ROLE OF TRYPTOPHAN OXIDATION IN PEROXYNITRITE-DEPENDENT PROTEIN CHEMILUMINESCENCE

Bovine serum albumin oxidation by peroxynitrite is accompanied by chem iluminescence (Watts ef al., Arch. Biochem. Biophys. 317, 324-330, 199 5). Peak chemiluminescence during the reaction between bovine serum al bumin (with or without fatty acids) and peroxynitrite was not modified in the presence of D2O, suggesting that light emission arising from L ipid or protein oxidation was not the result of singlet oxygen formati on. Light emission from fatty acid-free albumin increased in the prese nce of diphenylanthracene (DPA), suggesting that it is a consequence o f the fluorescent decay of excited species. Exposure of individual ami no acids to peroxynitrite in 50 mM potassium phosphate at pH 8.0 showe d that tryptophan is the one that emits most light during oxidation, f ollowed by phenylalanine. Tryptophan chemiluminescence correlated with oxygen consumption. The spin trap N-t-butyl- alpha-phenylnitrone (PBN ) inhibited both oxygen consumption and chemiluminescence during trypt ophan oxidation, suggesting that the reactions leading to light emissi on start with the abstraction of a H atom, forming a C-centered radica l which in turn adds oxygen. When the oxidation of tryptophan by perox ynitrite was carried out in Tris-HCl instead of potassium phosphate, t here was a second oxidative reaction between the peroxide and Tris. Ch emiluminescence and oxygen consumption during the oxidation of tryptop han by peroxynitrite was 50% lower in the presence of Tris and in this case PEN did not inhibit chemiluminescence, suggesting that the new r adicals formed during the reaction of Tris with peroxynitrite reacted with the amino acyl radicals inhibiting the formation of excited inter mediates. Exposure of Tris but not phosphate to peroxynitrite (in the absence of amino acids) also resulted in light emission. In summary, t hese results suggest that tryptophan is one of the amino acids respons ible for light emission during protein oxidation. in addition, this st udy confirms that Tris buffer is a target for strong oxidants and show s that its oxidation also is accompanied by light emission. (C) 1998 A cademic Press.