High-performance liquid chromatography of amino acids, peptides and proteins. XC. Investigations into the relationship between structure and reversed-phase high-performance liquid chromatography retention behaviour of peptides related to human growth hormone.
The gradient elution behaviour of eight synthetic peptides encompassing residues [6-13] of human growth hormone, i.e. Leu1-Ser-Arg-Leu-Phe-Asp-Asn-Ala8, has been investigated, by using an octadecylsilica, a butylsilica, and a polymeric fluorocarbon as stationary phases. Quantitative expressions, derived from the linear-solvent-strength theory and the general plate-height theory, were used to assess the influence of gradient time on the relative retention and bandwidths of these peptides. It was demonstrated that the chromatographic properties of the cyclised imide form involving Asp6 are consistent with the formation of a highly stabilised amphipathic helix, while the open-chain alpha- and beta-rearranged forms eluted as less rigid structures. The putative hydrophobic contact region consists of two leucine residues and one phenylalanine residue. From an analysis of the retention and bandwidth data obtained at pH 9, a surface-induced molecular reorientation of the beta-linked peptides was observed, in which the repulsion of the aspartyl carboxyl group from the hydrophobic stationary phase directs the C-terminal moiety away from the sorbent surface. Furthermore, the fluorocarbon sorbent exhibited characteristics favourable for use in preparative purification of these peptides. The present results demonstrate the sensitivity of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor small changes in the interactive behaviour of peptides with hydrocarbonaceous ligands and aquo-organic solvent combinations in reversed-phase systems. These observations further illustrate the general utility of HPLC for investigating the conformational behaviour of peptides at solid-liquid interfaces.
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
A W
AW
Purcell
M I
MI
Aguilar
M T
MT
Hearn
D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D013006:Growth Hormone / Q000032:analysis*; D006801:Humans; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D010455:Peptides; D011506:Proteins; D013006:Growth Hormone