Extraction, Isolation and Analysis of Chondroitin Sulfate Glycosaminoglycans (1)
Glycosaminoglycans (GAGs) are a straight chain anionic polysaccharide composed of repeating disaccharide units of hexosamine (glucosamine or galactosamine) and uronic acid (glucuronic acid or iduronic acid) or galactose (in keratan sulfate), such as include chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan (or hyaluronic acid), keratan sulfate, heparan sulfate and heparin.
Galactosaminoglycans can be prepared by proteolysis or alkaline treatment of proteoglycans. Guanidinium chloride (4 M) is commonly used to extract proteoglycans from tissues. Proteoglycans are then purified by using various techniques including anion exchange chromatography and gel filtration chromatography. Cartilage tissues with relatively low fat content may be used for GAG extraction without defatting. Bovine nasal and tracheal cartilage and shark cartilage are common sources of CS, while pig skin is the major source of CS/DS.
Proteolysis with papain or pronase yields a single GAG chain, to which a small peptide consisting of several amino acids is covalently attached. After proteolysis, trichloroacetic acid treatment is commonly used to precipitate residual proteins and peptides. The proteinase in the digest can be deactivated by heating in boiling water.
Incubation of bovine nasal cartilage in 0.1 M sodium acetate, pH 4.5 at 37C release approximately 80% of total CS uronic from the tissue. The GAGs liberated by proteolysis are recovered by one of the following methods: a) precipitation with ethanol in the presence of sodium or potassium acetate, For ethanol precipitation, 1-2% solution of GAG is suggested to be preferable to obtain high recovery of GAG; b) precipitation with quarternary ammonium compound (cetylpyridinium chloride, CPC or cetyltrimethylammonium bromide, CTAB); and c) adsorption on an anion exchanger [e.g. diethylaminoethyl (DEAE)-cellulose, Dowex, epichlorohydrin triethanolamine (Ecteola)-cellulose, Q-Sepharose etc.]
