Structure-function relationships in heparin cofactor II: Chemical modification of arginine and tryptophan and demonstration of a two-domain structure

Abstract

Heparin cofactor II and antithrombin III are plasma proteins functionally similar in their ability to inhibit thrombin at accelerated rates in the presence of heparin. To further characterize the structural and functional properties of human heparin cofactor II as compared to antithrombin III, we studied the possible significance of arginyl and tryptophanyl residues and the changes in protein structure and activity during guanidinium chloride (GdmCl) denaturation. Both antithrombin and heparin cofactor activities of heparin cofactor II are inactivated by the arginine-specific reagent, 2,3-butanedione. Saturation kinetics are observed during modification and suggest formation of a reversible protease inhibitor-butanedione complex. Quantitation of arginyl residues following butanedione modification shows a loss of about four residues for total inactivation, one of which is essential for antithrombin activity. Arginine-modified heparin cofactor II did not bind to heparin-agarose and implies a role for the other modified arginyl residues during heparin cofactor activity. N-Bromosuccinimide oxidation (20 mol of reagent/mol of protein) of heparin cofactor II results in modification of approximately two tryptophanyl residues with no concomitant loss of heparin cofactor activity. Moreover, there is no enhancement of intrinsic protein fluorescence during heparin binding to the native inhibitor. Circular dichroism measurements show that the structural transition of heparin cofactor II during denaturation is distinctly biphasic, yielding midpoints at 0.6 and 2.6 m GdmCl. Functional protease inhibitory activities are affected to the same extent following denaturation-renaturation at various GdmCl concentrations. The results indicate that arginyl residues are critical for both antithrombin and heparin binding activities. In contrast, tryptophanyl residues are apparently not essential for heparin-dependent interactions. The results also suggest that heparin cofactor II contains two structural domains which unfold at different GdmCl concentrations.