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Fractionation of heparin by affinity chromatography on covalently-bound human α-thrombin

1978; Elsevier BV; Volume: 83; Issue: 3 Linguagem: Inglês

10.1016/0006-291x(78)91522-x

1090-2104

Michael Griffith, Henry S. Kingdon, Roger L. Lundblad,

Glycosylation and Glycoproteins Research

Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of N-α-tosyl-L-glycyl-L-prolyl-L-arginine-p-nitroanilide hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.