GIANT HEMOGLOBIN LINKER CHAIN L1 DISULFIDE CONNECTIVITY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。环节动物和管虫的胞外血红蛋白是由200多个多肽和至少3900 kDa的分子量组成的多亚单位蛋白。它们与所有其他HBs的不同之处在于它们具有O2结合链和连接器链。陆虫血红蛋白连接链L1含有38-39个残基，具有半胱氨基残基(CysX6)3-CysX5-CysX10-Cys的重复模式。这种模式在任何珠蛋白序列中都不存在，恰好对应于人类和非洲爪哇低密度脂蛋白(LDL)受体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性在低密度脂蛋白受体中尚未确定。因此，连接链中二硫键的测定是了解载脂蛋白E和低密度脂蛋白受体相互作用的重要第一步。连接物链L1经多种酶消化后，用基质辅助激光解吸质谱仪进行分析。由于可能存在大量的二硫键，因此将峰指定给含有多肽的二硫键是非常困难的。为此，进行了多肽混合物的高效液相色谱分离。为了鉴定含有二硫键的多肽组分，进一步进行了酶反应和化学反应。对所得多肽产物的鉴定提供了有助于绘制二硫键图谱的信息。最终，我们发现酶消化蛋白质的MALDI-ITMS提供了最高质量的图谱数据。一篇描述这项技术并讨论其结果的论文正在出版中。

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.