Reversed-phase high-performance liquid chromatographic characterization of a novel proline-rich tryptophyllin

A tryptophan-containing tridecapeptide of amphibian origin (tryptophyllin-13, TPH-13), having the sequence < Glu-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH, was found to give peak splitting in isocratic and gradient reversed-phase high-performance liquid chromatography. The influence of many experimental conditions (column temperature, type of bonded phase, buffer pH, type of organic solvent, flow-rate, on-column incubation of the peptide, presence of a denaturant in the mobile phase, oxidation state of the peptide) on the chromatographic pattern of TPH-13 under selected gradient conditions, was studied. A pronounced conformational isomerism of the peptide chain, due to the presence of five proline residues, three of which are consecutively linked, is suggested to give rise to two major conformers of comparable stability and different hydrophobicity, detectable as a doublet by reversed-phase high-performance liquid chromatography.