Yamakura Fumiyuki, Matsumoto Takashi, Ikeda Keiichi, Taka Hikari, Fujimura Tsutomu, Murayama Kimie, Watanabe Eiji, Tamaki Makoto, Imai Takeo, Takamori Kenji
Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba 270-1695.
J Biochem. 2005 Jul;138(1):57-69. doi: 10.1093/jb/mvi095.
We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.
我们之前报道过，人铜锌超氧化物歧化酶中的单个色氨酸残基Trp32会被过氧亚硝酸根 - 二氧化碳特异性修饰[Yamakura等人（2001年），《生物化学与生物物理学报》1548卷，第38 - 46页]。在本研究中，我们使用髓过氧化物酶、过氧化氢和亚硝酸盐的组合对铜锌超氧化物歧化酶进行修饰。修饰后的酶没有铜和锌的损失，酶活性降低了15%。Trp32是唯一显著损失的氨基酸。在用过氧亚硝酸根 - 二氧化碳和髓过氧化物酶系统修饰超氧化物歧化酶后进行胰蛋白酶消化，在微液相色谱 - 电喷雾 - 四极杆 - 飞行时间质谱分析以及配备光电二极管阵列检测器的高效液相色谱中观察到六个新出现的含色氨酸衍生物的肽段。色氨酸残基的衍生物质量增加4、16（两个峰）(、32、45)（主要）和45道尔顿（次要），分别被鉴定为犬尿氨酸、羟吲哚 - 3 -丙氨酸及其衍生物、二羟基色氨酸、6 -硝基色氨酸和5 -硝基色氨酸。我们进一步从链霉蛋白酶消化产物的(^1H)核磁共振谱中鉴定出6 -硝基色氨酸，并计算出每种修饰方法中6 -硝基色氨酸的产率约为(30%)。在两种不同的修饰系统中，修饰后的人铜锌超氧化物歧化酶中的色氨酸残基产生的产物光谱相同，其中6 -硝基色氨酸是主要的硝化产物。
