insulin protease acts on insulin in an ordered sequence of steps to generate a number of intermediates that are precipitable by trichloroacetic acid, but are subsequently degraded to material that is soluble in trichloroacetic acid. Sulfitolysis of 5 major peaks and subsequent HPLC analysis of the fragments showed none of the peaks to possess intact A chains. Peptide sequencing of 2 of the peaks indicates that the A-chain is cleaved in at least 2 positions, one beyond the 14th position, and one between the 13th and 14th amino acids (leucine and tyrosine).
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