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首页> 外文期刊>Biochemistry >KINETICS OF THE INTERACTION BETWEEN THE HUMAN FACTOR VIIIA SUBUNITS - EFFECTS OF PH, IONIC STRENGTH, CA2+ CONCENTRATION, HEPARIN, AND ACTIVATED PROTEIN C-CATALYZED PROTEOLYSIS
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KINETICS OF THE INTERACTION BETWEEN THE HUMAN FACTOR VIIIA SUBUNITS - EFFECTS OF PH, IONIC STRENGTH, CA2+ CONCENTRATION, HEPARIN, AND ACTIVATED PROTEIN C-CATALYZED PROTEOLYSIS

机译：人因子VIIIA子之间相互作用的动力学-pH，离子强度，CA2 +浓度，肝素和活性蛋白C催化蛋白水解的影响

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Coagulation factor VIIIa consists of a heterotrimer in which the A2 subunit is bound to the A1/A3C1C2 dimer. The dissociation of this complex causes the spontaneous and reversible decay of factor VIIIa activity. In order to characterize the kinetics and affinity of the interaction between A2 and A1/A3C1C2, as well as the influence of different parameters on the interaction, the subunits were chromatographically separated and reassembled in a BIAcore instrument (Pharmacia Biosensor). In the binding experiments, A2 was free in solution, whereas A1/A3C1C2 was immobilized on the dextran surface by direct coupling or captured on an immobilized monoclonal anti-C2 antibody. At our chosen standard condition (pH = 6.0, I = 0.12, and [Ca2+] = 2 mM), the association rate constant, dissociation rate constant, and resulting equilibrium dissociation constant were ca. 1.4 x 10(4) M(-1) s(-1), 2.1 x 10(-4) s(-1), and 16 nM, respectively. Increasing the ionic strength or Ca2+ concentration resulted in both slower association and faster dissociation. At 0.3 M NaCl or 25 mM Ca2+, the dissociation constant was >1 mu M. This implies that electrostatic forces involved in the interaction contribute at least one-fourth of the total binding energy. Increasing pH caused a similar effect, yielding a dissociation constant of ca. 0.9 mu M at pH 7.5. In those cases where the equilibrium dissociation constants had been determined from solution phase experiments [Fay, P. J., & Smudzin, T. M. (1992) J. Biol. Chem. 267, 13246-13250; Lollar, P., Parker, E. T., & Fay, P. J. (1992) J. Biol. Chem. 267, 23652-23657], these constants agreed well with our results. In addition, direct immobilization of A1/A3C1C2 or capture on an antibody gave very similar rate constants, indicating that neither the immobilization per se nor the mode of immobilization affected the subunit interaction. Limited proteolysis of A1/A3C1C2 by activated protein C abolished its ability to bind A2, supporting the involvement of the negatively charged region containing residues 337-372. Heparin prevented A2/A1/A3C1C2 heterotrimer formation, presumably by binding to and blocking basic regions of importance for the interaction.
机译：凝血因子VIIIa由杂三聚体组成，其中A2亚基与A1 / A3C1C2二聚体结合。该复合物的解离导致因子VIIIa活性自发和可逆地衰减。为了表征A2和A1 / A3C1C2之间相互作用的动力学和亲和力以及不同参数对相互作用的影响，对亚基进行了色谱分离，并在BIAcore仪器（Pharmacia Biosensor）中重新组装。在结合实验中，A2在溶液中游离，而A1 / A3C1C2通过直接偶联固定在葡聚糖表面，或捕获在固定的单克隆抗C2抗体上。在我们选择的标准条件下（pH = 6.0，I = 0.12，[Ca2 +] = 2 mM），缔合速率常数，解离速率常数和所得的平衡解离常数均为ca。 1.4 x 10（4）M（-1）s（-1），2.1 x 10（-4）s（-1）和16 nM。离子强度或Ca2 +浓度的增加会导致缔合变慢和解离变快。在0.3 M NaCl或25 mM Ca2 +下，解离常数> 1μM。这意味着相互作用中涉及的静电力至少贡献了总结合能的四分之一。增加pH值会产生类似的效果，产生约解离常数。 pH 7.5时为0.9μM。在那些已经从溶液相实验中确定了平衡解离常数的情况下[Fay，P.J。，＆Smudzin，T.M。（1992）J.Biol.Chem.Soc.Sci。，1992，9，1897]。化学267，13246-13250; Lollar，P.，Parker，E.T。，＆Fay，P.J。（1992）J.Biol.Chem.Soc。，1992。化学267，23652-23657]，这些常数与我们的结果非常吻合。另外，直接固定化A1 / A3C1C2或捕获抗体可得到非常相似的速率常数，表明固定化本身或固定化模式均不会影响亚基相互作用。活化蛋白C对A1 / A3C1C2的有限蛋白水解消除了其结合A2的能力，从而支持了包含残基337-372的带负电荷的区域的参与。肝素阻止了A2 / A1 / A3C1C2异源三聚体的形成，大概是通过结合并阻断了相互作用的重要基本区域。

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