Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase
Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase
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DOI：
10.1021/bi972191x
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发表时间：
1997-12-02
期刊：
影响因子：
2.9
通讯作者：
Poole, LB
中科院分区：
文献类型：
--
作者：
Ellis, HR;Poole, LB
研究了鼠伤寒沙门氏菌AhpC过氧化物酶蛋白的完全活性C165 S突变体内次磺酸的捕获。在这些研究中使用的亲电试剂，7-氯-4-硝基苯-2-氧杂-1，3-二唑（NBD-Cl），以前已被用于修改蛋白质中的巯基，氨基和酪氨酸羟基;在中性pH值下，只有半胱氨酸残基的AhpC蛋白质被修改。与NBD-Cl孵育的AhpC的过氧化物氧化的C165 S突变体在347 nm处产生具有最大吸收的产物，而由还原的蛋白质形成的硫醇-NBD缀合物在420 nm处吸收最大。修饰蛋白质的电喷雾电离质谱允许鉴定在347 nm处吸收的物质作为相对于Cys-S-NBD产物含有一个额外氧的Cys-S（O）-NBD衍生物。当与未反应的C165 S和野生型AhpC相比时，具有Cys-S（O）-NBD和Cys-S-NBD的C165 S缀合物没有过氧化物酶活性，但两者都通过DTT去除NBD而被再活化。氧化的C165 S也被双甲酮（一种常见的次磺酸试剂）修饰，以得到预期的更高质量的灭活缀合物。该试剂未被DTT去除，并阻断了蛋白质与NBD-Cl的任何进一步反应。粪肠球菌NADH过氧化物酶的NBD修饰，一种具有活性位点次磺酸（Cys-SOH）的充分表征的黄素蛋白，也在NBD-Cl与变性过氧化物酶的氧化和还原形式孵育后产生光谱可区分的NBD缀合物，表明该试剂与其他含次磺酸蛋白的通用性。NBD-C1相对于以前使用的次磺酸试剂（如二甲酮）的显著优势在于保留了改性产物中的次磺酸氧;因此，现在可以通过NBD改性蛋白质的可见吸收特性和质量分析来区分蛋白质相关硫醇和次磺酸。
The trapping of a sulfenic acid within the fully active C165S mutant of the AhpC peroxidase protein from Salmonella typhimurium was investigated. The electrophilic reagent employed in these studies, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modify thiol, amino, and tyrosine hydroxyl groups in proteins; at neutral pH only cysteinyl residues of AhpC proteins are modified. The peroxide-oxidized C165S mutant of AhpC incubated with NBD-Cl gave a product with an absorbance maximum at 347 nm, whereas the thiol-NBD conjugate formed from the reduced protein absorbed maximally at 420 nm. Electrospray ionization mass spectrometry of the modified proteins allowed identification of the species absorbing at 347 nm as a Cys-S(O)-NBD derivative containing one additional oxygen relative to the Cys-S-NBD product. The C165S conjugates with Cys-S(O)-NBD and Cys-S-NBD had no peroxidase activity when compared to unreacted C165S and wild-type AhpC, but were both reactivated through removal of NBD by DTT. Oxidized C165S was also modified by dimedone, a common sulfenic acid reagent, to give the expected inactivated conjugate of higher mass. This reagent was not removed by DTT and blocked any further reaction of the protein with NBD-Cl. NBD modification of Enterococcus faecalis NADH peroxidase, a well-characterized flavoprotein with an active-site sulfenic acid (Cys-SOH), also yielded the spectrally-distinguishable NBD conjugates following incubation of NBD-Cl with oxidized and reduced forms of the denatured peroxidase, indicating a general utility for this reagent with other sulfenic acid-containing proteins. A significant advantage of NBD-C1 over previously-used sulfenic acid reagents such as dimedone is in the retention of the sulfenic acid oxygen in the modified product; differentiation between protein-associated thiols and sulfenic acids is therefore now possible by means of both visible absorbance properties and mass analyses of the NBD-modified proteins.