VON WILLEBRAND FACTOR--CHEMICAL CHARACTERIZATION

这是一项对人的结构特征进行研究的建议 Von Willebrand因子，一种异常大的寡聚糖蛋白(亚单位 大小约200,000道尔顿)，在止血和 血液凝结。将特别强调对其进行解释 完整的一级结构和与已定义的 具有结合血小板或胶原的能力的亚结构结构域。 将适当注意确定合作地点-- 或翻译后修饰，包括糖基化和二硫键 搭桥。这种蛋白质将从一种 商业第八因子浓缩和数字和身份 组成多肽链将通过化学手段建立。这个 整个蛋白质，在二硫化物还原之前或之后，将被 有限的蛋白质分解提供将被测试的亚结构结构域 用于残留或修饰的生物功能。整个变性蛋白质 也会在天冬酰胺-甘氨酸、天冬氨酸氨基-脯氨酸、甲硫基或 精氨酸键产生大片段。各式各样的产品 裂解反应将被提纯并进行序列分析。 这些结果的综合有望提供完整的氨基酸 序列。含有MET、Trp和Cys的多肽将被选择性地 分离和分析它们的设计潜力，以合成 寡核苷酸杂交探针。这项研究的长期目标是 寻找称为von的出血性疾病中功能障碍的化学基础 威尔布兰德病。拟议的结构分析应提供 超分子组织问题的化学框架 而功能障碍可以用具体的术语来回答。

This is a proposal to undertake a study of the structural features of human von Willebrand Factor, an unusually large oligomeric glycoprotein (subunit size ca 200,000 daltons) which play an important role in hemostasis and blood coagulation. Particular emphasis will be given to elucidation of its complete primary structure and to correlation of defined substructuraldomains with its capability to bind platelets or lolagen. Appropriate attention will be given to the identification of sites of co- or post-translational modification, including glycosylation and disulfide bridging. The protein will be purified in gram quantities from a commercial Factor VIII concentrate and the number and identity to constituent polypeptide chains will be established by chemical means. The whole protein, before or after disulfide reduction, will be fragmented by limited proteolysis to provide substructural domains which will be tested for residual or modified biological function. The whole denatured protein will aslo be cleaved at asparaginyl-glycine, aspartyl-proline, methionyl or arginyl bonds to generate large fragments. The products of the various cleavage reaction will be purified and subjected to sequence analysis. Integration of these results is expected to provide the complete amino acid sequence. Met, Trp and Cys containing peptides will be selectively isolated and analyzed for their design potential for synthetic oligonucleotide hybridization probes. A long-term goal of this study is to seek the chemical basis of dysfunction in the bleeding disorders termed von Willebrand's disease. The proposed structural analysis should provide a chemical framework within which questions of supramolecular organization and dysfunction can be answered in specific terms.