Daniel R. Doerge

Oxygenation of organosulfur compounds by peroxidases: evidence of an electron transfer mechanism for lactoperoxidase

The oxygenation of benzyl methylsulfide, thioanisole, and thiobenzamide to the respective sulfoxides was found to be catalyzed by chloroperoxidase, lactoperoxidase, and horseradish peroxidase. The activities of lactoperoxidase and horseradish peroxidase were similarly low toward benzyl methylsulfide and thioanisole but lactoperoxidase efficiently catalyzed the oxygenation of thiobenzamide while horseradish peroxidase showed low activity. Chloroperoxidase had high reactivity toward all three substrates tested in halide-independent reactions and only small differences in the rates of enzymatic sulfoxidation were observed. The logarithm of lactoperoxidase activity was found to linearly correlate with the voltammetric peak potentials for oxidation of the three substrates tested. The results of this study are consistent with a one-electron transfer mechanism for lactoperoxidase-mediated sulfoxidation.

Particle beam/liquid chromatography/mass spectrometry of national pesticide survey analytes

The U.S. Environmental Protection Agency (US EPA) lists 126 polar, water-soluble compounds in the National Pesticide Survey (NPS) because of their potential for contaminating ground water. From this list, approximately 100 compounds were analyzed by particle beam liquid chromatography/mass spectrometry (PB/LC/MS). The fragmentation patterns of the electron impact (EI) spectra were generally comparable to reference EI spectra and were useful for analyte confirmations. A study of four different commercial nebulizers was performed to assess the effect on chromatographic integrity. In addition to the use of PB/LC/MS as a qualitative confirmation tool, its use as a selective LC detector was evaluated. Forty three of the NPS analytes showed sufficient sensitivity for detection of and spectrum generation from 100 ng or less. Using published concentration and cleanup methods, this sensitivity is sufficient for confirming the presence of these analytes in ground water at the 0.1 ppb level.

Covalent binding of 14C- and 35S-labeled thiocarbamides in rat hepatic microsomes.

The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition. Heat treatment of microsomes to inactivate the flavin-containing monooxygenase (FMO) decreased the observed binding. Equivalent amounts of [35S]- and [14C]MBI were covalently bound to hepatic microsomal proteins, suggesting retention of both the carbon and sulfur portions of the molecule in the MBI/protein adduct. Thiophilic reagents effected release of covalently bound [14C]- and [35S]MBI in equal amounts suggesting the presence of disulfide bonds between an MBI-derived sulfenic acid and microsomal protein thiols. Coincubation with bovine serum albumin (BSA) resulted in NADPH-dependent binding of [14C]-MBI to BSA sulfhydryls which was blocked by prior treatment of BSA with iodoacetamide. 1-Methyl-benzimidazole-2-thione (MMBI) also covalently bound to microsomal proteins and BSA but at levels lower than with MBI. P450, however, appeared to be more important than FMO in the metabolism of MMBI based on the effects of microsome heat pretreatment or ABT addition. In addition, ca. 1.5-fold more 35S- than 14C-label became bound. The covalent binding of [35S]1,3-dimethyl-benzimidazole-2-thione (DMMBI) to microsomal proteins was ca. six times greater than that of [14C]DMMBI. ABT, catalase and superoxide dismutase had a minimal effect on [35S]DMMBI binding, while FMO inactivation decreased binding by ca. 30%. These findings suggest that both monooxygenases contribute significantly to the hepatic metabolism of thiocarbamides. However, FMO activates thiocarbamides primarily to sulfenic acids, whereas P450 appears to produce both sulfenic acid and other reactive sulfur-derived metabolites. Thiol groups of P450 and other proteins are the molecular targets for these reactive species formed during the hepatic metabolism of anti-hyperthyroid drugs.

Application of semiempirical molecular orbital techniques to the study of peroxidase-mediated oxidation of phenols, anilines, sulfides and thiobenzamides

Abstract Reaction rates for a variety of enzymatically mediated oxidations were obtained and related to semiempirical (AMI) ionization potentials and molecular orbital structures. Highly significant correlations were generated for both vertical and adiabatic ionization potentials and horseradish peroxidase-mediated oxidation of substituted phenols, anilines and thioanisoles. The highest occupied molecular orbital for all substrates were clearly π in nature and associated with significant contributions by component heteroatoms. The results suggest that the oxidations considered proceed via an initial electron abstraction giving rise to a substrate radical or radical cation. Finally, theoretical approaches were used in concert with experimental paradigms to define the mechanism of lactoperoxidase-catalyzed oxidation of thiobenzamides. The sultoxidative process was shown to proceed via a tightly coupled oxygen transfer from the enzyme to the substrate in which the initial step involved electron loss from the thiobenzamide. In addition, the thiobenzamide S-oxide oxygen was found to be exclusively derived from the hydrogen peroxide and not from other sources.

SYNTHESIS OF N-SUBSTITUTED BENZIMIDAZOLE-2-THIONES

Abstract General methods were developed for N-substitution of benzimidazoline-2-thione with alkyl, alkenyl, alkynyl and acyl groups using S-tritylation as a protecting reaction.

Liquid chromatographic determination of ethylenethiourea using pulsed amperometric detection

A liquid chromatographic method was developed using pulsed amperometric detection at a gold working electrode to measure residue levels of ethylenethiourea (ETU) in crops and groundwater. Use of the sequential pulsing program eliminates electrode fouling while preserving the sensitive and selective detection of ETU. Minimum detection limits in crops were 5-10 ppb (1.25-2.5 ng on-column) and 5 ppb (0.5 ng) in groundwater. The commercial availability of the pulsed electrochemical detector and its gold working electrode that remains functional with a minimum of conditioning is an improvement in method simplicity.

Hydroperoxide-Dependent Metabolism of Goitrogenic Thiocarbamides by Lactoperoxidase

1. Inhibition of lactoperoxidase by thiocarbamides is consistent with a suicide mechanism whereby enzyme-catalysed S-oxygenation produces reactive intermediates which covalently modify the active site haem. 2. The reaction of thiocarbamide goitrogens with lactoperoxidase in the presence of hydroperoxides results in time-dependent and irreversible enzyme inactivation and an altered visible spectrum of the haem prosthetic group of the inactivated enzyme. 3. A mechanism of S-oxygenation for the inactivation is suggested by lactoperoxidase-catalysed formation of stable S-oxides from thioamide and organosulphur functional groups, and by a common dependence of substrate and inhibitor binding constants on their electrochemical oxidation potentials. 4. Hydroperoxide-dependent inactivation of lactoperoxidase by benzimidazoline-2-thiones occurs concomitantly to the covalent binding of stoichiometric amounts of 14C- or 35S-labelled inhibitors per mole of enzyme, and the formation of turnover products derived from the hydroperoxide cosubstrate and inhibitor.