Interaction of thrombin with synthetic fluorescent substrate immobilized on polymer membrane

Synthetic fluorescent substrates for thrombin were immobilized on a polyurethane membrane. The interaction of thrombin with the immobilized substrates was investigated. Upon hydrolysis of immobilized substrates by thrombin, fluorescence was emitted, intensity increasing linearly with time. From a Lineweaver-Burk plot, kinetic parameters of the enzyme reaction on the membrane surface were determined. The dissociation constant (K) was increased and the catalytic constant (k) decreased markedly by immobilization. More reactive thrombin substrates in solution were also more reactive in the immobilized state. More thrombin was adsorbed and adsorbed thrombins were inactivated more strongly on the surface with more reactive substrates when immobilized. The immobilization of thrombin substrate markedly suppressed fibrin formation on the membrane surface and made the membrane non-thrombogenic.