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.
Ikeda
Keiichi
K
The Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Chiba 279-0021, Japan.
Yukihiro Hiraoka
B
B
Iwai
Hideaki
H
Matsumoto
Takashi
T
Mineki
Reiko
R
Taka
Hikari
H
Takamori
Kenji
K
Ogawa
Hideoki
H
Yamakura
Fumiyuki
F
D000818:Animals; D000906:Antibodies / Q000032:analysis; D000917:Antibody Formation; D000918:Antibody Specificity; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D016716:PC12 Cells; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D011506:Proteins; D030421:Peroxynitrous Acid; C055424:6-nitrotryptophan; D014364:Tryptophan