Peroxidase-catalyzed halide ion oxidation

Abstract

The first complete mechanistic analysis of halide ion oxidation by a peroxidase was that of iodide oxidation by horseradish peroxidase. It was shown conclusively that a two-electron oxidation of iodide by compound I was occurring. This implied that oxygen atom transfer was occurring from compound I to iodide, forming hypoiodous acid, HOI. Searches were conducted for other two-electron oxidations. It was found that sulfite was oxidized by a two-electron mechanism. Nitrite and sulfoxides were not. If a competing substrate reduces some compound I to compound II by the usual one-electron route, then compound II will compete for available halide. Thus compound II oxidizes iodide to an iodine atom, I^·, although at a slower rate than oxidation of I^- by compound I. An early hint that mammalian peroxidases were designed for halide ion oxidation was obtained in the reaction of lactoperoxidase compound II with iodide. The reaction was accelerated by excess iodide, indicating a co-operative effect. Among the heme peroxidases, only chloroperoxidase (for example from Caldariomyces fumago) and mammalian myeloperoxidase are able to oxidize chloride ion. There is not yet a consensus as to whether the chlorinating agent produced in a peroxidase-catalyzed reaction is hypochlorous acid (HOCl), enzyme-bound hypochlorous acid (either Fe–HOCl or X–HOCl where X is an amino acid residue), or molecular chlorine Cl₂. A study of the non-enzymatic iodination of tyrosine showed that the iodinating reagent was either HOI or I₂. It was impossible to tell which species because of the equilibria:

I₂+H₂O=HOI+I^-+H⁺</ p>

I^-+I₂=I₃^-

The same considerations apply to product analysis of an enzyme-catalyzed reaction. Detection of molecular chlorine Cl₂ does not prove it is the chlorinating species. If Cl₂ is in equilibrium with HOCl then one cannot tell which (if either) is the chlorinating reagent. Examples will be shown of evidence that peroxidase-bound hypochlorous acid is the chlorinating agent. Also a recent clarification of the mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride along with accurate determination of the elementary rate constants will be discussed.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

Abstract

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl^-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1×10⁷ M^-1s^-1 (pH 5), 2.0×10⁸ M^-1s^-1 (pH 7) and 2.0×10⁶ M^-1s^-1 (pH 9) at 15°C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H₂O₂, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

Abstract

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (k_I 2.1×10⁶ M^-1s^-1), it reacts sluggishly with compound II (k_II 7 M^-1s^-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I₅₀) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I₅₀ 80 µM) unless superoxide dismutase was present (I₅₀ 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

Engineering analysis of continuous production of L-aspartic acid by immobilized Escherichia coli cells in fixed beds

The reaction mechanism and decay behavior of aspartase activity for immobilized Escherichia coli cells were investigated by using a sectional packed column. Reaction within the immobilized cell column proceeded at zero-order on substrate solutions ranging in concentration from 0.1 to 1.0M, and the initial reaction rate was found to be 1.556 × 10^?2 mol/min/liter of immobilized cells. The effect of temperature on the reaction rate constant was investigated. The Arrhenius plot was straight line at temperatures below 43°C, and the activation energy for immobilized cells was calculated to be 12.36 kcal/mol. Asparatase activity in the immobilized cell column decayed exponentially and uniformly in all sections of a column. Its half-life was approximately 120 days. The rate of formation of L-aspartic acid was shown to be independent of column dimensions.     

Inhibition of the α-Ketoglutarate Dehydrogenase Complex from Bakers’ Yeast by Acetaldehyde and Glyoxylate

Gallic acid, methyl gallate, dehydrodigallic acid, three tannic constituents named MP–2, MP–3, MP–4 and a related substance MP–10 were isolated from chestnut galls by solvent fractionation and column chromatography. Hydrolysis with tannase revealed the components of these tannic substances as follows, MP–2: d-glucose, gallic acid and compound I (3,4, 5-trihydroxybenzyl alcohol); MP–3 and MP–4: d-glucose, compound I and compound II (dehydrodigallic acid); MP–10: d-glucose and compound I.     

Nucleic acid of flacherie viruses of the silkworm, Bombyx mori

It was reported previously that two spherical flacherie viruses of silkworm, FVS I and FVS II, had been isolated from flacherie silkworm larvae and the nucleic acid of FVS II was RNA as suggested by the experiments of incorporation of [³H]-uracil. In this paper, it has been confirmed by biochemical methods that the nucleic acid of FVS I and FVS II is RNA. FVS I and FVS II were labeled with ³²P in flacherie silkworms, and the viruses were analyzed by sucrose density gradient centrifugation. When the ³²P-labeled compound in the viruses was treated with 0.5 n KOH, the acid-insoluble ³²P-labeled compound changed to acid-soluble compounds. It was determined by paper chromatography and ion-exchange column chromatography that the alkali-decomposed compounds included four ribonucleotides. Therefore, the viral nucleic acid of FVS I and FVS II was determined to be RNA. The correlations between FVS I and FVS II particles were discussed, and it was suggested that FVS I and FVS II might be closely related or were the same viral species.     

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine in normal human urine

Summary A compound, which had the same mobility on a high-voltage paper electrophoretogram and the sameR _F value on a thin-layer chromatogram as those ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (I), was partially purified from human urine by ion-exchange column chromatography. The compound gave a signal at m/z 260 on its FAB mass spectrum, which was assigned as MH⁺ of compound I. These results suggest that the urinary compound is compound I and it is a physiological precursor of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid [Kinuta et al., (1991) Biochem J 275: 617–621].     

Structure of Xanthocidin

A burnt flavoring compound, which imparts to aged sake its characteristic and dominant flavor, was isolated by Diaion HP–20, silicic acid and Dowex 1–X8 (CH₃COO^?) column chromatography and chloroform extraction. Based on thin-layer and gas liquid chromatography, UV and GC–MS spectral data, it was identified as 3-hydroxy-4,5-dimethyl-2 (5H)-furanone and its structure was also confirmed by synthesis. It was suggested that this compound was formed by the condensation of a-ketobutyric acid with acetaldehyde which occurred from degradation of threonine.     

The Constituents of Petasites japonicus: Structures of Fukiic Acid and Fukinolic Acid

A major polyphenol from P. japonicus, named fukinolic acid (XII), was isolated and its structure was investigated. Fukinolic acid afforded a new polyphenol named fukiic acid (I) and caffeic acid on hydrolysis in molar ratio 1:1. The structure of fukiic acid was established as 2,3-dihydroxy-4-(3′,4′-dihydroxyphenyl)-3-carboxybutyric acid (I), and its methyl derivatives were described.

Based on evidences presented, the structure for fukinolic acid was proposed as XII. It was further demonstrated that fukinolic acid caused the brown discoloration on action of a polyphenoloxidase from the plant tissues.     

Peptide Analogues of DNA Consisting of l-α-Amino-γ-thymine Butyric Acid and l-Valine Subunits

Abstract

Reaction of N-Boc-L-homoserine benzylester with N³ -benzoylthymine under Mitsunobu conditions afforded N-Boc-L-α-amino-γ-N³ -benzoylthymine butyric acid benzylester. After removal of the N-benzoyl and O-benzyl protecting group, this compound was used in solution phase peptide synthesis.     

Oxidation of hydroquinone, 2,3-dimethylhydroquinone and 2,3,5-trimethylhydroquinone by human myeloperoxidase

Abstract

Myeloperoxidase is very susceptible to reducing radicals because the reduction potential of the ferric/ferrous redox couple is much higher compared with other peroxidases. Semiquinone radicals are known to reduce heme proteins. Therefore, the kinetics and spectra of the reactions of p-hydroquinone, 2,3-dimethylhydroquinone and 2,3,5-trimethylhydroquinone with compounds I and II were investi-gated using both sequential-mixing stopped-flow techniques and conventional spectrophotometric measurements. At pH 7 and 15°C the rate constants for compound I reacting with p-hydroquinone, 2,3-dimethylhydroquinone and 2,3,5-trimethylhydroquinone were determined to be 5.6±0.4×10⁷ M^-1s^-1, 1.3±0.1×10⁶ M^-1s^-1 and 3.1±0.3×10⁶ M^-1s^-1, respectively. The corresponding reaction rates for compound II reduction were calculated to be 4.5±0.3×10⁶ M^-1s^-1, 1.9±0.1×10⁵ M^-1s^-1 and 4.5±0.2×10⁴ M^-1s^-1, respectively. Semiquinone radicals, produced by compounds I and II in the classical peroxidation cycle, promote compound III (oxymyeloperoxidase) formation. We could monitor formation of ferrous myeloperoxidase as well as its direct transition to compound III by addition of molecular oxygen. Formation of ferrous myeloperoxidase is shown to depend strongly on the reduction potential of the corresponding redox couple benzoquinone/semiquinone. With 2,3-dimethylhydroquinone and 2,3,5-trimethylhydroquinone as substrate, myeloperoxidase is extremely quickly trapped as compound III. These MPO-typical features could have potential in designing specific drugs which inhibit the production of hypochlorous acid and consequently attenuate inflammatory tissue damage.     

Metabonomics with 1H-NMR spectroscopy and liquid chromatography-mass spectrometry applied to the investigation of metabolic changes caused by gentamicin-induced nephrotoxicity in the rat

Abstract

The model nephrotoxin gentamicin was administered to male Wistar-derived rats daily, for 7 days, at 60?mg?kg^?1?day^?1, subcutaneously, twice daily. Conventional clinical chemistry urinalysis showed a significant increase in N-acetyl-β-D-glucosaminidase (NAG) activity from day 3. At necropsy on day 9, clear histological damage to the kidney was noted with all animals showing a generally severe nephropathy primarily focused on the proximal convoluted tubules. The urinary excretion pattern of endogenous metabolites over the time course of the study was studied using a combination of ¹H-NMR spectroscopy and HPLC-TOF-MS/MS using electrospray ionization (ESI). Changes in the pattern of endogenous metabolites as a result of daily administration of gentamicin were readily detected by both techniques with significant perturbations of the urinary profile observed from day 7 onwards. The findings by ¹H-NMR included raised glucose and reduced trimethylamine N-oxide (TMAO). Changes in metabonomic profiles were observed by HPLC-MS in both positive and negative ESI. The MS data showed reduced xanthurenic acid and kynurenic acid, whilst neutral loss experiments also revealed a changed pattern of sulphate conjugation on gentamicin administration.     

l-Prolyl-l-leucine Anhydride A Bitter Compound Formed in Aged Sake

A cyclic dipeptide with a bitter taste, compound B–1, was isolated from sake and its properties were characterized.

Compound B–1 was chromatographically purified by the use of Amberlite XAD–2, silicic acid, Dowex 50–X4 (H type), Amberlite IR–45 (OH type) and silicic acid again, and crystallized from ethanol,

From the decomposition point (159~165°C) and the ultraviolet and infrared spectra, the compound was identified as l-prolyl-l-leucine anhydride.

The content of this compound in sake samples aged under various conditions was determined by gas chromatography and it was found that the amount of this compound increased with the time of storage of sake.

Furthermore, the contribution of the bitter compound to the flavor of sake was evaluated from its content and the threshold taste value.     

Demethylation of theophylline (1,3-dimethylxanthine) to 1-methylxanthine: the first step of an antioxidising cascade

Abstract

The reaction of theophylline with HO^? radical, produced by photolytic methods at pH 7, was studied in aqueous solution and the products characterised by HPLC and GC-MS. In addition to the expected 1,3-dimethyluric acid, the formation of 1-methylxanthine and, to a lesser extent, of 3-methylxanthine was observed. Theoretical calculations confirmed the preferred formation of the former compound. Both demethylated products were also observed upon reaction of theophylline with O^?– radical anion at pH～13, and 1-methylxanthine was consumed faster than 3-methylxanthine after its formation. Molecular oxygen had no significant effect on the formation of the mono-methylxanthine derivatives. A reaction mechanism for the demethylation of theophylline by oxidising radicals is proposed. This demethylation reaction can play an important role in the protection of biological targets against oxidative stress as the first step of an antioxidising cascade.     

Effects of Vitamin B6 Deficiency on the Conversion Ratio of Tryptophan to Niacin

To investigate how vitamin B₆ (B₆) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets; a complete 20% casein diet (control diet), the control diet without B₆, the control diet without nicotinic acid, and the control diet without nicotinic acid and B₆, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone3- carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B₆-deficient groups, when compared with the B₆-containing groups. These results indicate that the rats fed with the B₆-free diets were in the vitamin-deficient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5carboxamide, and N¹-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B₆-free diet than in the B₆-containing diet under the niacin-free conditions.     

Antibacterial activity of sugar maple autumn-shed leaf extract: Identification of the active compound

Ethanolic crude extract prepared from autumn-shed leaves of sugar maple (Acer saccharum Marsh.) was recently shown to have antibacterial activity against Pseudomonas cichorii and Xanthomonas campestris pv. vitians, two bacteria causing diseases in lettuce production. In this study, antibacterial activity of sugar maple autumn-shed leaves (SMASL) extract was further investigated. SMASL ethanolic crude extract was fractionated using HPLC system and geraniin was identified as the antibacterial compound by UPLC/Q-Tof-MS system. Geraniin, an ellagitannin, was then purified from SMASL crude extract using a glass chromatographic C18-reversed phase silica gel column (purification Step 1) and a semi-preparative HPLC system equipped with 5 μm XTerra Prep MS C18 column (purification Step 2). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of purified geraniin (purity of 96%) against P. cichorii and X. campestris pv. vitians were determined. X. campestris pv. vitians (MIC of 0.024 mg ml⁻¹ and MBC of 3.125 mg ml⁻¹) was more sensitive to geraniin than P. cichorii (MIC of 0.781 mg ml⁻¹ and MBC of 6.25 mg ml⁻¹). In the present study, geraniin is reported for the first time as the main antibacterial compound present in SMASL.     

Isolation of the Melanization and Reddish Coloration Hormone (MRCH) of the Armyworm,Leucania separata,from the Silkworm,Bombyx mori

Two types of glycerol dehydrogenase (GDH) were found on DEAE-cellulose column chromatography of cell-free extracts of methylotrophic yeasts. One type, designated as GDH I, showed only the reductive activity which was detected in the reaction system containing dihydroxyacetone and NADH, at pH 6.0. The other type, designated as GDH II, showed the oxidative activity which was detected in the system containing glycerol and NAD ⁺, at pH 9.0, together with the reductive activity.

Candida boidinii No. 2201, which possesses the phosphorylative pathway for glycerol dissimilation, had only GDH I when grown on glycerol or methanol as the carbon source. Hansenula ofunaensis, which has the oxidative pathway, had both GDH I and GDH II when grown on glycerol, but only GDH I when grown on methanol. Hansenula polymorpha Dl-1, which has both pathways, had both GDH I and GDH II when grown on glycerol or methanol.     

Protonation and dimerization reactions of cyclopentadienylrhodiumcarbonylphosphine and phosphite derivatives

Eight cyclopentadienylrhodiumcarbonylphosphine and phosphite complexes have been prepared and their IR, ¹H, and ³¹P NMR spectra recorded. A good correlation between carbonyl stretching frequencies and rhodium-phosphorous coupling constants has been observed. Reaction of these compounds with trifluorosulfonic acid, HCF₃SO₃, forms the expected cationic rhodium-hydride species which were examined using ¹H and ³¹P NMR spectroscopy. Similar reactions between trifluoroacetic acid, HCF₃CO₂, and the phosphine compounds gave evidence of rapid proton exchange at the metal. Reaction between trifluoroacetic acid and cyclopentadienylrhodiumcarbonylphosphite compounds yielded new sets of rhodium-hydride resonances which were shown to be due to the formation of dinuclear rhodium complexes and cyclopentadienylrhodiumbis(phosphite) complexes which arise under the reaction conditions. A Scheme for the formation of these reaction products is presented which is consistent with all of the experimental data.     

Enzyme-facilitated synthesis of 1-nonene oxide and simple GC-MS SIM method for rapid screening of epoxidation processes

Abstract

Here, we describe an improved enzyme-facilitated epoxidation of 1-nonene using a conventional water bath shaker at ambient temperature. Enzymes were used to produce peroxy acids instantly from hydrogen peroxide (H₂O₂) and various perhydrolysis substrates. The peroxy acid generated was then utilised directly for in-situ oxidation of 1-nonene to 1-nonene oxide. Various parameters affecting the reaction were studied such as the nature of the peroxy acids, organic solvents, enzyme sources and enzyme concentrations. The highest conversion rate was achieved using phenylacetic acid as an oxygen carrier. 1-Nonene was converted most efficiently with 95% of the maximum yield by Novozym 435, an immobilised Candida antarctica lipase B, using dichloromethane as the reaction media. A minimum amount (16 mg, 1.4% w/w) of Novozym 435 was needed to maintain catalytic activity (160.0 Ug^?1). In addition, a simple and rapid Gas chromatography mass spectroscopy selective ion monitoring (GC-MS SIM) method was developed using a HP-5ms column for determining 1-nonene oxide. The method was found to be linear in the range of 29.9 to 298.8 mg/L with R² = 0.9981.