Comparison of the amino acid sequences of human, porcine and bovine proinsulins reveals a high degree of variance within the respective connecting peptide segments. This is particularly reflected by species-specific guinea pig antisera which indicate that the proinsulin antigenic determinants are contained within the connecting peptides. In porcine proinsulin, the major antigenic determinant appears to be a hydrophobic region within the 41–54 amino acid sequence. Collectively, proinsulin and proinsulin-related intermediates may account for 3 to 6 per cent of the protein in commercial insulin preparations, which explains most of the heterogeneity observed by the sensitive polyacrylamide disc-gel electrophoresis technic. Theoretically, many of these intermediates may be formed from proinsulin by the combined actions of trypsin and carboxypeptidase B or by enzymes of similar specificity; this implies that such enzymes comprise the conversion system in the beta cells. On the basis of structure-activity studies with these unique insulin analogs, the connecting peptide probably interferes with insulininsulin antibody interactions, and a free NH2-terminal glycine on the A chain is needed for full biological activity.

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