Separation of heparin into fractions with different anticoagulant activity by hydrophobic interaction chromatography.

Hog mucosal heparin purified on Sephadex G-100 (anticoagulant activity assayed by the method of the United States Pharmacopoeia, 179 units/mg) was separated by hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B into two groups, one with high affinity and another with low affinity for the gels. The former group was further separated into three fractions differing in hydrophobicity. The anticoagulant activities of the fractions with higher hydrophobicity ranged from 210 to 254 units/mg, whereas that of the fraction with lower hydrophobicity was 100 units/mg. The difference in antithrombin III-activation potency was much more prominent. The data obtained from affinity chromatography of these fractions on antithrombin III-Sepharose also substantiated the observed difference in anticoagulant activity. Analytical data of the fractions revealed a characteristic difference in both N-acetyl content and molecular size. While the N-acetyl content (mol/mol of hexosamine) and Kav value (on Ultrogel AcA44) of the fraction with the lowest hydrophobicity were 0.12 mol and 0.48, those of the fractions with higher hydrophobicity were 0.15-0.17 mol and 0.35-0.23, respectively.