Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl)
sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety
per molecule of antithrombin III. Heparin protects against tryptophan modification,
particularly at low reagent concentrations. Unlike native antithrombin, which has
high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity
column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained
with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti-
thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of
thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native
antithrombin possess progressive antithrombin activity, inactivating these proteases
at identical rates. These results indicate that the integrity of a specific tryptophan
residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic
cleavage techniques have been used to isolate peptides containing this tryptophan
from both HNB-labeled and native antithrombin and to identify this critical tryptophan
residue within the amino acid sequence of the antithrombin molecule.
