Ikeda Keiichi, Yukihiro Hiraoka B, Iwai Hideaki, Matsumoto Takashi, Mineki Reiko, Taka Hikari, Takamori Kenji, Ogawa Hideoki, Yamakura Fumiyuki
The Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Chiba 279-0021, Japan.
Nitric Oxide. 2007 Feb;16(1):18-28. doi: 10.1016/j.niox.2006.04.263. Epub 2006 May 4.
We have previously reported on the formation of 6-nitrotryptophan by the reaction of reactive nitrogen species with a tryptophan residue in human Cu, Zn-superoxide dismutase (SOD) (F. Yamakura et al., J. Biochem. 138 (2005) 57-69). Here, we report on the preparation of anti-6-nitrotryptophan antiserum by using synthesized 6-nitrotryptophan-conjugated keyhole limpet hemocyanin as an antigen and the purification of the antibody by using a 6-nitrotryptophan-conjugated affinity column. The purified antibody was immunoreactive with 6-nitrotryptophan residue containing Cu, Zn-SOD but not immunoreactive with Cu, Zn-SOD, Mn-SOD, bovine serum albumin, and 3-nitrotyrosine residue containing Mn-SOD. Nitro group of 6-nitrotryptophan was reduced by sodium hydrosulfite to form 6-aminotryptophan as a major product. The reduced 6-nitrotryptophan residues lost its immunoreactivity with the antibody. We detected different immunoreactive bands between using antibody for 6-nitrotryptophan residues and that for 3-nitrotyrosine residues in crude extracts of neuron-like PC12 cells treated with peroxynitrite by a Western blot analysis. Western blot analysis for two-dimensional gel electrophoresis showed nine intensively stained immunoreactive spots for 6-nitrotryptophan residues in the peroxynitrite-treated PC12 cells, which were subjected to trypsin digestion and LC-ESI-MS/MS analysis. We identified M2 pyruvate kinase, elongation factor 2, mitochondrial aconitase, pyruvate carboxylase, and heat shock protein HSP90alpha as candidates for 6-nitrotryptophan residues containing proteins, with peptide coverage over 10%, in crude extracts of peroxynitrite-treated PC12 cells.
我们之前曾报道过，活性氮物种与人铜锌超氧化物歧化酶（SOD）中的色氨酸残基反应会形成6-硝基色氨酸（F. Yamakura等人，《生物化学杂志》138 (2005) 57-69）。在此，我们报告了以合成的6-硝基色氨酸偶联的钥孔血蓝蛋白作为抗原制备抗6-硝基色氨酸抗血清，以及使用6-硝基色氨酸偶联的亲和柱纯化抗体的过程。纯化后的抗体与含6-硝基色氨酸残基的铜锌SOD具有免疫反应性，但与铜锌SOD、锰SOD、牛血清白蛋白以及含3-硝基酪氨酸残基的锰SOD无免疫反应性。6-硝基色氨酸的硝基被连二亚硫酸钠还原，主要产物为6-氨基色氨酸。还原后的6-硝基色氨酸残基失去了与抗体的免疫反应性。通过蛋白质印迹分析，我们在经过氧亚硝酸盐处理的神经元样PC12细胞粗提物中检测到了针对6-硝基色氨酸残基的抗体和针对3-硝基酪氨酸残基的抗体之间不同的免疫反应条带。对二维凝胶电泳进行的蛋白质印迹分析显示，在经过氧亚硝酸盐处理的PC12细胞中，有9个针对6-硝基色氨酸残基的强烈染色免疫反应斑点，这些斑点经过胰蛋白酶消化和液相色谱-电喷雾电离串联质谱（LC-ESI-MS/MS）分析。我们确定M2丙酮酸激酶、延伸因子2、线粒体乌头酸酶、丙酮酸羧化酶和热休克蛋白HSP90α为经过氧亚硝酸盐处理的PC12细胞粗提物中含6-硝基色氨酸残基蛋白质的候选物，其肽段覆盖率超过10%。
