Evidence for a Covalent Linkage between Heteropolysaccharide and an Hydroxyproline-containing Peptide from Metridium dianthus Connective Tissue

Pepsin-solubilized collagen from the sea anemone Metridium dianthus was prepared and purified by the procedure of Rubin et al. (Biochemistry, 4, 181 (1965)). It contained 8% carbohydrate consisting of 0.3% fucose, 0.02% xylose, 0.35% mannose, 3.2% galactose, 3.8% glucose, 0.1% galactosamine, and 0.4% glucosamine. The collagen (44 g) was digested with Clostridium histolyticum collagenase and fractionated by gel filtration on P-100 and P-30 resins, by ion exchange chromatography on DEAE-cellulose, and by preparative high voltage paper electrophoresis. A homogeneous peptide was obtained (yield: 0.13% by weight, 3.8% by mannose content). The peptide contained 30% of carbohydrate consisting primarily of fucose, mannose, and glucosamine, and 47 amino acid residues, 4 of which were hydroxyproline. A partial structure was determined by Edman degradation, hydrazinolysis, and by controlled acid hydrolysis. [see PDF for sequence]