Guo Qiong, Detweiler Charles D, Mason Ronald P
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle Park, NC 27709, USA.
J Biol Chem. 2004 Mar 26;279(13):13272-83. doi: 10.1074/jbc.M310034200. Epub 2004 Jan 14.
A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H(2)O(2). The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.
一种新型的抗5,5-二甲基-1-吡咯啉N-氧化物（DMPO）多克隆抗血清已被研制出来，它能特异性识别蛋白质自由基衍生的DMPO硝酮加合物。在本研究中，我们采用了这种结合自旋捕获特异性和抗原-抗体相互作用敏感性的新方法，来研究乳过氧化物酶（LPO）产生蛋白质自由基的情况。当LPO在DMPO存在下与谷胱甘肽（GSH）反应时，我们使用酶联免疫吸附测定法和蛋白质印迹法检测到了一种LPO自由基衍生的DMPO硝酮加合物。这种硝酮加合物的形成取决于GSH、LPO和DMPO的浓度以及pH值，并且H(2)O(2)不能替代GSH。叠氮化物、过氧化氢酶、抗坏血酸盐、碘化物、硫氰酸盐、苯酚或亚硝酸盐可显著降低这种硝酮加合物的水平。然而，LPO上的游离半胱氨酸、酪氨酸和色氨酸残基的化学修饰对其形成没有影响。电子顺磁共振（ESR）光谱表明，LPO/GSH/DMPO系统形成了谷胱甘肽硫自由基，但ESR检测不到蛋白质自由基加合物。叠氮化物、过氧化氢酶、抗坏血酸盐、碘化物或硫氰酸盐可降低其形成，而苯酚或亚硝酸盐则增加其形成。GSH导致LPO化合物II的光谱发生明显变化，表明GSH与化合物II的血红素结合，而苯酚或亚硝酸盐可阻止这些变化并将化合物II还原回天然酶。通过测量2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）氧化来确定，GSH还剂量依赖性地抑制LPO的过氧化物酶活性。综上所述，这些结果表明，GSH依赖性的LPO自由基形成是由谷胱甘肽硫自由基介导的，可能是通过谷胱甘肽硫自由基与化合物II的血红素反应形成一个被DMPO捕获以血红素为中心的自由基。
