The use of spin labels in looking at subtle conformational changes in blood coagulation proteins

Spin labels have now enjoyed a history of close to twenty-five years in demonstrating the applicability of paramagnetic nitroxides to biochemical problems of structure and function in enzymes, membranes, cells and animals. These have been employed traditionally as reporter groups; that is, the nitroxide spin label serves as a physical probe which reports aspects of its structure and environment at a localized molecular site. The work presented here describes the use of active-site directed fluorosulfonylphenyl nitroxide spin label inhibitors for the blood clotting enzyme, thrombin. The results describe a topographical mapping of the extended active site regions of coagulant human a-thrombin, noncoagulant human y-thrombin, and bovine a-thrombin. Subtle differences are revealed between all of these species and forms. Lastly, interactions between the regulatory protein thrombomodulin and spin labeled a -thrombin are described which confirms the propagation of subtle conformational changes to the catalytic site in the thrombin molecule upon complexation.