VON WILLEBRAND FACTOR--CHEMICAL CHARACTERIZATION

血管性血友病因子--化学特性

• 批准号: 3340719
• 负责人: KOITI TITANI
• 金额: $ 18.95万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 1986-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3340719
• 关键词: chemical cleavage; chemical structure function; chromatography; coagulation factor VIII; disulfide bond; electrophoresis; human tissue; ligands; pathologic process; platelets; proteolysis; von Willebrand's disease

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.

这是一项对人类的结构特征进行研究的建议， 血管性血友病因子，一种异常大的寡聚糖蛋白（亚单位 大小约200，000道尔顿），其在止血中起重要作用， 血液凝固 将特别强调阐明其 完整的一级结构和定义的相关性 substructuraldomains与它的能力，结合血小板或胶原。 将适当注意确定共同使用的地点， 或翻译后修饰，包括糖基化和二硫键 桥接 蛋白质将以克为单位从一个 商业凝血因子VIII浓缩物以及 组成多肽链将通过化学方法建立。 的 在二硫键还原之前或之后，整个蛋白质将被 有限的蛋白水解以提供将被测试的亚结构域 残留或改变的生物功能。 整个变性蛋白 也将在天冬酰胺基-甘氨酸、天冬氨酰-脯氨酸、甲硫氨酰或 乙酰基键生成大片段。 各种产品 将裂解反应纯化并进行序列分析。 整合这些结果有望提供完整的氨基酸 顺序 含有Met、Trp和Cys的肽将被选择性地 分离并分析其合成的设计潜力 寡核苷酸杂交探针。 这项研究的长期目标是 寻找出血性疾病中功能障碍的化学基础， Willebrand氏病 拟议的结构分析应提供 在这个化学框架中， 和功能障碍可以用具体的术语来回答。