FUNCTIONAL DOMAINS IN PROTHROMBIN AND FACTOR X

这个建议是基于这样的假设，即凝血酶原和因子X （因子Xa）包含参与以下的离散功能结构域： 这些生物活性表达的离散部分， 复杂蛋白质 建议通过合并的 使用特定的化学修饰和有限的蛋白水解。 凝血酶原 将用2-羟基-5-硝基苄基溴或用 四硝基甲烷 将分析经修饰的蛋白质， 达到功能性质的改性和改变的程度。 的 选用的两种试剂可分别鉴定功能性酪氨酰和酪氨酰 此外，还提供了 介绍了光谱和荧光 属性可以测量修改后的 残基 在存在下天然凝血酶原的有限蛋白水解 并且不存在配体如二价阳离子、磷脂和其它 凝血酶原酶复合物的组分将提供 与天然凝血酶原比较。 类似的研究建议， 阐明因子X和因子Xa中的功能结构域。 主 我们将致力于Xa因子的研究，因为我们的重点是 针对凝血酶原酶复合物的研究。 拟议 实验将提供凝血酶原酶的分子理解 复杂. 这种理解对于阐明 正常的血液凝固机制。

This proposal is based on the hypothesis that prothrombin and factor X (factor Xa) contain discrete functional domains which are involved in discrete portions of the expression of the biological activity of these complex proteins. It is proposed to identify these regions by the combined use of specific chemical modification and limited proteolysis. Prothrombin will be modified with 2-hydroxy-5-nitrobenzyl bromide or with tetranitromethane. The modified protein(s) will be analyzed with respect to the extent of modification and changes in functional properties. The two reagents selected can identify functional tryptophanyl and tyrosyl residues respectively and, in addition, provide the basis for the introduction of structural probes whose spectral and fluorescence properties can measure changes in the microenvironment around the modified residues. Limited proteolysis of the native prothrombin in the presence and absence of ligands such as divalent cations, phospholipid and other components of the prothrombinase complex will provide derivatives for comparison with native prothrombin. Similar studies are proposed for the elucidation of functional domains in factor X and factor Xa. The primary effort will be directed toward the study of factor Xa since our emphasis is directed toward the study of the prothrombinase complex. The proposed experiments will provide a molecular understanding of prothrombinase complex. Such understanding is critical to the elucidation of the mechanism of normal blood coagulation.