PURIFICATION AND ANALYSIS OF SYNTHETIC, TRIPLE-HELICAL MINICOLLAGENS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

To better study collagen-mediated cellular and enzymatic activities, a generally applicable solid-phase methodology has been developed by wh ich aligned triple-helical peptides (designated THPs or ''minicollagen s'') ranging from 79 to 124 residues can be assembled. Reversed-phase HPLC is typically the purification method of choice following chemical synthesis of small proteins of this size, as well as one of the analy tical techniques used to verify product purity. We have thus compared the effects of different stationary phases (C-18, C-4, Or diphenyl), o rganic modifiers (acetonitrile or isopropanol), support pore sizes (12 0 Angstrom 300 Angstrom or nonporous), and counterions for the reverse d-phase HPLC analysis of THPs. Large-pore C-18 or C-4 reversed-phase H PLC gave broad THP peaks, resulting in poor resolution of the desired THP from synthetic impurities. Broad peaks were presumably due to conf ormational instability of THPs to reversed-phase HPLC conditions and s ubsequent slow cis-trans isomerization of the peptide bonds. Peak shar pness was improved greatly by using large-pore diphenyl reversed-phase HPLC. We found that THPs can be best resolved from synthetic impuriti es by diphenyl or nonporous C-18 reversed-phase HPLC using water-aceto nitrile gradients. These results most likely reflect conditions which maintain the native conformation of collagen-like triple-helices. (C) 1995 Academic Press, Inc.