Ito Y, Liu L S, Imanishi Y
Department of Polymer Chemistry, Kyoto University, Japan.
Biomaterials. 1992;13(6):375-81. doi: 10.1016/0142-9612(92)90043-n.
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.
凝血酶的合成荧光底物被固定在聚氨酯膜上。研究了凝血酶与固定化底物的相互作用。当固定化底物被凝血酶水解时，会发出荧光，其强度随时间呈线性增加。通过Lineweaver-Burk图，确定了酶在膜表面反应的动力学参数。固定化使解离常数（K）增加，催化常数（k）显著降低。溶液中反应性更强的凝血酶底物在固定化状态下也更具反应性。固定化时，更多的凝血酶被吸附，且在具有更高反应性底物的表面上，吸附的凝血酶失活更强。凝血酶底物的固定化显著抑制了膜表面纤维蛋白的形成，使膜具有抗血栓形成性。
