Mauk M R, Mauk A G
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Eur J Biochem. 1989 Dec 22;186(3):473-86. doi: 10.1111/j.1432-1033.1989.tb15231.x.
A water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), has been used to crosslink horse heart cytochrome c and trypsin-solubilized bovine liver microsomal cytochrome b5. The reaction was conducted under a variety of solution conditions, and the products were purified by a combination of gel filtration and ion-exchange chromatography. Under all conditions of pH, ionic strength, EDC/protein ratio and reaction time that were studied, multiple 1:1 crosslinked complexes were observed with no evidence of a single, dominant species. Acetate, which is often used as a quencher of such reactions, was found to increase the complexity of the reaction products, presumably through EDC-promoted coupling to cytochrome c. Hydroxylamine treatment of the crosslinked complexes, a procedure frequently used to reverse EDC modification of tyrosyl residues, did not reduce the number of crosslinked components observed. The cytochrome b5 heme group was readily extracted from each of the 1:1 crosslinked complexes by standard techniques, so the crosslinking of heme propionate 7 with Lys79 of cytochrome c that might have been anticipated on the basis of molecular graphics modeling [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was not evident from this analysis. Analysis of HPLC tryptic peptide maps produced from crosslinked complexes revealed reduced specificity of trypsin in hydrolysis of EDC-crosslinked protein-protein complexes and unsatisfactory resolution of crosslinked or branched peptides. Nevertheless, it was possible to demonstrate that residues 52-72 of cytochrome b5, a region predicted to be critical to interaction with cytochrome b5 [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568] was absent from all peptide maps of 1:1 cytochrome c.cytochrome b5 complexes. Based on these results and a review of the literature involving EDC crosslinking of electron transfer proteins, we conclude that the techniques available for specific protein hydrolysis and separation of crosslinked peptides are not adequate to permit routine unambiguous identification of crosslinking sites in carbodiimide-crosslinked complexes.
一种水溶性碳二亚胺，1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC），已被用于使马心脏细胞色素c与胰蛋白酶溶解的牛肝微粒体细胞色素b5交联。反应在多种溶液条件下进行，产物通过凝胶过滤和离子交换色谱相结合的方法进行纯化。在所研究的所有pH、离子强度、EDC/蛋白质比例和反应时间条件下，均观察到多个1:1交联复合物，没有证据表明存在单一的主要物种。通常用作此类反应淬灭剂的乙酸盐，被发现会增加反应产物的复杂性，推测是通过EDC促进与细胞色素c的偶联。对交联复合物进行羟胺处理，这是一种常用于逆转酪氨酸残基的EDC修饰的方法，并没有减少观察到的交联成分的数量。细胞色素b5血红素基团很容易通过标准技术从每个1:1交联复合物中提取出来，因此基于分子图形建模[萨勒姆，F.R.（1976年）《分子生物学杂志》102卷，563 - 568页]预期的血红素丙酸酯7与细胞色素c的Lys79的交联在该分析中并不明显。对交联复合物产生的高效液相色谱胰蛋白酶肽图的分析表明，胰蛋白酶在水解EDC交联的蛋白质 - 蛋白质复合物时特异性降低，并且交联或分支肽的分辨率不理想。然而，有可能证明细胞色素b5的52 - 72位残基，一个预计对与细胞色素b5相互作用至关重要的区域[萨勒姆，F.R.（1976年）《分子生物学杂志》102卷，563 - 568页]，在所有1:1细胞色素c·细胞色素b5复合物的肽图中均不存在。基于这些结果以及对涉及电子传递蛋白的EDC交联的文献综述，我们得出结论，现有的用于特定蛋白质水解和交联肽分离的技术不足以允许常规明确鉴定碳二亚胺交联复合物中的交联位点。
