Effects of high-performance liquid chromatographic solvents and hydrophobic matrices on the secondary and quarternary structure of a model protein : Reversed-phase and size-exclusion high-performance liquid chromatography

Abstract

A series of five synthetic peptide polymers of 8, 15, 22, 29 and 36 residues with the sequence Ac-(Lys-Leu-Glu-Gly) n -Lys-amide where n = 1–5 was used to examine protein denaturation during reversed-phase high-performance liquid chromatography (HPLC). Size-exclusion HPLC on a TSK G3000SW column was used to show that the 8-, 15- and 22-residue peptides are monomers in the starting solvent for reversed-phase chromatography, 0.1% aqueous trifluoroacetic acid (TFA) while the 29- and 36-residue peptides are dimers. These dimers have been shown to be extremely stable two-stranded α-helical coiled coils where the subunits are held together by hydrophobic interactions [S. Y. M. Lau, A. K. Taneja and R. S. Hodges, J. Biol. Chem., (1984) in press]. In contrast, all five peptides are monomers in acetonitrile—0.1% aq. TFA (1:1). These five peptides were separated by reversed-phase chromatography with an increasing gradient of 0.7% acetonitrile per minute on three matrices of varying carbon loading, pore size, and alkyl chain length (C 3, C 8 and C 18). In all cases a linear relationship between the natural logarithm of the monomeric molecular weight and retention volume was obtained for these peptides, indicating that the 29- and 36-residue dimers had been dissociated on binding to the reversed-phase columns. These results strongly suggest that the vast majority of proteins are denatured upon binding to the hydrophobic matrix. This series of peptides, 900–8000 molecular weight range, was also used for two additional purposes; firstly, to evaluate the reversed-phase columns and, secondly, to evaluate the relationship of ln molecular weight versus retention volume on the TSK G3000SW column.