Interactions of Hypochlorous Acid with Pyrimidine Nucleotides, and Secondary Reactions of Chlorinated Pyrimidines with GSH, NADH, and Other Substrates

doi:10.1006/abbi.1997.0440

Archives of Biochemistry and Biophysics

Volume 349, Issue 1, 1 January 1998, Pages 183-191

https://doi.org/10.1006/abbi.1997.0440 Get rights and content

Abstract

HOCl-induced chlorination of pyrimidine nucleotides, PyNH, strikingly depends on the nature of the available chlorine acceptor group. For CMP, with an −NH₂group as acceptor, the reaction is slow and involves predominantly the acid [k(CMP + HOCl) ≈ 100 M⁻¹s⁻¹at pH 6]; apparent rate constants of the reaction decrease around the pK_a(HOCl), to 0 in alkaline solution. For TMP and UMP, with a heterocyclic >NH group (at³N) as acceptor, the reaction is faster and involves mainly the conjugated ClO⁻anion [e.g.,k(UMP + ClO⁻) ≈ 3 × 10⁴M⁻¹s⁻¹andk(UMP + HOCl) ≈ 200 M⁻¹s⁻¹]. The 3-N-methylthymidine derivative is inert toward HOCl. Reactions of ClO⁻with TMP, UMP, and poly(U) are shown to be reversible, PyNH + ClO⁻⇌ PyNCl + OH⁻; an increase in pH due to this reaction was confirmed, and equilibrium constants have been estimated. The chlorinated derivatives of TMP and UMP are very reactive toward GSH, disulfide, aliphatic amines, and NADH. In contrast, the PyNCl derivative of CMP is unreactive, except with GSH. Rate constants of reactions of PyNCl species with various substrates are presented. Oxidation of NADH, by both HOCl and PyNCl derivatives, leads to a stable product (not NAD⁺) which is irreversibly degraded by reaction with excess HOCl, but inert toward acsorbate, GSH, and H₂O₂. Thiols (GSH) and disulfides (DTPA) were previously found capable of scavenging up to four HOCl molecules (Prütz, W. A.,Arch. Biochem. Biophys.332, 110–120, 1996). In the present study it was established that reactions of GSH or DTPA with excess HOCl give rise to a rapid drop in the pH by release of up to four HCl molecules per GSH or DTPA, as expected for a sequence of consecutive sulfoxidations. Reactions of GSH and DTPA with PyNCl efficiently regenerate PyNH, namely up to four molecules per GSH or DTPA in the case of TMP and UMP, but only one molecule per GSH in the case of CMP. The PyNCl derivatives of TMP and UMP transfer chlorine slowly but completely to CMP or AMP. Such chlorine transfer between nucleic acid bases is likely to occur also in DNA; it is shown that HOCl in fact induces a complex series of reactions on interaction with native DNA.

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HOCl reacts with the amino groups in amino acid side chains (e.g., Lys or His) as well as the α-amino group of free amino acids resulting in the formation of reactive N-chloramines (Pattison & Davies, 2001, 2005; Thomas, Grisham, & Jefferson, 1986). Chloramines preserve the oxidizing capacity of HOCl as they contain the moderately reactive nitrogen‑chlorine bond (RNHCl), which can mediate secondary oxidative and biological reactions including changes in endothelial signaling and function (Pattison & Davies, 2005; Prütz, 1996, 1998). For example, exposure of cultured endothelial cells to taurine chloramine activates ERK1/2 signaling via activation of the epidermal growth factor receptor independent of changes to the intracellular redox status, supporting initiation of signaling by taurine chloramine at the plasma membrane (Midwinter, Peskin, Vissers, & Winterbourn, 2004).
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These reactions are considerably slower than with HOCl (by 102 – 104) (Nagy, Jameson, & Winterbourn, 2009; Skaff et al., 2009)), and therefore HOSCN is less damaging overall than HOCl or HOBr, and the damage it induces is predominantly reversible, rather than irreversible (Barrett et al., 2012; Chandler, Nichols, Nick, Hondal, & Day, 2013). Reaction of HOCl and HOBr (and possibly also other hypohalous acids) with nitrogen atoms (e.g. those present on the the Lys side-chain, the guanidine group of Arg, the imidazole ring of His, the N-terminus of proteins/peptides/amino acids, and the amine groups of phospholipids and nucleobases) gives chloramines and bromamines, respectively [i.e. RNHCl and RNHBr species with weak N-X bonds] (Flemmig, Spalteholz, Schubert, Meier, & Arnhold, 2009; Hawkins & Davies, 2001; C.L. Hawkins & Davies, 2002; Kawai et al., 2006; Ohnishi, Murata, & Kawanishi, 2002; Prutz, 1998; Test, Lampert, Ossanna, Thoene, & Weiss, 1984). These species are also oxidants, but less powerful than HOCl and HOBr, and react more slowly (Pattison & Davies, 2006b; Peskin & Winterbourn, 2001; Peskin & Winterbourn, 2003, 2006), though this is structure-dependent, with some species showing considerable reactivity (e.g. His chloramines (Pattison & Davies, 2005, 2006a; Peskin & Winterbourn, 2003)) whereas others are only weakly reactive.
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^☆: Florkin, M.Stotz, E. H.

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Regular ArticleInteractions of Hypochlorous Acid with Pyrimidine Nucleotides, and Secondary Reactions of Chlorinated Pyrimidines with GSH, NADH, and Other Substrates☆

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Regular Article
Interactions of Hypochlorous Acid with Pyrimidine Nucleotides, and Secondary Reactions of Chlorinated Pyrimidines with GSH, NADH, and Other Substrates☆