Role of tryptophan oxidation in peroxynitrite-dependent protein chemiluminescence.
Bovine serum albumin oxidation by peroxynitrite is accompanied by chemiluminescence (Watts et al., Arch. Biochem. Biophys. 317, 324-330, 1995). Peak chemiluminescence during the reaction between bovine serum albumin (with or without fatty acids) and peroxynitrite was not modified in the presence of D2O, suggesting that light emission arising from lipid or protein oxidation was not the result of singlet oxygen formation. Light emission from fatty acid-free albumin increased in the presence of diphenylanthracene (DPA), suggesting that it is a consequence of the fluorescent decay of excited species. Exposure of individual amino acids to peroxynitrite in 50 mM potassium phosphate at pH 8.0 showed that tryptophan is the one that emits most light during oxidation, followed by phenylalanine. Tryptophan chemiluminescence correlated with oxygen consumption. The spin trap N-t-butyl-alpha-phenylnitrone (PBN) inhibited both oxygen consumption and chemiluminescence during tryptophan oxidation, suggesting that the reactions leading to light emission start with the abstraction of a H atom, forming a C-centered radical which in turn adds oxygen. When the oxidation of tryptophan by peroxynitrite was carried out in Tris-HCl instead of potassium phosphate, there was a second oxidative reaction between the peroxide and Tris. Chemiluminescence and oxygen consumption during the oxidation of tryptophan by peroxynitrite was 50% lower in the presence of Tris and in this case PBN did not inhibit chemiluminescence, suggesting that the new radicals formed during the reaction of Tris with peroxynitrite reacted with the amino acyl radicals inhibiting the formation of excited intermediates. Exposure of Tris but not phosphate to peroxynitrite (in the absence of amino acids) also resulted in light emission. In summary, these results suggest that tryptophan is one of the amino acids responsible for light emission during protein oxidation. In addition, this study confirms that Tris buffer is a target for strong oxidants and shows that its oxidation also is accompanied by light emission.
Pollet
E
E
Department of Biomedical Sciences, University of South Alabama, Mobile 36688, USA.
Martínez
J A
JA
Metha
B
B
Watts
B P
BP
Turrens
J F
JF
D000818:Animals; D002417:Cattle; D008163:Luminescent Measurements; D009566:Nitrates / Q000737:chemistry*; D010084:Oxidation-Reduction; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
D009566:Nitrates; C039642:peroxynitric acid; D012710:Serum Albumin, Bovine; D014364:Tryptophan