Isolation of proteoglycans from tendon.

Abstract

Proteoglycans make up less than 1% of the dry weight of a dense connective tissue such as tendon (1). Most of this proteoglycan is a small molecule called decorin. Because decorin has only one glycosaminoglycan chain, it cannot be separated from other proteins by the CsCl density-gradient centrifugation method that was originally used to purify aggrecan from cartilage. The basic approach described in this chapter is to extract proteoglycans from the tissue with 4 M guanidine, a solution that will denature collagen and disrupt most kinds of noncovalent molecular interactions. The cross-linked collagen of adult tendon remains insoluble during this extraction, allowing the proteoglycans and other soluble proteins to be separated from the bulk of the tissue by centrifugation. Proteoglycans are then separated from other extracted proteins by ion-exchange chromatography, taking advantage of the anionic nature of the glycosaminoglycan chain. This method has been used to quantitate and isolate proteoglycans from the tensile (proximal) and compressed (distal) regions of bovine deep flexor tendon. These mechanically distinct regions of flexor tendon are characterized by differences in proteoglycan amount and type (2). The method is equally applicable to isolation of proteoglycans from human tendon or from other dense connective tissues (3). Once isolated, the large proteoglycans can be separated from smaller ones by sieve chromatography. These isolated proteoglycans and their unique core proteins and glycosaminoglycan chains are of sufficient purity to then be examined by specific analytical techniques or used in functional assays.