Chlorination Of Monochlorodimedone Research Articles

Microbicidal Activity of Vascular Peroxidase 1 in Human Plasma via Generation of Hypochlorous Acid

Members of the heme peroxidase family play an important role in host defense. Myeloperoxidase (MPO) is expressed in phagocytes and is the only animal heme peroxidase previously reported to be capable of using chloride ion as a substrate to form the highly microbicidal species hypochlorous acid (HOCl) at neutral pH. Despite the potent bacterial killing activity of HOCl, individuals who fail to express MPO typically show only a modest increase in some fungal infections. This may point to the existence of redundant host defense mechanisms. Vascular peroxidase 1 (VPO1) is newly discovered member of the heme peroxidase family. VPO1 is expressed in cells of the cardiovascular system and is secreted into the bloodstream. In the present study, we investigate whether VPO1 is capable of generating HOCl and its role in host defense. Like MPO, VPO1 in the presence of H₂O₂ and chloride generates HOCl. VPO1-dependent HOCl generation was demonstrated by chlorination of taurine and tyrosine using mass spectrometry. In addition, the VPO1/H₂O₂/Cl⁻ system can cause the chlorination of monochlorodimedone and the oxidation of 5-thio-2-nitrobenzoic acid. Purified VPO1 and VPO1 in plasma mediate bacterial killing that is dependent on chloride and H₂O₂; killing is inhibited by peroxidase inhibitors and by the H₂O₂ scavenger catalase. In the presence of erythrocytes, bacterial killing by VPO1 is slightly reduced. Thus, VPO1, in addition to MPO, is the second member of the heme peroxidase family capable of generating HOCl under physiological conditions. VPO1 is likely to participate in host defense, with bactericidal activity mediated through the generation of HOCl.

Enhanced catalytic performance and stability of chloroperoxidase from Caldariomyces fumago in surfactant free ternary water–organic solvent systems

Surfactant free ternary systems formulated with organic solvents such as α-pinene or n-hexane, short chain alcohols and water, were used as new reaction media to maximize the catalytic performance of chloroperoxidase from Caldariomyces fumago (CPO) towards the oxidation of hydrophobic compounds including olefins and terpenols. The influence of the composition and the hydration state of the system, the nature of the oxidizing agent as well as substrate availability on the catalytic behavior and stability of the enzyme was investigated, using the chlorination of monochlorodimedone (MCD) and the epoxidation of styrene as model reactions. Based on residual chlorination activity measurements, which were in accordance with differential scanning calorimetry (DSC) findings, it was concluded that in low water systems formulated with α-pinene CPO stability is significantly increased compared to other reaction media (up to 65% residual activity after 24 h of incubation at 30 °C). Electron paramagnetic resonance (EPR) spectroscopy indicated that in low water content systems the ferric environment of the active site of CPO does not undergo severe modifications. Total turnover numbers (TTN) obtained for the oxidation of various olefins and terpenols in α-pinene-based systems, were up to 25,000 which is 2–170 times higher than previously reported values. The results indicate that such ternary systems can be efficiently employed as reaction media leading to highly enhanced stability and increase CPO potential as practical catalyst for oxidation of large amounts of hydrophobic compounds in a single-step biocatalytic process.

Chlorination of Monochlorodimedone with Chloramines. II. Chlorination Rate Constants for Chlorinated Nitrogenous Compounds.

N-Chloro nitrogenous compounds were formed by addition of HOCl to an aqueous solution of ammonia, various amines, imines, amides, imides, and amino acids. The chlorination reactivities of these N-chloro compounds were evaluated with monochlorodimedone (MCD) chlorination rate constants, (kinetics was defined in a previous paper), at pH 7 and at room temperature. The rate constants varied with the structure of the nitrogenous compounds and with the number of chlorine atoms (Cl+) combined with the nitrogen atom in the compound. The rate constants (min-1) for chlorinated secondary amines such as dimethylamine, diethylamine and N-methylglycine (0.338-1.540) were higher than those for monochlorinated primary amines methylamine, ethylamine, and glycine (0.169-0.464). The rate constants for strong base imines like piperazine and morpholine (0.025 and 0.031, respectively) were much lower than those for weak base imines like piperidine and piperazinedione (0.924 and 0.441, respectively). The rate constants for these monochlorinated primary amines (0.123-0.464) were higher than those for dichlorinated primary amines (0.061-0.087). In ammonia chloramines, the rate constant of NH2Cl (0.027) at 12°C was about ten times higher than that of NHCl2 (0.0025) at 12°C.

A chloroperoxidase-like catalyst in soil: Detection and characterization of some properties

The objectives of the present study were: (i) to evaluate methods for detection of low rates of chloroperoxidase activity in aqueous solutions; (ii) to investigate the ability of soil extracts to catalyze chlorination; (iii) to investigate if the catalyzing ability of soil extracts is due to enzymatic activity. Initially, solutions of a commercially-available chloroperoxidase (EC 1.11.1.10, CPO) were studied, and it was found that chlorination of monochlorodimedone (MCD) and anisole followed by gas chomatographic analysis of chlorinated reaction products represented sensitive assay methods for determination of chloroperoxidase activity. Using these methods, eight out of nine soil extracts, obtained by performing a modified peroxidase extraction procedure, were found to catalyze chlorination. Further experimentation provided the following information: (i) the reaction did not proceed in the absence of hydrogen peroxide; (ii) the catalyst had a molecular weight larger than 10,000; (iii) the incorporation of chlorine was strongly pH-dependent, with an optimum at pH 3.0–3.5; (iv) the reaction was inhibited by orcinol, resorcinol and phloroglucinol. All these properties are similar to those of the commercial chloroperoxidase. It was therefore concluded that a chloroperoxidase-like catalyst was present in the investigated soils.

Chlorination of Monochlorodimedone with Chloramines. I. Kinetics of the Chlorination.

The kinetics of monochlorodimedone (MCD) chlorination with sodium hypochlorite (NaOCl), chloroisocyanurates (2Cl-and 3Cl-ICA), N-chlorosuccinimide (Cl-succinimide), and chloramine T and B were studied. The chlorination of MCD was recognized by a decrease in absorbance at 290 nm and at pH 7. Instantaneous chlorinations occurred with NaOCl and chloroisocyanurates. The rate constants for the chlorination with Cl-succinimide and chloramine T and B were determined. The reactions were determined to be second order-first order in each reactant, and were dependent on temperature. The observed second order rate constants at 15°C for Cl-succinimide, and at 25°C for chloramine T and B were 1500, 61.8 and 41.5 M-1·S-1, respectively.

The chlorinating activity of human myeloperoxidase: high initial activity at neutral pH value and activation by electron donors

The steady-state activity of myeloperoxidase in the chlorination of monochlorodimedone at neutral pH was investigated. Using a stopped-flow spectrophotometer we were able to show that the enzymic activity at pH 7.2 rapidly declined in time. During the first 50–100 ms after addition of H2O2 to the enzyme, a turnover number of about 320 s−1 per haem was observed. However, this activity decreased rapidly to a value of about 25 s−1 after 1 s. This shows that in classical steady-state activity measurements, the real activity of the enzyme at neutral pH is grossly underestimated. By following the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity was probably caused by the formation of an enzymically inactive form of the enzyme, Compound II. As demonstrated before (Bolscher, B.G.J.M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1984) Biochim. Biophys. Acta 784, 189–191) reductants such as ascorbic acid and ferrocyanide convert Compound II, which accumulates during turnover, into active myeloperoxidase. Activity measurements in the presence of ascorbic acid showed, indeed, that the moderate enzymic activity was higher than in the absence of ascorbic acid. With 5-aminosalicylic acid present, however, the myeloperoxidase activity remained at a much higher level, namely about 150 s−1 per haem during the time interval from 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it became clear that in the presence of 5-aminosalicylic acid the initially formed Compound II was rapidly converted back to native enzyme. Presteady-state experiments showed that 5-aminosalicylic acid reacted with Compound II with a K2 of 3.2·105 M−1·s−1, whereas for ascorbic acid a K2 of 1.5·104 M−1·s−1 was measured at pH 7.2. In the presence of 5-aminosalicylic acid during the time interval in which the myeloperoxidase activity remained constant, a Km for H2O2 at pH 7.2 was determined of about 30 μM at 200 mM chloride. In the absence of reductants the same value was found during the first 100 ms after addition of H2O2 to the enzyme. The physiological consequences of these findings are discussed.

The mechanism of myeloperoxidase-catalysed oxidation of aminopyrine

1. Myeloperoxidase catalysed the H2O2-supported oxidation of aminopyrine in the presence of Cl-, generating the aminopyrine cation radical (AP+.). The rate of AP+. formation was determined by monitoring the absorbance at 565 nm. The pH optimum of the reaction was around 5.0. The rate of AP+. formation increased with increasing concentration of aminopyrine. Inhibition by excess H2O2 was seen at pH 4.5-5.5. 2. When Cl- was replaced by Br-, the rate of AP+. formation increased significantly, and inhibition by H2O2 became less evident and was observed only at pH 5.5. The rate of chemical oxidation of aminopyrine by HOCl was much slower than that by Br2. 3. Compared with the chlorination of monochlorodimedone (MCD), the reaction between aminopyrine and HOCl, produced by the enzymic peroxidation of Cl-, is rate-limiting in the myeloperoxidase-catalysed oxidation of aminopyrine. The differences in kinetic behaviour between the myeloperoxidase-catalysed chlorination of MCD and oxidation of aminopyrine are explained by the low reactivity of HOCl towards aminopyrine.

Chemical reactivities of bleomycin.

Ferric bleomycin was tested for its ability to catalyze a set of six oxidative reactions characteristic of the heme-containing proteins, cytochrome P-450 and chloroperoxidase. These reactions included peroxyacid decarboxylation and aliphatic hydroxylation as typical cytochrome P-450 chemistries. Peroxyacid-supported oxygen evolution and hydrogen peroxide-mediated chlorination were utilized as characteristic chloroperoxidase reactivities. A typical peroxidative reaction and heteroatom dealkylation, common to both O2 activating enzymes, were also studied. Bleomycin was found to catalyze peroxidation of o-dianisidine. The ferric drug complex was found competent in carrying out N-demethylation of N,N-dimethylaniline when peroxides or peroxyacids or iodosobenzene were used as the oxidants. N-Demethylation was not achieved when N,N-dimethylaniline-N-oxide was substituted as the oxidant under similar conditions. Hydroxylation of cumene and decarboxylation of phenylperacetic acid were not found to be catalyzed by bleomycin. Oxygen evolution from m-chloroperbenzoic acid and chlorination of monochlorodimedone from chloride ion and hydrogen peroxide were found to be catalyzed by bleomycin. Cytochrome P-450cam was also evaluated for O2 evolution, and halogenation activity and was found not to demonstrate such reactivities. The results of this initial survey, along with those of previous studies, appear to indicate that the chemical reactivity of bleomycin can be more closely aligned with the reactivities demonstrated by chloroperoxidase than those of cytochrome P-450.