Demonstration of a two-domain structure of antithrombin III during its denaturation in guanidinium chloride.

The denaturation of human antithrombin III in urea and guanidinium chloride (GdmCl) was investigated by intrinsic fluorescence, circular dichroism, and absorption difference spectroscopy. Results suggest the existence of two structural domains as evidenced by the occurrence of two structural transitions, one at 0.7 M GdmCl and the other at 2.3 M GdmCl. This multistate unfolding transition has been confirmed under various protein concentrations and incubation times using different antithrombin III preparations, including a preparation that had been heated at 60 degrees C in the presence of 0.5 M citrate. The persistence of the multistate transition under these conditions rules out the possibilities that it could be due to the presence of different antithrombin populations in a mixture or the presence of a prosthetic group in the molecule. The first unfolding domain (0.7 M GdmCl) is stabilized by heparin and citrate. These protections suggest that the first unfolding domain of low stability may have functional relevance and important implications with respect to antithrombin III-heparin and antithrombin III-protease interaction.