GIANT HEMOGLOBIN LINKER CHAIN L1 DISULPHIDE CONNECTIVITY

环节动物和管蠕虫的细胞外血红蛋白是 多达200个多肽和分子量的多亚基蛋白质 至至少3,900 kDa。 它们与所有其他血红蛋白的不同之处在于， 结合链和“接头”链。 本发明的连接体链L1 蚯蚓血红蛋白含有38-39个残基片段 具有重复模式的半胱氨酸残基（CysX 6）3 -CysX5-CysX10-Cys。 这种模式，不存在于任何珠蛋白序列中， 恰好对应于配体的富含半胱氨酸的重复序列 人低密度脂蛋白受体结合区 和非洲爪蟾。 这些结构域的二硫键连接具有 在LDL受体中尚未确定。 的确定 因此，接头链中的二硫键是重要的第一步， 在理解载脂蛋白E之间的相互作用的一步 和LDL受体。 连接链L1用许多酶消化， 酶和产生的肽。续混合物进行了分析， 基质辅助激光解吸质谱法 由于巨大的 可能的二硫键的数量，峰的归属 含有肽的二硫键是极其困难的。 因此，进行肽混合物的HPLC分级分离。 用于鉴定含二硫键的肽级分， 进行感兴趣进一步的酶促和化学反应。 对所得肽产物的鉴定提供信息 这对绘制二硫键图谱很有用。 最终，我们发现 酶消化蛋白质的MALDI-ITMS提供了最高的 质量映射数据。 本文描述了该技术并讨论了 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.