Pearce K H, Hof M, Lentz B R, Thompson N L
Department of Chemistry, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1993 Nov 5;268(31):22984-91.
Total internal reflection fluorescence microscopy has been used to compare the membrane binding characteristics of fluorescein-labeled bovine prothrombin and fluorescein-labeled bovine prothrombin fragment 1. The Ca(2+)-dependent association of these proteins with quartz-supported planar membranes composed of mixtures of phosphatidylserine (2-10 mol%) and phosphatidylcholine was examined. Equilibrium binding measurements showed that the apparent equilibrium dissociation constants increased with decreasing molar fractions of phosphatidylserine and that the dissociation constants were somewhat lower for intact prothrombin. Kinetic measurements, using fluorescence photobleaching recovery, showed that the measured dissociation rates were approximately equivalent for prothrombin and fragment 1 and did not change with the protein solution concentration or the molar fraction of phosphatidylserine. The kinetic data also implied that the surface binding mechanism for both proteins is more complex than a simple reversible reaction between monovalent proteins and monovalent surface sites. Measured equilibrium and kinetic constants are reported and compared for prothrombin and fragment 1 on planar membranes.
全内反射荧光显微镜已被用于比较荧光素标记的牛凝血酶原和荧光素标记的牛凝血酶原片段1的膜结合特性。研究了这些蛋白质与由磷脂酰丝氨酸（2 - 10摩尔%）和磷脂酰胆碱混合物组成的石英支撑平面膜的钙依赖性结合。平衡结合测量表明，表观平衡解离常数随着磷脂酰丝氨酸摩尔分数的降低而增加，并且完整凝血酶原的解离常数略低。使用荧光光漂白恢复的动力学测量表明，凝血酶原和片段1的测量解离速率大致相当，并且不随蛋白质溶液浓度或磷脂酰丝氨酸的摩尔分数而变化。动力学数据还表明，两种蛋白质的表面结合机制比单价蛋白质与单价表面位点之间的简单可逆反应更为复杂。报告并比较了凝血酶原和片段1在平面膜上的测量平衡常数和动力学常数。
