细胞色素P-450侧链裂解酶与肾上腺皮质铁氧化还原蛋白之间相互作用的定量分析——通过二阶导数光谱法在紫外中值区域进行分析。

Quantitation of interaction between cytochrome P-450scc and adrenodoxin--analysis in the median UV-region by second derivative spectroscopy.

作者信息

Shkumatov V M, Smettan G, Ristau O, Rein H, Ruckpaul K, Chaschin V L, Akhrem A A

机构信息

Institute of Bioorganic Chemistry, Academy of Sciences of the BSSR, Minsk, U.S.S.R.

出版信息

Chem Biol Interact. 1988;68(1-2):71-83. doi: 10.1016/0009-2797(88)90007-5.

DOI:10.1016/0009-2797(88)90007-5

PMID:3203409

Abstract

Interaction between the essential protein components of the bovine adrenal mitochondrial enzyme system (cytochrome P-450scc, adrenodoxin and adrenodoxin reductase) were studied in the median UV-region utilizing second derivative difference spectroscopy. Complex formation of cytochrome P-450scc with adrenodoxin induces a signal in the second derivative difference spectrum which can be attributed to tyrosine due to its minimum at 283 nm. Based on this signal cytochrome P-450scc was titrated with adrenodoxin in dependence on different effectors (reductase, phospholipid, cholesterol). The dissociation constants (Kd) of the P-450scc/adrenodoxin complexes derived therefrom revealed an increasing affinity between both components starting from titrations in buffer solution without additional components up to the completely reconstituted system. A high affinity between P-450scc and adrenodoxin corresponds to a high turnover rate of cholesterol. Dissociation constants of the P-450scc/adrenodoxin complex were also derived from spectral changes in the Soret region. But these data do not correlate with the substrate turnover.

摘要

利用二阶导数差示光谱法，在中紫外区域研究了牛肾上腺线粒体酶系统（细胞色素P-450scc、肾上腺铁氧还蛋白和肾上腺铁氧还蛋白还原酶）的必需蛋白质成分之间的相互作用。细胞色素P-450scc与肾上腺铁氧还蛋白形成复合物会在二阶导数差示光谱中产生一个信号，该信号可归因于酪氨酸，因为其在283nm处有最小值。基于此信号，根据不同的效应物（还原酶、磷脂、胆固醇），用肾上腺铁氧还蛋白对细胞色素P-450scc进行滴定。由此得出的P-450scc/肾上腺铁氧还蛋白复合物的解离常数（Kd）显示，从无其他成分的缓冲溶液滴定到完全重组系统，两种成分之间的亲和力不断增加。P-450scc与肾上腺铁氧还蛋白之间的高亲和力对应着胆固醇的高周转率。P-450scc/肾上腺铁氧还蛋白复合物的解离常数也可从索雷特区域的光谱变化得出。但这些数据与底物周转率无关。