Peroxyl Radical Is Produced upon the Interaction of Hypochlorite with tert-Butyl Hydroperoxide

O. M. Panasenko^1*, A. N. Osipov², A. V. Chekanov¹, J. Arnhold³, and V. I. Sergienko¹

¹Research Institute of Physico-Chemical Medicine, ul. Malaya Pirogovskaya 1a, Moscow, 119828 Russia; fax: (095) 246-4490; E-mail: oleg-panasenko@newmail.ru

²Russian State Medical University, ul. Ostrovityanova 1, Moscow, 117513 Russia; E-mail: an-osipov@mail.ru

³Institute of Medical Physics and Biophysics, University of Leipzig, Liebigstrasse 27, Leipzig, 04103, Germany; fax: (49-341) 971-5709; E-mail: arnj@server3.medizin.uni-leipzig.de

^* To whom correspondence should be addressed.

Received May 4, 2001; Revision received June 9, 2001

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As we reported previously, hypochlorite interacting with organic hydroperoxides causes their decomposition ((1995) Biochemistry (Moscow), 60, 1079-1086). This interaction was supposed to be a free-radical process and serve as a source of free radicals initiating lipid peroxidation (LP). The present study is the first attempt to detect and identify free radicals produced in the reaction of hypochlorite with tert-butyl hydroperoxide, (CH₃)₃COOH, which we have used as an example of organic hydroperoxides. We have used a direct method for free radical detection, EPR of spin trapping, and the following spin traps: N-tert-butyl-alpha-phenylnitrone (PBN) and alpha-(4-pyridyl-1-oxyl)-N-tert-butylnitrone (4-POBN). When hypochlorite was added to (CH₃)₃COOH in the presence of a spin trap, an EPR spectrum appeared representing a superposition of two signals. One of them belonged to a spin adduct formed as a result of direct interaction of hypochlorite with the spin trap (hyperfine splitting constants were: a^beta_H = 0.148 mT; a_N = 1.537 mT; and DeltaH_PP = 0.042 mT for 4-POBN and a^beta_H = 0.190 mT; a_N = 1.558 mT; and DeltaH_PP = 0.074 mT for PBN). The other signal was produced by hypochlorite interactions with (CH₃)₃COOH itself (hyperfine splitting constants were: a^beta_H = 0.233 mT; a_N = 1.484 mT; DeltaH_PP = 0.063 mT and a^beta_H = 0.360 mT; a_N = 1.547 mT; DeltaH_PP = 0.063 mT for 4-POBN and PBN, respectively). Comparison of spectral characteristics of this spin adduct with those of tert-butoxyl or tert-butyl peroxyl radicals produced in known reactions of (CH₃)₃COOH with Fe²⁺ and Ce⁴⁺, respectively, showed that the radical (CH₃)₃COO^· is produced from the interaction of hypochlorite with (CH₃)₃COOH. Like Ce⁴⁺ but not Fe²⁺, hypochlorite addition to (CH₃)₃COOH was accompanied by a bright flash of chemiluminescence characteristic of the reactions in which peroxyl radicals are produced. Thus, all these results suggest peroxyl radical production in the reaction of hypochlorite with hydroperoxide. This reaction is one of the most possible ways for the initiation of free-radical LP that occurs in vivo, when hypochlorite interacts with unsaturated lipids comprising natural protein-lipid complexes, such as lipoproteins and biological membranes.

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KEY WORDS: hypochlorite, hydroperoxide, tert-butyl hydroperoxide, free radicals, peroxyl radical, lipid peroxidation, spin traps

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