FORMATION OF NITRATING AND CHLORINATING SPECIES BY REACTION OF NITRITE WITH HYPOCHLOROUS ACID - A NOVEL MECHANISM FOR NITRIC OXIDE-MEDIATEDPROTEIN MODIFICATION

Detection of 3-nitrotyrosine has served as an in vivo marker for the p roduction of the cytotoxic species peroxynitrite (ONOO-). We show here that reaction of nitrite (NO2-), the autoxidation product of nitric o xide ((NO)-N-.), with hypochlorous acid (HOCl) forms reactive intermed iate species that are also capable of nitrating phenolic substrates su ch as tyrosine and 4-hydroxy-phenylacetic acid, with maximum yields ob tained at physiological pH. Monitoring the reaction of NO2- with HOCl by continuous flow photodiode array spectrophotometry indicates the fo rmation of a transient species with spectral characteristics similar t o those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO 2-/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO2-/HOCl and synthetic Cl-NO2. The r eaction of N-acetyl-L-tyrosine with NO2-/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are i nvolved in the reaction mechanism. Our data indicate that while chlori nation reactions of Cl-NO2 are mediated by direct electrophilic additi on to the aromatic ring, a free radical mechanism appears to be operat ive in nitrations mediated by NO,1 HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide ((NO2)-N-.) and tyrosyl radical. W e propose that NO2- reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and CI-NO2 as intermediates that modify tyrosine by either direct re action or after decomposition to r eactive free and solvent-caged Cl-. and (NO2)-N-. as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously un recognized mediators of inflammation-mediate protein modification and tissue injury, and offers an additional mechanism of tyrosine nitratio n independent of ONOO-.