GIANT HEMOGLOBIN LINKER CHAIN L1 DISULFIDE CONNECTIVITY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 环节动物和管蠕虫的细胞外血红蛋白是多亚基蛋白质，最多有200个多肽，分子量至少为3，900 kDa。 它们与所有其他血红蛋白的不同之处在于具有O2结合链和“接头”链。 蚯蚓血红蛋白的连接链L1含有38-39个残基片段，具有半胱氨酰残基（CysX 6）3 -CysX 5-CysX 10-Cys的重复模式。 这种模式，不存在于任何珠蛋白序列，正好对应于人和非洲爪蟾的低密度脂蛋白（LDL）受体的配体结合域的富含半胱氨酸的重复序列。 这些结构域的二硫键连接尚未在LDL受体中确定。 因此，确定连接链中的二硫键是理解载脂蛋白E和LDL受体之间相互作用的重要第一步。 用多种酶消化接头链L1，并通过基质辅助激光解吸质谱法分析所得肽混合物。 由于可能的二硫键数量巨大，因此将峰归属于含二硫键的肽非常困难。因此，进行肽混合物的HPLC分级分离。 为了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。 所得肽产物的鉴定提供了可用于绘制二硫键的信息。 最终，我们发现酶消化蛋白质的MALDI-ITMS提供了最高质量的映射数据。 已经发表了一篇描述该技术并讨论结果的论文。

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 has been published.