Fractionation of Heparin on an Anion Exchanger

Abstract

PREPARATIONS of heparin have been shown to be heterogeneous by a variety of techniques^1,2, but no method is available for the fractionation of bulk quantities of heparin or of other sulphomucopolysaccharides. These compounds carry a strong negative charge over a wide range of pH, and, therefore, it seemed possible that they could be purified on basic resins. Five anion-exchange resins (‘Dowex–1’, ‘Dowex–2’, ‘Dowex–3’, ‘Amberlite CG–45’, and ‘Duolite A–4’), each buffered at pH 6.0, readily adsorbed heparin, which was easily eluted with strong salt solutions, but the eluates differed from the original material in having greater absorption at 400 mµ than at 535 mµ in the carbazole reaction³. However, ‘Ecteola’^4,5 (exchange capacity = 0.3 m.equiv./gm.) resolved a commercial preparation of bovine heparin (obtained from Nutritional Biochemicals Corp.) into four major and one minor carbazole-positive fractions (Fig. 1), each with maximum absorption at 535 mµ, and all showing metachromasia⁶. 90 per cent of the original material was recovered. Heparin-³⁵SO₄, extracted from a mast-cell tumour of the mouse⁷, gave only one peak on chromatography on ‘Ecteola’ (Fig. 2), and only 50 per cent of the material was eluted.