Collagen type II is prepared from bovine calf articular joint by modifications of published methods (22, 23, 24).
Chromatographically purified, salt-free, lyophilized acid soluble powder. Free of type I collagen. Contains less than 0.4% proteoglycan. Soluble in 0.01 M to 0.5 M HOAc.
In our method of purification, starting tissue is extracted with guanidine-HC1 to remove the bulk of proteoglycan. The residue is extracted with pepsinized acetic acid at pH 3.0 to solubilize collagens. Type II collagen is purified by fractional precipitations with NaCl, precipitations by dialysis against Na2HPO4 and column chromatography at pH 4.0 on CM-cellulose and at pH 7.5 on DEAE-cellulose to remove trace quantities of residual proteoglycans and types I and III collagens.
Purity is determined by SDS-PAGE electrophoresis in a reducing gel containing 5% 2-mercaptoethanol. Type I collagen is simultaneously ran as a control. The type II collagen is free of type I as indicated by the absence of alpha-2 chains. Uronic acid assay (25) is performed using glucuronolactone as standard.