Evidence by chemical modification for the involvement of one or more tryptophanyl residues of bovine antithrombin in the binding of high-affinity heparin.

Tryptophanyl residues of bovine antithrombin were modified with N-bromosuccinimide at near-neutral pH. The reaction was found to be specific for tryptophan at low levels of modification, i.e. when only up to 1--1.3 mol tryptophan/mol protein were oxidized. Further modification led to extensive side reactions. Modification of an average of about one tryptophanyl residue per protein molecule did not affect antithrombin activity measured in the absence of heparin, but decreased the activity assayed in the presence of heparin to about half the value given by unmodified antithrombin. Addition of an excess of high-affinity heparin to a similarly modified antithrombin sample resulted in much smaller circular dichroism, ultraviolet absorption and fluorescence changes than those observed with the intact protein. Modification experiments in the presence of excess high-affinity heparin gave a definitely lower extent of modification than when heparin was excluded. These studies thus reinforce the conclusion from previous spectroscopic analyses that one or more tryptophanyl residues of antithrombin are involved in the binding of high-affinity heparin, presumably by being located at or close to the heparin binding site.