A study of equilibrium binding of link protein to hyaluronate

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Abstract
Link protein was extracted from bovine femoral-head cartilage, radiolabeled while in the proteoglycan aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis and 2 species with MW .apprx. 45,000 and 48,000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an .**GRAPHIC**. of 4.75 S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high MW hyaluronate was studied by frontal-gel chromatography on Sepahrose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10.degree., 17.degree. and 25.degree. C. Dissociation constants of .apprx. (1-4) .times. 10-8 M were obtained. The length of hyaluronate occupied 1 link protein molecule was determined to be 6-7 disaccharides.