Recent studies indicate the formation of protein nitrosamines in vivo and tryptophan residues in proteins might represent important targets of nitrosative and oxidative stress. In the present work, we examined the mechanism by which xanthine oxidase (XO) denitrosates N-nitroso Trp residues and determined the applicability of the reactions involved to the detection of nitrosated Trp residues by tri-iodide-based chemiluminescence. We found that - in addition to superoxide - denitrosation of N-acetyl-nitroso Trp (NANT) by hypoxanthine and XO occurred via the intermediacy of uric acid. Zero-order dependence of NANT decay rate with uric acid was achieved with increasing concentrations of uric acid (k(0)∼6.0×10(-4)s(-1)) and generated nitric oxide. In contrast, S-nitrosoglutathione and nitrosyl-myoglobin were stable in the presence of uric acid. NANT decomposition by uric acid could be reproducibly measured using the tri-iodide-based chemiluminescence assay in the presence of excess nitrite upon pre-treatment with acidified sulfanilamide. N-nitrosated albumin was sensitive to uric acid-induced decomposition only after proteolytic degradation. In conclusion, XO decomposes nitrosated Trp through superoxide and uric acid pathways and in the case of uric acid generates free nitric oxide. Site-specificity of this reaction may possibly be used in combination with the tri-iodide-based chemiluminescence assay to discern between nitrosated Trp, S-nitrosothiols, and nitrosylated heme proteins.
Copyright © 2012 Elsevier Inc. All rights reserved.