Conformational effects in reversed-phase high-performance liquid chromatography of polypeptides II. The role of insulin A and B chains in the chromatographic behaviour of insulin

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The contribution of the insulin A- and B-chain to the retention and bandwidth behaviour of bovine insulin has been investigated. The influence of temperature and residence time on the logarithmic capacity factor (log k) versus the mole fraction of organic modifier ψ, i.e. the effect of temperature and ligand residency on the S and log k0 values of the individual peptide chains, were assessed at temperatures between 5 and 85°C and elution times between 30 to 90 min with an n-octadecyl (C18) and an n-butyl (C4) sorbent. Analysis of these log k versus ψ dependencies revealed that the insulin A-chain exhibits retention behaviour significantly different to the intact insulin molecule whilst the B-chain exhibits retention behaviour which is remarkably similar to the parent protein. However, in terms of kinetic processes, the A-chain exhibited a peak-splitting phenomenon at higher temperatures which was similar to the behaviour of the intact insulin molecule, whilst only bandbroadening with no peak splitting was apparent for the B-chain. Overall, the similarity of the retention behaviour of the insulin B-chain and the intact insulin molecule with regard to their temperature and residency dependencies suggests that the insulin B-chain makes a significant contribution to the chromatographic contact region of the insulin molecule when this polypeptide is exposed to hydrocarbonaceous ligands at low to intermediate temperatures due to the progressive unfolding of the molecule and greater accessibility of the previously buried B-chain residues. Under these same conditions, the A-chain contributes a relatively smaller proportion to the contact region but strongly influences the conformational stability of insulin at these lower temperatures. However, at higher temperatures the unfolding of the A-chain helices leads to significant bandspreading and eventually to the emergence of multiple peaks for the insulin molecule in the presence of both C18 and C4 ligands.

Original languageEnglish
Pages (from-to)71-79
Number of pages9
JournalJournal of Chromatography A
Issue number1
Publication statusPublished - 8 Sep 1995

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