Tyrosine Nitration by Simultaneous Generation of ⋅NO and O⨪2 under Physiological Conditions

Abstract

Radiation chemical experiments demonstrate that the reaction of tyrosyl radical (TyrO ⅐ ) with ⅐ NO 2 yields 45 ؎ 3% 3-nitrotyrosine and that a major product of the reaction of TyrO ⅐ with ⅐ NO is 3,3-dityrosine. Radiolysis was used to generate ⅐ NO and O 2 . in the presence of tyrosine and bicarbonate at pH 7.5 ؎ 0.1. The nitration yield was found to be dose rate-dependent, and the yield per radical produced by pulse radiolysis was identical to that obtained with authentic peroxynitrite. .(1, 2), and this reaction is currently accepted as the main biological source of peroxynitrite (3). Peroxynitrite has been shown to nitrate tyrosine and tyrosine residues in proteins under physiological conditions (3-8), and the nitration yield increases in the presence of bicarbonate (7,8). 3-Nitrotyrosine (3-NT) 1 was found in human tissues and fluids and in animal models and cellular models of disease, and it appears to be associated with enzymatically produced ⅐ NO (9). Therefore, it has been suggested that peroxynitrite can operate as a nitrating agent in vivo (3, 9). In many cases the evidence that implicates ONOO Ϫ in a large variety of diseases is the detection of 3-NT in the injured tissues (3, 9 -14). However, it has been recently shown (15) that the in vitro nitration yield of tyrosine at pH 7.4 following the simultaneous generation of ⅐ NO and O 2. was considerably lower than that obtained upon bolus addition of peroxynitrite. Furthermore, addition of bicarbonate to the former system had hardly any effect on the nitration yield (15), whereas it doubled the yield in the latter case (7,8). Therefore, it has been suggested (15) that the reaction of ⅐ NO with O 2 . forms a species different from the one present in alkaline bolus additions, although flash photolysis (1) and pulse radiolysis (2) studies have demonstrated the formation of peroxynitrite via the combination of these radicals. Nevertheless, these experimental results (15) seem to present a problem for the hypothesis that 3-NT is a product of ONOO Ϫ in biological systems, given that the main biological source of ONOO Ϫ is the reaction of ⅐ NO with O 2 . .In the present study we used radiolysis to generate ⅐ NO and O 2. in the presence of tyrosine and bicarbonate at pH 7.5. Pulse radiolysis mimics bolus addition of peroxynitrite, whereas with ␥-radiolysis the radicals are continuously formed and the flux is determined by the dose rate. We shall show that maximum nitration yield is obtained for essentially equal fluxes of O 2 . and ⅐ NO and that the yield decreases upon decreasing the flux of these radicals. The biological implications of these observations will be discussed. EXPERIMENTAL PROCEDURESChemicals-All chemicals were purchased from Sigma and were used as received. Solutions were prepared with distilled water, which was further purified using a Milli-Q water purification system. Peroxynitrite was prepared by having nitrite react with acidified hydrogen peroxide at room temperature in a quenched flow system (16). The yield of ONOO Ϫ was determined from ...

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