Insulin, peptide sequence

Modem methods of peptide sequencing follow a strategy similar to that used to sequence insulin but are automated and can be carried out on a small scale A key feature is repetitive N terminal identification using the Edman degradation... 

Although a high degree of homology is evident between insulins from various species, the same is not true for proinsulins, as the C peptide sequence can vary considerably. This has therapeutic implications, as the presence of proinsulin in animal-derived insulin preparations can potentially elicit an immune response in humans. 

Tumell, W. G., and Finch, J. T. (1992). Binding of the dye Congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences./. Mol. Biol. 227, 1205-1223. 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

Godkin A, Friede T, Davenport M, Stevanovic S, Willis A, Jewell D, et al. Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2). Int Immunol 1997 9 905-911. 

The most valuable confirmation of this view to date is, without doubt, to be found in the known structures of homologous proteins and peptide hormones, that is compounds of identical biological function isolated from different species. As is well known, the primary structures of the homologous insulins, corticotropins, hypertensins, posterior pituitary hormones, and heme peptide sequences from cytochromes c are closely similar and differ only at certain definite sites in the peptide chains. These can, in particular, serve as a useful point of departure in a search for more general principles governing protein structure, and in the comparison of different proteins. 

It is clear that the estimation of the amount of a particular peptide in a partial hydrolyzate may in certain cases yield considerably more information concerning the structure of the protein than its mere identification. Unfortunately it is impossible to determine directly the total amount of a particular peptide sequence in a protein molecule. Only a minimal value can be obtained from the composition of a partial hydrolyzate. In the case of the A-terminal peptides of insulin it was possible to estimate certain sequences in the protein from the yields of the peptides by allowing for the rate of breakdown of the bonds involved (p. 52). Such an approach may clearly be useful in the future where the yield of a peptide can be determined. 

The techniques for synthesis of peptides have been developed to a stage of refinement such that in the mid-1960 s, several groups of workers were able to report total syntheses of insulin. The sequence of 51 amino acids present in insulin is given in Figure 11.1. Still larger polypeptides have subsequently been synthesized. 

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