Proton NMR and electrophoretic studies of the covalent complex formed by cross-linking yeast cytochrome c peroxidase and horse cytochrome c with a water-soluble carbodiimide.
Proton NMR and electrophoretic studies of the covalent complex formed by cross-linking yeast cytochrome c peroxidase and horse cytochrome c with a water-soluble carbodiimide.
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对酵母细胞色素 c 过氧化物酶和马细胞色素 c 与水溶性碳二亚胺交联形成的共价复合物进行质子 NMR 和电泳研究。
DOI：
10.1021/bi00387a013
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Erman,JE
中科院分区：
文献类型：
--
作者：
Moench,SJ;Satterlee,JD;Erman,JE
1986年12月18日收到的修订版摘要：通过对Waldmeyer和Bosshard [Waldmeyer，B.，& Bosshard，H. R.（1985）J.Biol.Chem.260，5184-5190]，这一早期研究已经扩展到表明两种蛋白质的有效交联可以在宽的离子强度范围内在各种缓冲液中发生。在研究的条件下，在交联研究中用亚铁细胞色素c取代亚铁细胞色素c导致1：1复合物的产率增加（约10-20%）。本文提出了一种改进的方法，用于纯化相对大量的共价复合物，并对该复合物进行了电泳和质子NMR研究。电泳和核磁共振研究表明，碳二亚胺修饰的共价复合物中的一些表面酸性氨基酸。与游离细胞色素c相比，共价复合物中细胞色素c的质子超精细位移共振峰变宽，而与细胞色素c血红素3-CH 3和8-CH 3基团相对应的共振峰移近。NMR共振的积分证实1：1复合物为主要交联反应产物。然而，我们也证明，共价复合物可以进一步耦合到CCP和细胞色素C，形成更高分子量的聚集体。
Revised Manuscript Received December 18, 1986 abstract: The 1: 1 covalently cross-linked complex betweenhorse cytochrome c and yeast cytochrome c peroxidase (ccp) has been formed by a slight modification of the method of Waldmeyer and Bosshard [Waldmeyer, B., & Bosshard, H. R.(1985) J. Biol. Chem. 260, 5184-5190], This earlier study has been extended to show that efficient cross-linking of thetwo proteins can occur in a variety of buffers over a broad ionic strength range. The substitutionof ferrocytochrome c for ferricytochrome c in the cross-linking studies resulted in an increased yield of 1: 1 complex (~ 10-20%) under the conditions studied. An improved method for purifying the covalent complex in relatively large quantities is presented here as are the results of electrophoresis and proton NMR studies of the complex. Both electrophoresis and NMR studies indicate modification of some surface acidic amino acids in the covalent complex by the carbodiimide. The proton hyperfine-shifted resonances of cytochrome c are broadened in the covalent complex relative to free cytochrome c, and the resonances corresponding to thecytochrome c heme 3-CH3 and 8-CH3 groups are shifted closer together in the complex. Integration of NMR resonances confirms a 1: 1 complex as the primary cross-linking reaction product. However, we also demonstrate that the covalent complex can be further coupled to ccp and to cytochrome c to form higher molecular weight aggregates.