Isolation and characterization of dermatan sulphate proteoglycans from bovine sclera

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Abstract
Proteoglycans were extracted from sclera with 4 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulfate-chondroitin sulfate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. The scleral proteodermatan sulfates were fractionated into a larger (I) and a smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M sodium acetate than in 4.0 M guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M sodium acetate) as was the starting material (in 4.0 M guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had .**GRAPHIC**. values of 2.8 .times. 10-13 and 2.2 .times. 10-13 s, respectively, in 6.0 M guanidine hydrochloride. The 2 proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulfate side chains of proteoglycan I were D-glucuronic acid-rich (80%); those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. The co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was larger than that of proteoglycan II. The latter had an apparent MW of 46,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis); the former was > 100,000. In addition, the amino-acid composition of the 2 core preparations was different. As proteoglycan I altered its elution position on gel chromatography in 4 M guanidine hydrochloride compared with 0.5 M sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favored, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.

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