TYPE-II COLLAGEN QUANTIFICATION IN EXPERIMENTAL CHONDROGENESIS

Type II collagen is an excellent indicator of the cartilage phenotype. Accurate quantification with existing methods requires about 100 mu g of collagen. We have developed a method which allows for accurate quantification of type II collagen in samples as small as 1 mu g (and sample volumes as little as 1 mu l). Types I and II collagen were pepsin purified, cleaved with cyanogen bromide, and dissolved in sample buffer at concentrations of 0.125-200 mu g/mu l. Volumes of 1 mu l were analyzed by electrophoresis on microgels. The gels were scanned on a laser densitometer and the ratios of the alpha 1(II)CB10 to the alpha 1(I)CB7,8 plus alpha 1(II)CB11 determined. The cyanogen bromide-derived (CNBr) peptides could be resolved at concentrations as low as 0.25 mu g/mu l. The ideal working concentration for purified collagens was 1-8 mu g/mu l. Standard mixtures of both purified and non-purified types I and II collagen were analyzed. At a concentration of 1 mu g/mu l the ratio of the bands referred to above was closely related to the relative proportion of type II collagen, in a polynomial fashion. At 8 mu g/mu l there was an almost perfect linear relationship. The presence of 15-30% type III collagen had <5% effect on the measurements of type II collagen. The method is simple, reliable, fast and automated. It should have good potential for application in cartilage research as it permits quantitation of type II collagen in extremely small samples of tissue.