Studies on the polydispersity and heterogeneity of proteokeratan sulfate from calf and porcine cornea.

After proteolysis of the calf and porcine corneae with papain 66 (calf) and 56 (hog) polysaccharide-containing fractions were obtained by chromatography on Dowex 1X2 and fractionating precipitation with ethanol (calf) and by chromatography on Dowex 1X2 and CPC-cellulose (hog). The sulfatation degrees (mol sulfate/mol hexosamine) and molecular weights (Mw) of 13 peptidokeratan sulfate fractions from calf cornea and of 9 such fractions from porcine cornea were 0.41-1.25 (Mw 3200-21 500), and 0.42-1.41 (Mw = 4900-25 600), respectively. It was found that the sulfatation degree increases more than proportionally with the chain-length. More than 90% of total keratan sulfate in both cases contain 2-3 mannose and 5-9 amino acid molecules per peptidokeratan sulfate molecule. About 30% of the corepeptide amino acids were asparagine or aspartic acid. More than 90% of the peptidokeratan sulfates from porcine cornea exhibits chain lengths of 31-47 disaccharide units. Two oligosaccharide peptides were isolated from porcine cornea, both being sulfate-free. Besides a core peptide of about 9 amino acids (2 Asx) one of them showed a sugar composition very similar to that of the binding-region from corneal keratan sulfate accord. to Keller, R., Stein, T., Stuhlsatz, H.W., Greiling, H., Ohst, E., Müller, E., & Scharf, H.-D. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, 327-336: 3 Man, 2 Gal, 4 GlcNAc. The calculated molecular weight based on 3 mannose residues (Mw = 2 600) agrees with that determined by gel chromatography. The second oligosaccharide peptide was homogeneous on electrophoresis and contained the constituents of the linkage region from chondroitin sulfate beside those from keratan sulfate. Based on 4 mannose residues the calculated molecular weight (4 300) is in agreement with that from gel chromatography. The sequence Asn-X-Asx starting with the binding-Asn is postulated in the core protein of proteokeratan sulfate from amino acid analyses of the corneal peptidokeratan sulfate fractions.