Hypochlorous Acid Interactions with Thiols, Nucleotides, DNA, and Other Biological Substrates

Abstract

HOC1-induced one-electron oxidation of Fe(CN)(6)4- was used as a reference reaction to investigate the stoichiometry of interaction of HOCl with a variety of biological substrates. GSH and GSSG were both found capable of reacting with four and 2-mercaptoethanol with three HOCl molecules. Stopped-flow investigations, with HOCl in excess, indicate that very fast primary reactions of HOCl with GSH and DTPA are followed by slower secondary reactions. In the case of GSH we propose that one HOCl reacts at the terminal alpha-amino-group and three HOCl react at the -SH group to generate the sulfonylchloride GSO2Cl. This assignment is supported by the finding that reaction of HOCl (in excess) with 2-mercaptonaphthalene generates the absorption spectrum of authentic naphthalene-2-sulfonylchloride. NADH reacts with at least two HOCl molecules. A very fast primary reaction of HOCl was followed by a slower secondary reaction at HOCl/NADH > 2, but neither the primary nor the secondary reaction led to NAD+. Stopped-flow investigations of reactions of HOCl with nucleotides indicate that HOCl reacts slowly with the amino-groups of AMP, CMP, and GMP but very fast with the heterocyclic NH-groups of GMP, inosine, and TMP. AMP and CMP promote, but GMP, inosine, and TMP retard HOCl-induced oxidation of Fe(CN)(6)4-. At present we have no convincing evidence, however, that products of interaction of HOCl with nucleotides are capable of one-electron oxidation of Fe(CN)6(-4), with generation of free radical intermediates. HOCl causes slow but very efficient denaturation of native DNA, in our opinion not by oxidative fragmentation, but due to chlorination of amino- and heterocyclic NH-groups of the DNA-bases, which leads to dissociation of the double strand by the loss of hydrogen bonding. HOCl-induced oxidation of Fe(CN)(6)4- is promoted very efficiently by catalytic amounts of Cu2+. Catalysis is explainable by formation of a CuIFeIII(CN)(6)2- complex, with CuI acting as electron donor in a propagating Fenton-like reaction, CuIFeIII(CN)(6)2- +HOCl-->Cu2+ + Fe(CN)(6)3- + Cl- + OH, the rate constant of which was estimated as k = 1.8 x 10(5) M-1 s-1. HOCl is inactivated by Tris, but Hepes promotes HOCl-induced oxidation of Fe(CN)(6)4- very efficiently; this is a warning against application of such buffers in investigations of HOCl- or myeloperoxidase-induced reactions. Anthranilic acid was found to interact with four HOCl molecules to yield highly reactive (unidentified) one-electron oxidants.