GIANT HEMOGLOBIN LINKER CHAIN L I DISULPHIDE CONNECTIVITY

巨型血红蛋白连接链 L I 二硫化物连接

• 批准号: 6279501
• 负责人: AUSTEN F RIGGS
• 金额: $ 0.08万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6279501
• 关键词: Invertebrata; biological products; biomedical resource; proteins; spectrometry

The extracellular hemoglobin of annelids and tube worms are multisubunit proteins of up to 200 polypeptides and molecular masses to at least 3,900 kDa. They differ from all other Hbs in having 02 binding chains and "linker" chains. The linker chain LI of the hemoglobin of Lumbricus terrestris contains a 38-39 residue segment with a repeating pattern of cysteinyl residues: (CYSX6)3 -CysX5CysX I O-Cys. This pattern, not present in any globin sequence, corresponds exactly to the cystein rich repeats of the ligand binding domain of the low density lipoprotein (LDL) receptor of man and Xenopus laevis. The disulfide connectivity of these domains has not been determined in the LDL receptor. The determination of the disulfide bonds in the linker chains is therefore an important first step in the understanding of the interaction between apolipoprotein E and the LDL receptor. Linker chain L I was digested with a number of enzymes and theresulting peptide mixtures were analysed by matrix-assisted laser desorption mass spectrometry. Due to the huge number of possible disulfide bridges, the assignment of peaks to disulfide bridges containing peptides was extremely difficult. Therefore HPLC fractionation of peptide mixtures were carried out. For the identification of disulfide containing peptides fractions of interest further enzymatic and chemical reactions are performed. Identification of the resulting peptide products provide information that is useful for mapping the disulfide linkages. Ultimately, we determined that MALDI-ITMS of the enzyme digested protein provided the highest quality mapping data. A paper describing the technique and discussing the results is in preparation.

环节动物和管蠕虫的细胞外血红蛋白是 多达200种多肽和分子量的多亚基蛋白质 至至少3,900 kDa。 他们与所有其他血红蛋白的不同之处在于， 结合链和“接头”链。 所述多核苷酸的接头链Ll 蚯蚓血红蛋白含有38-39个残基片段 具有重复模式的半胱氨酰残基：（CYSX 6）3 -CysX 5CysX I O-Cys 这种模式，不存在于任何珠蛋白序列，对应于 精确地与配体结合结构域的富含半胱氨酸的重复序列 人类和非洲爪蟾的低密度脂蛋白（LDL）受体。 这些结构域的二硫键连接性尚未确定， LDL受体。 二硫键的测定 因此，连接链是重要的第一步 了解载脂蛋白E和LDL之间的相互作用 受体的 用多种酶消化接头链L1， 用基质辅助激光分析所得肽混合物 解吸质谱法 由于大量的可能 二硫键，峰与二硫键的归属 含有肽的蛋白质是非常困难的。 因此HPLC 进行肽混合物的分级分离。 为 鉴定含有二硫化物的感兴趣的肽级分 进行进一步的酶促和化学反应。 对所得肽产物的鉴定提供信息 这对绘制二硫键图谱很有用。 最终我们 确定酶消化的蛋白质的MALDI-ITMS提供了 最高质量的地图数据。 一份描述该技术的文件， 正在准备讨论结果。