Detection of 6-nitrotryptophan in proteins by Western blot analysis and its application for peroxynitrite-treated PC12 cells.

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.

[The dynamic state of the concentrations of copper and histidine, and the complex effects of histidine and copper on the formation of lipid peroxidation in sera of undernourished young women].

OBJECTIVES: First, we observed that copper and histidine levels were increased in the sera of undernourished women. The objective of this study was to clarify the reason for the increase in copper and histidine. Furthermore, we tried to determine the compound(s) to which the increased copper was binding, and examined the effect of the increased copper and histidine on lipid peroxidation. METHODS: We investigated young women's diets and took blood samples, and the contents of histidine in the sera were determined by HPLC. The contents of copper were determined by flameless atomic absorption spectroscopy. The subjects were classified into three groups according to the concentration of histidine in the sera. The contents of copper and lipid peroxide in the sera were compared among the high histidine group and the other groups. We also examined the ability of the complexes to prevent LDL oxidation induced by copper, using an in vitro assay. RESULTS: The contents of lipid peroxide were lower in the high histidine group than in the other groups. Furthermore, the complexes of histidine and copper inhibited the formation of peroxidized lipids in an in vitro assay. CONCLUSIONS: These results indicate that histidine masks copper, reducing oxidation reaction. They also suggest that the complexes are suited for plasma antioxidation, preventing oxidative modification of lipids in the sera of undernourished women. The increased histidine appeared to be an effective trap for active oxygen.