Characterisation of silica-based heparin affinity sorbents from equilibrium binding studies on plasma fractions...

The binding properties of rigid heparin sorbents, synthesised by end-point-attachment of heparin onto aminopropylderivatised silica through reductive amination, were characterised through batch-adsorption studies employing human plasma fractions containing thrombin. Thrombin was quantified using a chromogenic assay that had been specially modified for these studies. These investigations yielded information regarding the maximum adsorption capacities/stoichiometries and binding affinities for thrombin present in complex protein mixtures. Of the two types of heparin-silica evaluated, heparin-Fractosil 1000, with a pore size of 1000 A, displayed a capacity of 2.4 mol of thrombin/mol of heparin (mol T-mol H). This stoichiometry was significantly higher than the value of 1.8 mol T-mol H obtained for the commercial soft gel heparin-Sepharose CL-6B. Furthermore, the heparin-Fractosil 1000 sorbents were superior in capacity and binding site accessibility to heparin-LiChroprep Si60 sorbents, where the smaller pore size of 60 A largely restricts the ligand-protein interactions to the outer surface of the sorbent particles. Nevertheless, heparin-LiChroprep Si60 sorbents were useful, in that they simulated a non-porous particle system, in which intra-pore diffusion effects are eliminated. The batch adsorption results with these sorbents indicated that the adsorption involved both high and low binding affinity characteristics. This bimodal binding mechanism was also evident with the commercial heparin-Sepharose sorbent. Binding stoichiometries and affinities in the high concentration range were similar to values reported for a largely non-specific electrostatic thrombin-heparin interaction. Dissociation constants in the nanomolar range were observed in the low concentrations range. This stronger binding affinity is more similar to highly specific bio-affinity interactions. Thus, the results indicated that heparin-thrombin interactions with these systems involve both a weak electrostatic and a strong biospecific interaction component.