Crosslinking of cytochrome c and cytochrome b5 with a water-soluble carbodiimide. Reaction conditions, product analysis and critique of the technique.
Crosslinking of cytochrome c and cytochrome b5 with a water-soluble carbodiimide. Reaction conditions, product analysis and critique of the technique.
复制标题
细胞色素 c 和细胞色素 b5 与水溶性碳二亚胺的交联。
DOI：
10.1111/j.1432-1033.1989.tb15231.x
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Mauk,AG
中科院分区：
文献类型：
--
作者：
Mauk,MR;Mauk,AG
A water soluble carbodiimide, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC), has been used to crosslink horse heart cytochromecand trypsin‐solubilized bovine liver microsomal cytochromeb5. 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 cytochromec. 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 cytochromeb5heme 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 cytochromecthat 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 cytochromeb5, a region predicted to be critical to interaction with cytochromeb5[Salemme, F. R. (1976)J. Mol. Biol. 102, 563–568] was absent from all peptide maps of 1:1 cytochromec· cytochromeb5complexes. 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.
