Reversed-phase high-performance liquid chromatography of some ultra-heterogeneous and covalently-modified proteins...

A new procedure for the fractionation of the heterogeneous cystine-rich proteins from human hair, utilizing reversed-phase high-performance liquid chromatography, is described. Of these proteins 27 fractions have been collected and analyzed for amino acid composition. There seems to be little correlation between the elution order and the hydrophobicity of the fraction constituents except for the late-eluting fractions. Based on the elution profiles and amino acid contents, these fractions appear to fall into four families. The effects of alkyl chain length, flow-rate and gradient slope, as well as various additives to the organic modifier on the separation have also been investigated. A low flow-rate (0.4 ml/min) and a shallow gradient were essential for the separation of these proteins as was the use of short alkyl chain (C4) or medium alkyl chain (C8) columns. However, with the C4 column reproducibility and recovery were excellent.