Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis

Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70 °C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50 °C. Overnight digestion with thermolysin completely solubilized cartilage, whereas collagenase with >10-times higher proteolytic activity digested 98% extraction efficiencies of several GAG classes from thermolysin-treated cartilage, compared with