A Comprehensive Approach to the Study of Collagen Primary Structure Based on High‐Performance Liquid Chromatography

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Abstract
A comprehensive approach for the structural microanalysis of collagen based on high-performance liquid chromatography (h.p.1.c.) has been developed using calf skin type I collagen as a model system. The α, β and γ components were separated, after heat denaturation, on a TSK 4000 SW gel permeation column, using a nonvolatile buffer. Monitoring at 210 nm permits the detection of 1 μg of a single chain. The α1(I) and α2(I) chains were completely resolved using a large-pore reversed-phase column (Vydac 201 TP 4.6) eluted by an aqueous acetonitrile gradient (24-48 %) containing 0.01 M heptafluorabutyric acid as an ion-pairing agent. The purified α1(I) chain was digested with CNBr and the resulting fragments separated in the same chromatography system with a gradient containing a 12.8-44.8%, acetonitrile gradient. The purified α1(I)CB 3 peptide was further cleaved with trypsin and the resulting peptides separated first by a similar chromatography with a 4-32 % acetonitrile gradient. Resolution of some poorly separated peptides was obtained by a rechromatography using trifluoroacetic acid as counterion. The isolated peptides were hydrolyzed and identified by their amino-acid composition. Sequencing of h.p.l.c.-purified α1(I)CB 3 was also performed to demonstrate the suitability of the technique for the preparation of peptides for amino-acid sequencing. This study demonstrates that detailed structural analysis can be performed on 3 mg of a purified collagen.