Analysis of Native Collagen Monomers and Oligomers by Size-Exclusion High-Performance Liquid Chromatography and Its Application

Abstract

Collagen extracted from tissues by pepsin digestion is a mixture of monomeric and oligomeric molecules. The oligomers are held together by covalent crosslinks between molecules. A simple size-exclusion high-performance liquid chromatography (HPLC) method for separation of collagen monomers and oligomers using a Bio-Gel TSK 60XL column has been developed for type I collagen from calfskin. Factors influencing the resolution include flow rate, the protein concentration of the sample, and the injection volume. To overcome electrostatic interactions between the protein and the column packing, addition of sodium chloride to the mobile phase was required to recover protein from the HPLC column when using 5 mM acetic acid as the mobile phase. Optimum recovery and oligomer content were obtained at 0.25 M NaCl. Component peaks eluting from the column were identified as monomers and oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the method showed that increasing the oligomer content of collagen preparations accelerated fibrillogenesis in vitro and decreased the ultimate fibril size produced. Also during fibrillogenesis, collagen oligomers were preferentially incorporated into fibrils, leaving only monomeric collagen in the soluble supernatant fraction. The assay was also shown to be useful for measurement of conversion of monomers to oligomers during accelerated aging of collagen fibrils at 30°C in vitro.