GIANT HEMOGLOBIN LINKER CHAIN L1 DISULFIDE CONNECTIVITY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。环节动物和管虫的细胞外血红蛋白是由多达200个多肽组成的多亚基蛋白，分子质量至少为3,900 kDa。它们与所有其他Hbs的不同之处在于具有O2结合链和“连接”链。地蚓血红蛋白连接链L1含有38-39个残基片段，残基重复模式为半胱氨酸残基（CysX6）3 -CysX5-CysX10-Cys。这种模式不存在于任何珠蛋白序列中，与人类和非洲爪蟾低密度脂蛋白（LDL）受体的配体结合域的富含半胱氨酸的重复完全对应。这些结构域的二硫连通性在LDL受体中尚未确定。因此，确定连接链中的二硫键是了解载脂蛋白E与LDL受体之间相互作用的重要的第一步。连接链L1用多种酶消化，所得肽混合物用基质辅助激光解吸质谱法分析。由于可能的二硫桥的数量巨大，峰分配到含肽的二硫桥是非常困难的。为此，采用高效液相色谱法对多肽混合物进行了分离。为了鉴定含二硫的多肽，进一步进行酶和化学反应。鉴定所产生的肽产物为绘制二硫键提供了有用的信息。最终，我们发现酶解蛋白的MALDI-ITMS提供了最高质量的制图数据。一篇描述该技术和讨论结果的论文即将出版。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 mixtures were a nalysed 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 was 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 provides information that is useful for mapping the disulfide 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 press.