GIANT HEMOGLOBIN LINKER CHAIN L1 DISULPHIDE CONNECTIVITY

环节动物和管虫的胞外血红蛋白是 最多200个多肽和分子质量的多亚基蛋白质 至少3900 kDa。它们与所有其他HBs的不同之处在于它含有O2 结合链和“连接”链。的链接器链L1 陆虫血红蛋白含有38-39个残基片段 具有半胱氨基残基的重复模式(CysX6)3 -CysX5-CysX10-Cys。这种模式在任何珠蛋白序列中都不存在， 确切地对应于陶，富含半胱氨酸的配体的重复序列 人低密度脂蛋白受体的结合域 和非洲爪哇。这些结构域的二硫键连接性具有 在低密度脂蛋白受体中未被检测到。这一决定决定了 因此，连接链中的二硫键是一个重要的第一步 了解载脂蛋白E之间相互作用的步骤 和低密度脂蛋白受体。链接器链L1被多个 酶和生成的多肽..续。对混合物进行了分析 基质辅助激光解吸质谱。由于巨大的 可能的二硫键的数量，峰的分配到 含有多肽的二硫键是极其困难的。 为此，进行了多肽混合物的高效液相色谱分离。 用于鉴定含二硫化物的多肽组分 进一步进行酶反应和化学反应。 对所得多肽产品的鉴定提供了信息 这对于绘制二硫键的联系很有用。最终，我们发现 酶消化蛋白质的MALDI-ITMS提供了最高的 质量测绘数据。一篇描述这项技术的论文并进行了讨论 结果正在准备中。

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 O2 binding chains and "linker" chains. The linker chain L1 of the hemoglobin of Lumbricus terrestris contains a 38-39 residue segment with a repeating pattern of cysteinyl residues (CysX6)3 -CysX5-CysX10-Cys. This pattern, not present in any globin sequence, corresponds exactly tao the cysteine 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 L1 was digested with a number of enzymes and the resulting peptide ..cont. 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 disulfe linkages. Ultimately, we found 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.