Structural characteristics of heparan sulfates with varying sulfate contents.

Structural properties of heparan sulfate preparations from hog mucosa and beef lung sources were obtained by application of Smith degradation and nitrous acid reactions. Products formed by these reactions indicated that most of the iduronic acid present in these mucopolysaccharides is ester sulfated, whereas N-sulfated glucosamine residues are ester sulfated much less frequently. Repeating units with sulfated iduronic acid found to occur almost entirely in single sequences. Futhermore, the iduronic acid moieties may be bound to either N-acetylated or N-sulfated glucosamine units, with these occuring at either end of the uronic acid unit.

Preparation of carcinoembryonic antigen (CEA) containing significantly increased amounts of galactose and galactosamine.

CEA was prepared by combined isoelectric precipitation, ultrafiltration and column chromatography under controlled conditions of pH. The resulting immunologically active materials were higher in carbohydrate (85-87%), N-acetyl galactosamine (10-11.5%) and galactose (28-32%) content than that previously reported. Differences in amino acid yield were also noted; the concentrations of aspartate, serine, glycine and alanine being higher and that of lysine, histidine, arginine, proline, valine, isoleucine, leucine and tyrosine were lower than that reported for CEA prepared by previous methods. The tumor tissues for CEA extraction were obtained from two Group O Rh positive deceased. Neither preparation showed Group A or B activity as measured by hemagglutination inhibition. It is suggested that the method of purification influences the carbohydrate and amino acid yields.

The distribution of sulfated uronic acid and hexosamine residues in heparin and heparan sulfate.

Heparins from various sources and heparan sulfate from umbilical cords have been subjected to Smith-degradation and reaction with nitrites. These procedures were effective for providing data relating to the distribution of sulfated iduronic acid residues in the molecule. Results indicated that heparins may have, as a prominent structural feature of the molecule, non-sulfated uronic acid distributed in single sequences, much as had been shown previously for N-acetylglucosamine residues. Sulfated uronic acid, however, may occur in multiple sequences of up to 5 or 6 residues. Heparan sulfate was found to have a major proportion of its ester sulfate on iduronic acid rather than hexosamine units, thereby having sections similar to those in heparins, though in considerably lower proportion.

The linkage of corneal keratan sulfate to protein.

The linkage of corneal keratan sulfate to protein has been investigated. After exhaustive digestion of bovine corneas with papain and pronase, a product was obtained in which aspartic acid was the predominant amino acid and constituted 59% of the total amino acids. A carbohydrate-protein linkage fragment was isolated from this preparation by a relatively simple procedure involving the following steps: (1) partial acid hydrolysis, adsorption of glycopeptides and other cationic material on Dowex 50-X2 (H+) and elution with 0.25 M HCl: (2) paper electrophoresis of the eluted fraction at pH 6.5 and pH 1.9; (3) paper chromatography; and (4) final purification by column chromatography on Aminex A"-5 resin. The structure of the linkage fragment was established as 2-acetamido-1-(L-beta-aspartamido)-1,2-dideoxy-beta-D-glucose (Asn-GlcNAc). Evidence for this structure was obtained from qualitative and quantitative analyses as well as from the migration characteristics in several chromatographic anc electrophoretic systems. Further support for the identity of the isolated compound was provided by treatment with beta-aspartyl N-acetylglucosyl-amine amidohydrolase which specifically cleaves Asn-GlcNAc or asparaginyl-oligosaccharides. It is concluded that corneal keratan sulfate is bound to protein via a N-glycosylamine linkage between N-acetylglucosamine and asparagine: this type of linkage is common to many glycoproteins.