NADPH oxidation catalyzed by the peroxidase/H2O2 system. Iodide-mediated oxidation of NADPH to iodinated NADP

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADPH+

We have examined the respective roles played by guaiacol and scopoletin in NADPH oxidation catalyzed by the peroxidase/H2O2 system. It was shown that NADPH was not oxidized by either the horseradish or lactoperoxidase/H2O2 systems alone; oxidation occurred immediately after the addition of guaiacol or scopoletin. In both cases, the oxidation product was enzymatically active NADP+. Differences were observed in the NADPH oxidation mechanism depending on whether guaiacol or scopoletin was the mediator molecule. In guaiacol-mediated NADPH oxidation, the stoichiometry between H2O2 and oxidized NADPH was about 1; superoxide dismutase did not affect the oxidation rate. In scopoletin-mediated oxidation, the stoichiometry was much higher (1:14 in the present experiments); superoxide dismutase considerably increased the oxidation rate. It is concluded that catalysis of NADPH oxidation by the horse radish peroxidase/H2O2 system requires the presence of a mediator molecule. The NADPH oxidation mechanism depends on the intermediary oxidation state of this molecule.     

The oxidation of oxytocin and vasopressin by peroxidase/H2O2 system

Summary Oxytocin and vasopressin are oxidized by horseradish peroxidase and by lactoperoxidase, in the presence of hydrogen peroxide. Spectrophotometric measurements are indicative of the formation of dityrosine. Kinetic parameters indicate that the affinity of horseradish peroxidase is slightly higher for oxytocin with respect to vasopressin and that the two hormones are better substrates for both peroxidases than free tyrosine.     

Design of H2O2-dependent oxidation catalyzed by hemoproteins

The monooxygenese activity of cytochrome P450 is successfully introduced into myoglobin by rational design of its active site. Introduction of an aromatic ring, tryptophan, near the heme by site-directed mutagenesis resulted in the hydroxylation of tryptophan at the C6 position by using an almost stoichiometric amount of H(2)O(2). We also altered the substrate specificity of H(2)O(2)-dependent P450 by employing a simple substrate trick. Although P450(BSβ) exclusively catalyzes peroxygenation of long-alkyl-chain fatty acids, oxidation of non-natural substrates such as styrene, ethylbenzene, and 1-methoxynaphthalen are catalyzed by P450(BSβ) in the presence of decoy molecules having a carboxyl group. Advantageously, the substrate specificity of P450(BSβ) can be altered by simply adding the decoy molecule without replacing any amino acid residues. Moreover, the stereoselectivity can be controlled by changing the structure of the decoy molecule. The crystal structure analysis of the decoy molecule bound-form of P450(BSβ) shows that P450(BSβ) accepts the decoy molecule, whose carboxylate is located at the same position to that of long-alkyl-chain fatty acid.     

A novel and efficient polymerization of lignosulfonates by horseradish peroxidase/H2O2 incubation

Lignosulfonates(LSs), by-products from chemical pulping processes, are low-value products with limited dispersion properties. The ability of commercially available horseradish peroxidase (HRP) to polymerize LS macromolecules and improve the dispersion properties of LSs was investigated. The polymerization of LSs proceeded efficiently under mild reaction conditions in an aqueous solution with HRP/H₂O₂. Gel permeation chromatography showed a significant increase in weight-average molecular weight (M _w ) of sulfonated kraft lignin and sodium lignosulfonate (NaLS) by 8.5-fold and 4.7-fold, respectively. The mechanism of polymerization was investigated by elemental analysis, surface charge measurement, headspace gas chromatography, infrared spectroscopy (IR), and hydrogen nuclear magnetic resonance spectrometry (¹H-NMR). The functional group measurements indicated that HRP incubation did not reduce the sulfonic group content. However, it decreased the phenolic and methoxyl group contents. As the phenolic group content decreased, M _w increased as a power function. The polymerization was proposed to involve the random coupling of phenoxy radical intermediates. The radicals coupled with each other to form different inter-unit linkages, most of which were the β-O-4’ type, as the ¹H-NMR spectra indicated. Moreover, the HRP/H₂O₂ incubation induced a significant improvement in the adsorption and dispersion properties of LSs. Therefore, the HRP/H₂O₂ incubation is a promising approach for industrial applications of LSs.     

Subnanogram estimation of the proximate carcinogen N-hydroxy-2-fluorenylacetamide by gas-liquid chromatography

A very sensitive method, using electron-capture gas chromatography, has been developed for the quantitative estimation of N-hydroxy-2-fluorenylacetamide, the proximal carcinogenic metabolite of N-2-fluorenylacetamide. After incubation of the carcinogenic parent arylamide with rat liver microsomes, the N-hydroxy derivative produced is converted into N-chloro-2-fluorenylamine by treatment with hydrochloric acid; the amine is extracted with cyclohexane and transformed into N-chloro-2-fluorenyltrifluoroacetamide with trifluoroacetic anhydride. As little as 0.06 ng of the latter compound can be readily detected by gas-liquid chromatography using an electron-capture detector.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

A novel assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide

A method for the assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide is described. The method employs UDP-[U-14C ))glucuronic acid and Baker C18 extraction columns for separation of the glucuronides from their aglycones and from the glucuronic acid. The 14C-labeled glucuronides, generated by rat liver microsomes, are eluted from the columns with 30% (v/v) methanol after prewashing the columns and elution of the radioactivity of 14C-glucuronic acid with 1 mM ammonium acetate, pH 6.9. The radioactivity of the eluates is measured by scintillation counting. The method is modified for assays of glucuronidation of alpha-naphthol and p-nitrophenol in that 1 mM phosphoric acid is used instead of 1 mM ammonium acetate, and the method is potentially adaptable to other aglycones. By monitoring radioactivity or uv absorbance of the column eluates, it is shown that all aglycones, except p-nitrophenol, are retained on the columns during elution of their glucuronides with 30% (v/v) methanol and are eluted only when absolute methanol is used. The identity of the glucuronides is shown by their response to hydrolysis by beta-glucuronidase in the presence and absence of D-saccharic acid-1,4-lactone and, in some instances, by chromatographic and spectral analyses of the released aglycones.     

Stopped-flow kinetic study of the H2O2 oxidation of substrates catalyzed by microperoxidase-8

We have studied the oxidation of microperoxidase-8 (MP-8) by H2O2 and the subsequent reaction of the intermediates with substrate by stopped-flow experiments. Oxidation of MP-8 by H2O2 gives two intermediates, I and II. The observed rate constant for the formation of I is linearly dependent on [H2O2] and exhibits a bell-shaped dependence on pH with pKa values of 8.90 and 10.60, which are attributed to the deprotonation of MP-bound H2O2 and H2O, respectively. The observed rate constant for the conversion of I to II is independent of [H2O2], but increases sharply at pH>9.0. The predominant forms of the intermediate at pH 7.0 and 10.7 are I and II, respectively. Addition of substrate to the intermediates at pH 9.0 gives rise to three distinct stages, corresponding to the three steps (in decreasing order of rate): I-->II*, II-->MP, and II*-->MP. The rates of these steps are all linearly dependent on the substrate concentration and each individual rate constant has been determined. Substrate reactivity at pH 10.7 covers over two orders of magnitude, ranging from 1.36 x 10(7) M(-1) s(-1) for 1-naphthol to 4.03 x 10(4) M(-1) s(-1) for ferrocyanide. The substrate reactivity is linearly correlated with its reduction potential, indicating that an electron transfer process is involved in the rate-limiting step.     

Arylamidation and arylation by the carcinogen N-2-fluorenylacetamide: A sensitive and rapid radiochemical assay

N-acetoxy-N-arylacetamides, which are generally considered as an ultimate carcinogenic form of the corresponding N-arylacetamides, react with the cellular macromolecules (nucleic acids, proteins, etc.) to give two types of adducts: (I) arylamidation and (II) arylation addition products. In this paper, we present a radiochemical determination of the amount of N-2-fluorenylacetamide bound to DNA via arylamidation or arylation, respectively. This assay is based upon the difference of stability under weak alkali hydrolysis conditions (0.1 N NaOH, 75°C, 2 h) of the specifically ¹⁴C-labeled N-acetyl group of the N-2-fluorenylacetamide residue linked to the macromolecule either via arylamidation or arylation. Native DNA which has been reacted with N-acetoxy-N-2-[¹⁴C]acetylaminofluorene exhibits 16% of the fluorene adducts linked to the bases via arylation. On the other hand, denatured DNA reacts with the fluorene derivative to give almost only arylamidation addition products.     

Luminol oxidation catalyzed by royal palm leaf peroxidase

We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 micrograms/ml (26 microM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of beta-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.     

Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase

The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.     

Free radical intermediates formed during the oxidation of cyanide by horseradish peroxidase/H2O2 as detected with nitroso spin traps

Aqueous solutions of cyanide react with hydrogen peroxide/horseradish peroxidase and form the cyanyl radical, which can be trapped by 2-methyl-2-nitrosopropane (t-nitrosobutane, tNB) at pH 9.8. At lower pH a variety of radical adducts are formed; at higher pH, the main product was the spin adduct of the formamide radical with tNB. The use of deuterated tNB and 15N-labeled potassium cyanide allowed the observation of the very small nitrogen coupling of this radical adduct. Experiments using 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) as the spin trap yielded only the formamide radical adduct, which was identified by an independent synthesis starting from formamide. Both hydrogen splittings of its amino group could be resolved using deuterated DBNBS as the spin trap.     

Generation of excited species catalyzed by horseradish peroxidase or hemin in the presence of reduced glutathione and H2O2

Horseradish peroxidase (HRP) (EC 1.11.1.7) catalyzes the oxidation of reduced glutathione. This reaction is accompanied by light emission, which is attributed to the generation of singlet oxygen. The chemiluminescence is directly related to thiyl radical formation, as deduced from the correlation between the time course of HRP-compound II formation and light emission in the presence of different amounts of H2O2. Superoxide dismutase has an inhibitory effect on the chemiluminescence without affecting the HRP-compound II formation. This indicates the direct involvement of superoxide radicals in the production of photoemissive species. Replacement of HRP by hemin is also accompanied by chemiluminescence.     

Steady-state kinetics of thiocyanate oxidation catalyzed by human salivary peroxidase

A steady-state kinetic analysis was made of thiocyanate (SCN-) oxidation catalyzed by human peroxidase (SPO) isolated from parotid saliva. For comparative purposes, bovine lactoperoxidase (LPO) was also studied. Both enzymes followed the classical Theorell-Chance mechanism under the initial conditions [H2O2] less than 0.2mM, [SCN-] less than 10mM, and pH greater than 6.0. The pH-independent rate constants (k1) for the formation of compound I were estimated to be 8 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 5 X 10(6) M-1 s-1 (SD = 1, n = 11) for SPO. The pH-independent second-order rate constants (k4) for the oxidation of thiocyanate by compound I were estimated to be 5 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 9 X 10(6) M-1 s-1 (SD = 2, n = 11) for SPO. Both enzymes were inhibited by SCN- at pH less than 6. The pH-independent equilibrium constant (Ki) for the formation of the inhibited enzyme-SCN- complex was estimated to be 24 M-1 (SD = 12, n = 8) for LPO and 44 M-1 (SD = 4, n = 10) for SPO. An apparent pH dependence of the estimated values for k4 and Ki for both LPO and SPO was consistent with a mechanism based on assumptions that protonation of compound I was necessary for the SCN- peroxidation step, that a second protonation of compound I gave an inactive form, and that the inhibited enzyme-SCN- complex could be further protonated to give another inactive form.(ABSTRACT TRUNCATED AT 250 WORDS)