Whale heparin was separated by affinity chromatography on an antithrombin III-Sepharose column into two distinct fractions. The high-affinity fraction accounted for most of the anticoagulant activity of the unfractionated material, while the low-affinity fraction was relatively inactive. The yields of the two fractions were substantially equivalent. No significant difference was observed between these fractions in terms of electrophoretic mobilities on cellulose acetate membrane and analytical data except for the contents of N-acetylglucosamine and N-sulfoglucosamine. The highly active form contained more N-acetylglucosamine and less N-sulfoglucosamine than the relatively inactive form.
The two fractions were separately subjected to sequential digestion with purified heparinase and heparitinase, and the oligosaccharide fractions were isolated from the digests by DEAE-cellulose column chromatography, followed by preparative paper chromatography. The purified compounds were then characterized by routine chemical and physical methods. Compound 1, Δ_{4, 5}hexosyluronic acidl→4 N-acetylglucosamine, was exclusively obtained from the highly active form, whereas compound 3 a, Δ_{4, 5}hexosyluronic acidl→4 N-acetylglucosamine 6-sulfate, and compound 3 b Δ_{4, 5}hexosyluronic acidl→4 N-sulfoglucosamine, were the only ones obtained from the relatively inactive form. The yields of other oligosaccharide fractions from both forms were comparable. The present data suggest that an N-acetylglucosamine-containing oligosaccharide structure in whale heparin is essential for binding to antithrombin III.