Separation of proinsulin conversion intermediates

Abstract

The processing of proinsulin (PI) to its final product, insulin, involves three different proteases (proprotein convertase 1 (PC1), PC2 and carboxypeptidase E) and a number of possible cleavage intermediates. The two major intermediates, des‐(31,32)‐PI and des‐(64,65)‐PI are of interest as potential biomarkers for predicting diabetes and as substrates for examining the specificity of prohormone processing. Unfortunately, these intermediates are not commercially available and they are extremely difficult to purify because they differ from the precursor by the absence of two basic amino acids, and from each other only in the identity of the two missing residues.We have combined trypsin/carboxypeptidase B digestion to generate the intermediates, with anion‐exchange chromatography to separate the reaction products. We were able to obtain three peaks of PI‐related material. Each of these peaks was characterized by performing reduction and alkylation of the peptides, followed by reversed‐phase high performance liquid chromatography. Peak 1 contains only the substrate, PI; peak 2 contains only the final products, insulin and C peptide; and peak 3 contains only the two cleavage intermediates, des‐(31,32)‐PI and des‐(64,65)‐PI. While we are continuing our efforts to separate the intermediates from each another, we are currently using the combined intermediates to examine the specificity of PC1 and PC2.