Chromatography of heparin on Sepharose–lysine: molecular size fractionation and its relevance to thrombin and antithrombin III binding

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Abstract
Batches of Sepharose-lysine, which varied in lysine content from 35-430 .mu.mol/g of dry gel, were prepared by varying quantity of CNBr in the activation reaction. The batches were tested for heparin binding by using a controlled chromatographic procedure. Sepharose-lysine, containing < 150 .mu.mol of lysine/g, did not significantly bind heparin whereas conjugates with > 400 .mu.mol/g retained the entire heparin load. For intermediate batches of Sepharose-lysine (150-400 .mu.mol/g) the quantity of heparin bound largely paralleled the lysine content. Sepharose-lysine of an intermediate lysine content separated heparin into an unretained fraction and a bound fraction which was recovered from the column by eluting with 1 M NaCl. On testing for anticoagulant activity by factor Xa inhibition assay, no significant difference in specific anticoagulant activity was observed between these heparin fractions and the heparin load. From gel filtration studies, a substantial difference in molecular size was noted. An unretained heparin fraction from Sepharose-lysine was of a lower average MW than the parent heparin. A retained heparin peak was of a higher average MW compared with the parent heparin. These observations were confirmed by studying the chromatographic properties of low (10,000) and high (23,000) MW heparin samples on various Sepharose-lysine batches. A model was proposed to explain this discriminating property of Sepharose-lysine. For a conjugate containing 400 .mu.mol/g, the mean lysine spacing was calculated at 47 .ANG. (1 .ANG. = 0.1 nm), which is approximately equivalent to 5-6 disaccharide units in heparin. The property of Sepharose-lysine to bind heparin was compared with the affinities of the mucopolysaccharide for [bovine] thrombin and antithrombin III. Evidence was proposed for the involvement of lysine residues of antithrombin III in this process. The lysine and arginine groups of thrombin are apparently also involved in heparin binding. By specifically modifying 2-4 lysine residues using nitrous acid, the heparin-binding capacity of the enzyme and its plasma clotting activity were largely destroyed, although the esterase activity was retained.