GIANT HEMOGLOBIN LINKER CHAIN L I 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。它们与所有其他哈佛商学院的不同之处在于有02 结合链和“连接”链。的链接器链Li 陆虫血红蛋白含有38-39个残基片段 半胱氨基残基重复模式：(CysX6)3-CysX5CysX I O-Cys。这种模式在任何珠蛋白序列中都不存在，与 准确到富含半胱氨酸的配体结合域的重复序列 人和非洲爪哇的低密度脂蛋白受体。 这些结构域的二硫键连接性尚未在 低密度脂蛋白受体。分子中二硫键的测定 因此，链接器链是 载脂蛋白E与低密度脂蛋白相互作用的认识 受体。连接物链L I被一些酶和酶消化 用基质辅助激光分析了生成的多肽混合物 解吸质谱。由于可能的数量巨大 二硫键，将峰分配给二硫键 含有多肽是极其困难的。因此高效液相色谱仪 对多肽混合物进行了分级。对于 含有二硫键的目标多肽部分的鉴定 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图谱是有用的。最终，我们 确定酶消化蛋白质的MALDI-ITMS提供了 最高质量的地图数据。一篇描述该技术的论文和 对结果的讨论正在准备中。

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 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.