Peroxidase activity as a tool for studying the folding of c-type cytochromes
Peroxidase activity as a tool for studying the folding of c-type cytochromes
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DOI：
10.1021/bi0260841
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发表时间：
2002-10-29
期刊：
影响因子：
2.9
通讯作者：
Canters, GW
中科院分区：
文献类型：
--
作者：
Diederix, REM;Ubbink, M;Canters, GW
c型细胞色素的过氧化物酶活性通过解折叠而显著增加。利用这一现象研究了铁细胞色素C的平衡去折叠。过氧化物酶活性在低变性剂浓度下已经增强。最低的自由能折叠中间体很容易通过这种方法检测到，而它是不可见的使用荧光或光学光谱。这种折叠中间体和天然状态之间的自由能差取决于血红素铁的第六配体的强度，并且在展开时过氧化物酶活性的增加被证明是这种配体的强度的敏感指标。在完全变性条件下，过氧化物酶活性被基于蛋白质的配体抑制。结果表明，至少有三种不同的配体基团可以负责这种抑制，在中性或碱性pH值，主要的配体不是组氨酸。使用过氧化物酶活性测定作为一种方法来研究细胞色素c的展开进行评估。
The peroxidase activity of c-type cytochromes increases substantially by unfolding. This phenomenon was used to study the equilibrium unfolding of ferricytochrome c. The peroxidase activity is already enhanced at low denaturant concentrations. The lowest free energy folding intermediate is easily detected by this method, while it is invisible using fluorescence or optical spectroscopy. The free energy difference between this folding intermediate and the native state depends on the strength of the sixth ligand of the heme-iron and the increase in peroxidase activity upon unfolding is shown to be a sensitive indicator of the strength of this ligand. Under fully denaturing conditions, the peroxidase activity is inhibited by protein-based ligands. It is shown that at least three different ligand groups can be responsible for this inhibition, and that at neutral or alkaline pH, the predominant ligand is not histidine. The use of peroxidase activity assays as a method to study the unfolding of cytochrome c is evaluated.