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At least 19 recordsLinked to original sources
PubMed1985

High-resolution proton nuclear magnetic resonance spectroscopy of chloride peroxidase: identification of new forms of the enzyme.

Chloride peroxidase from the mold Caldariomyces fumago in the native high-spin iron(III) and low-spin cyanoiron (III) states has been subjected to high-field proton nuclear magnetic resonance spectroscopic measurements. Signals shifted well outside the diamagnetic envelope by the paramagnetic iron(III) center are surprisingly insensitive to pH changes over the range from pH 3 to pH 7. The previously identified major form of chloride peroxidase (form A) and the minor form (B) show very similar chemical shift patterns. Of greatest significance, however, is the discovery that each of the separable forms of the enzyme exhibits splitting of porphyrin ring methyl resonances. The appearance of two sets of signals in both native and cyanide-complexed enzyme is best explained by the existence of two additional forms of the A and B isoenzymes. Structural differences for the newly identified forms of chloride peroxidase must be located in the vicinity of the heme prosthetic group.

Chloride Peroxidase↗
PubMed1980

The action of chloride peroxidase on 4-chloroaniline. N-oxidation and ring halogenation.

Chloride peroxidase catalyses both the ring halogenation and N-oxidation reactions of 4-chloroaniline by H2O2 and either KCl or KBr. In the absence of any halide salt only the N-oxidation reaction was observed, with the resulting conversion of 4-chloroaniline into 4-chloronitrosobenzene. The N-oxidation reaction proceeded even more rapidly in the presence of Cl- or Br-, in spite of the fact that ring halogenation was also a rapid reaction. The enhancement of N-oxidation was highly dependent on the pH of the media and displayed an optimum in the region of pH 3.5-4.0. No rate enhancement was observed above pH 5.5. KF partially inhibited the rate of N-oxidation in a pH-dependent manner. On the basis of calculated catalytic-centre activity the N-oxidation reaction was the major reaction at pH 3.5 or higher, whereas the ring-halogenation reaction became the major reaction below pH 3.5. In the presence of high concentrations of 4-chloroaniline relative to H2O2 the reaction intermediate, 4-chlorophenylhydroxylamine, was detected for the first time in a chloride peroxidase-catalysed reaction with this arylamine substrate. These findings were interpreted on the basis of current knowledge concerning the mechanism of action of chloride peroxidase.

Aniline Compounds↗
PubMedDate not supplied

[Hydrolysis of phosphoether bonds of heme-independent chloride peroxidase from Serratia marcescens].

Heme- and metal-independent chloroperoxidase from Serratia marcescens W 250 is shown to be capable of catalyzing the p-nitrophenyl phosphate hydrolysis. The parameters of the phosphatase reaction are determined and inhibitors and activators of the process are found. A hypothetical mechanism of the hydrolysis of phosphoesters by heme- and metal-independent haloperoxidases is suggested. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

Chloride Peroxidase↗
PubMed1999

Effects of age and manganese (II) chloride on peroxidase activity of brain and liver of the teleost, Channa punctatus.

Fish provide enormous spectrum of longevity and thus present the possibility of multiple mechanisms of senescence. Oxidative stress as a causative agent of senescence and the protective role of antioxidant enzymes were tested in the teleost, Channa punctatus taking peroxidase (POD) (EC 1.1 1.1.7) as the representative enzyme. The activity of POD in brain and liver declined during maturation phase (young vs middle-aged). During senescence phase (middle-aged to old) the enzyme activity increased in liver but remained stabilized in brain. The degree of increase in peroxidase activity following in vitro MnCl2 treatment was always higher in liver than in brain. The rate of MnCl2 induced increase in POD activity of both tissues showed an increasing trend with age. However statistical significance was observed only in brain during senescence phase. No significant loss of enzyme activity in both the tissues and greater degree of increase by MnCl2 in brain suggest that antioxidant capacity is not impaired in old murrels.

Aging↗
PubMed1986

The fate of electron opaque tracers (horseradish peroxidase and lanthanum chloride) during valproic acid-induced choleresis.

Horseradish peroxidase (HRP) and lanthanum chloride (LaCl3) are useful tools for, respectively, the study of vesicular transport through the hepatocyte and the study of the permeability of junctional complexes. These tracers have been used to detect the changes associated with the choleresis independent of bile acids induced by valproic acid (VPA) in rats. The animals were given a single dose of VPA (600 mg/kg, ip). HRP (100 mg/kg) or 5 mM LaCl3 were given intraportally after 1 h, when bile flow had increased twofold. The excretion of HRP in bile was measured colorimetrically up to 2 h after HRP. Ultrastructural morphometry was conducted on liver of intact rats taken from 1 to 40 min after HRP. The volume density (VD) of HRP-containing vesicles and of HRP-containing multivesicular bodies (MVB) was counted. In VPA-treated rats, HRP appeared in bile with a peak showing at 5 min against 20 min in controls, but the total amount of HRP excreted was less than in controls. The intrahepatocytic vesicular transport of HRP was also modified, showing a peak at 3 min in VPA-treated rats compared to 10 min in controls, together with a decreased VD of pericanalicular vesicles. This was accompanied by an increase of HRP-containing MVB, already evident at 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

On the molecular composition and physico-chemical properties of the pseudo-exfoliation material.

The protein tracer peroxidase has been found to be excluded from pseudoexfoliation (PE) material. But after the treatment of the PE material with cetylpyridinium-chloride, peroxidase was found to penetrate into the material. This observation seems to support the concept that the PE material is a gel of proteoglycans, from which peroxidase is excluded by an excluded volume effect. Microperoxidase was found to penetrate into the superficial parts of the (untreated) PE material, and to be regularly distributed according to two different patterns: 1) along the PE fibrils, and 2) along lines in the interfibrillar matrix; lines which can not be seen without the presence of microperoxidase. At both locations the microperoxidase reaction product was found at regular intervals, with spacings of about 53 nm. The interpretation is that both the fibrils and the interfibrillar matrix are composed of the same kind of long extended linear proteoglycan complexes, but with a different arrangement and density.

Eye Diseases↗
PubMedDate not supplied

Horseradish peroxidase and cobalt chloride as neuromarkers in the hypoglossal nucleus.

The hypoglossal nucleus in 129 REJ normal mouse strains was investigated using two neuroanatomical markers, namely the cobalt chloride (CoCl2) and the horseradish peroxidase (HRP) techniques. CoCl2 was introduced through the cut end of the hypoglossal nerve. In one set of experiments HRP was injected into the hypoglossal nerve, while in the other it was injected into the tongue musculature. Results show that with these techniques the hypoglossal neurons are conspicuously stained and can be easily located among series of brainstem sections. The mean number +/- SD of neurons in the hypoglossal nucleus was 1,417 +/- 37, 846 +/- 28 and 1,272 +/- 42 using CoCl2 and HRP injected into the tongue musculature or the hypoglossal nerve, respectively. The estimated length of the nucleus was 0.92 mm with the CoCl2 technique.

Animals↗
PubMed1989

Chloroperoxidase-catalyzed oxidation of aminopyrine.

Although in the absence of halide ion chloroperoxidase did not catalyze the ethylhydroperoxide (EHP)-supported oxidation of aminopyrine, in the presence of Br- or Cl-, chloroperoxidase did catalyze the oxidation of aminopyrine, generating the aminopyrine cation radical (AP+). The initial rate of AP+ formation was determined by monitoring the absorbance at 565 nm. The pH optimum of the reaction was centered around 5.0. The rate of AP+ formation showed typical Michaelis-Menten saturation kinetics with respect to EHP, aminopyrine and Br-. The rate of formation of bromine in the chloroperoxidase-EHP-Br- system was also determined by measuring the change in absorbance at 267 nm. In the system containing 1 mM EHP and 0.2 M KBr at pH 5.0, the rate was 1.8 nmol of bromine/s/micrograms of chloroperoxidase, which was slower than that of AP+ formation under the same conditions. The present results suggest that the formation of AP+ is initiated by the halogenation of the N,N-dimethylamino group followed by the homolysis of the haloammonium cation, and that the most likely halogenating reagent is an enzyme-bound halogenating intermediate.

Aminopyrine↗
PubMed1984

Singlet oxygen production by chloroperoxidase-hydrogen peroxide-halide systems.

Singlet oxygen production in the chloroperoxidase-hydrogen peroxide-halide system was studied using 1268 nm chemiluminescence. With chloride or bromide ions, singlet oxygen is produced by the mechanism (formula; see text) (formula; see text) where X- is chloride or bromide ion. Under conditions where there is high enzyme activity and when Reaction B is fast relative to Reaction A, singlet oxygen is produced in near stoichiometric amounts. In contrast, when Reaction A is fast relative to Reaction B, oxidized halogen species (chlorine and hypochlorous acid for chloride ion; bromide, tribromide ion, and hypobromous acid for bromide ion) are the principle reaction products. With iodide ion, no 1268 nm chemiluminescence was detected. Past studies have shown that iodine and iodate ion are the major end products of this system.

Bromides↗
PubMed2006

Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns.

OBJECTIVES: To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl(-)) on plasma unbound bilirubin (B(f)) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns. DESIGN AND METHODS: B(f) was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with B(f) measured at a 2-fold sample dilution using a FloPro Analyzer. B(f) was measured at two peroxidase concentrations to determine whether the peroxidase steady state B(f) (B(fss)) measurements were significantly less than the equilibrium B(f) (B(feq)), in which case it was necessary to calculate B(feq) from the two B(fss) measurements. B(f) was also measured before and after adding 100 mmol/L Cl(-) to the UB Analyzer assay buffer. RESULTS: B(feq) at the 42-fold dilution was nearly 10-fold less than but it correlated significantly with B(feq) at the 2-fold dilution (mean 8.2+/-5.2 nmol/L versus 73.5+/-70 nmol/L, respectively, p<0.0001; correlation r=0.6). The two UB Analyzer B(fss) measurements were significantly less than B(feq) in 42 of 74 (57%) samples, and Cl(-) increased B(feq) in 66 of 74 (89%) samples by a mean of 82+/-67%. CONCLUSIONS: B(fss) measured by the UB Analyzer at the standard 42-fold sample dilution using assay buffer without Cl(-) and a single peroxidase concentration is significantly less than the B(feq) in undiluted plasma. Accurate B(f) measurements can be made only in minimally diluted serum or plasma.

Analytic Sample Preparation Methods↗
PubMed1987

On the mechanism of chlorination by chloroperoxidase.

Spectral-scan results obtained on the millisecond time scale are reported for reactions of chloroperoxidase with peracetic acid and chloride ion in both the presence and the absence of monochlorodimedone. A multimixing experiment is performed in which stoichiometric amounts of chloroperoxidase and peracetic acid are premixed for 0.7 s before the resultant compound I is reacted with chloride ion. The combined results show that the only detectable enzyme intermediate species is compound I (except in very late stages of the reaction), that the disappearance of compound I is accelerated by the presence of chloride ion, and that it is further accelerated if both chloride and monochlorodimedone are present. It is concluded that compound I is an obligate intermediate species in the reaction. Experiments are performed on the reaction of monochlorodimedone with hypochlorous acid in both the presence and the absence of added chloride ion, but in the absence of chloroperoxidase. The presence of chloride ion greatly accelerates the reaction rate apparently by setting off a chlorine chain reaction. This reaction would be important in the enzyme-catalyzed reaction if hypochlorous acid were liberated into the solution. A careful analysis of steady-state kinetic results shows that in the chlorination of monochlorodimedone at least, liberation of free hypochlorous acid is not important in the enzyme-catalyzed pathway. Rather the reaction proceeds from compound I to formation of iron(III)-OCl by chloride ion addition to the ferryl oxygen atom. This obligate intermediate species then chlorinates the substrate. It is well described as enzyme-activated hypochlorous acid, in which replacement of the proton in HOCl by the heme iron ion produces a Cl+ species of great potency. Thus the enzyme controls chlorination of monochlorodimedone rather than unleashing an uncontrolled chain reaction in which it would be rapidly destroyed.

Chemical Phenomena↗
PubMed1985

Chlorination of NADH: similarities of the HOCl-supported and chloroperoxidase-catalyzed reactions.

The chloroperoxidase-catalyzed reactions of NAD(P)H with H2O2 in the presence of Cl- or Br- have been characterized. With 1 mol H2O2 per mol of NADH, one atom of 36Cl was incorporated into the 264-nm-absorbing intermediate product. This species was oxidized enzymatically by a second mole of H2O2 to a species distinct from NAD+, which retained one Cl atom. Spectroscopically identical species were also produced by reaction of NADH with one and two molar ratios of HOCl, respectively. These data indicate that, with respect to halogenation activities, chloroperoxidase functions similarly to myeloperoxidase, i.e., produces HOCl as the first product of Cl- oxidation by H2O2. Moreover, rapid chlorination of NAD(P)H followed by oxidation may be an important and highly lethal microbicidal effect of HOCl produced by myeloperoxidase in activated neutrophils.

Catalysis↗
PubMed1989

The chloride-activated peroxidation of catechol as a mechanistic probe of chloroperoxidase reactions. Competitive activation as evidence for a catalytic chloride binding site on compound I.

Chloride ion (Cl-) effects on chloroperoxidase (CPO)-catalyzed peroxidation of catechol were used to probe the involvement of Cl- in CPO reactions. High concentrations of Cl- inhibit catechol peroxidation by competing with hydrogen peroxide (KI = 370 mM). However, at lower concentrations, Cl- is a linear competitive activator versus catechol (KDC = 35 mM). Addition of good halogenation substrates to the peroxidatic reaction mixture converts Cl- from a competitive activator to a competitive inhibitor. The KI (10 mM) for this halogenation substrate promoted Cl- inhibition is equivalent to the KM (11 mM) for Cl- in CPO-catalyzed halogenation reactions. During this inhibition, the halogenation substrate is consumed and, at the point where its consumption is complete, Cl- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its Cl- -activated peroxidatic reaction have similar apparent kcat values. All data are consistent with a mechanism in which Cl- competes with catechol for binding to CPO Compound I. Catechol binding initiates the Cl- -independent path, in which Compound I acts as the oxidizing agent for catechol. When Cl- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the Cl- -dependent path or the chlorination of substrates in the halogenation pathway. Cl- activation of the peroxidatic reaction is due to a shift from the Cl- -independent pathway to the Cl- -dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol-Cl- system also offers further potential for probing the specificity and chemistry of the key enzymatic intermediates in haloperoxidase-catalyzed reactions.

Catechols↗
PubMed1981

The reaction of chloroperoxidase with chlorite and chlorine dioxide.

Chloroperoxidase catalyzes the dismutation of chlorite-forming chloride, chlorine dioxide, chlorate, and oxygen as products. The yields of chlorine dioxide are variable because chloroperoxidase also catalyzes the decomposition of this compound and, in addition, moderate concentrations of chlorine dioxide inactivate the enzyme. Chloride, chlorate, and oxygen are the products of the decomposition of chlorine dioxide. The optimum pH for the enzymic of decomposition of both chlorite and chlorine dioxide is approximately pH 2.75. At this pH, 1 mole of chlorine dioxide is dismutated to 0.3 mole of chloride, 0.7 mol of chlorate, and 0.17 mole of oxygen. At the same pH, the complete decomposition of 1 mole of chlorite yields 0.4 mole of chloride, 0.6 mole of chlorate, and 0.13 mole of oxygen. During the inactivation of chloroperoxidase by chlorine dioxide, the Soret absorption band of the native enzyme is completely lost, and the enzyme becomes chlorinated. Kinetic parameters for the chlorite reaction have been determined. The Km value for chlorite obtained from various kinetic plots was about 10 mM. The catalytic rate constant for the formation of chlorine dioxide from chlorite was about 70,000 s-1.

Chloride Peroxidase↗
PubMed2005

Efficient labelling of antibodies with horseradish peroxidase using cyanuric chloride.

An efficient and mild method for labelling of immunoglobulin G (IgG) with horseradish peroxidase (HRP) using cyanuric chloride (2,4,6-trichloro-1,3,5-triazine, CC) as a bridging molecule is described. The enzyme was treated first with cyanuric chloride to introduce dichloro triazine and after removal of excess reagent, the activated enzyme was mixed with the IgG preparation and incubated to effect linkages with amine groups in the antibody protein. Various amounts of coupling reagent were tested to optimise the conjugation method using commercially available enzyme and affinity-purified sheep IgG antibody preparations to three different test haptens. The conjugates were assessed by solid phase Enzyme Linked Immunosorbent Assays (ELISA) and commonly used peroxidase substrate preparations. The binding activity of the conjugates rose with increasing coupling reagent added during the enzyme activation step. Use of the conjugates prepared by the new method gave comparable sensitivity in direct competitive ELISAs for the three test haptens to assays carried out using indirect ELISA with commercial anti-sheep-HRP conjugates. No deterioration of enzyme activity or hapten-binding activity in the conjugates was observed after storage in 50% glycerol at -70 degrees C for up to 18 months. This study presents a relatively simple and efficient conjugating method for labelling antibodies with HRP and provides an additional and probably a better alternative to the periodate, glutaraldehyde and succinimide-maleimide procedures.

Animals↗
PubMed2006

Monoterpenes as novel substrates for oxidation and halo-hydroxylation with chloroperoxidase from Caldariomyces fumago.

Chloroperoxidase (CPO) from Caldariomyces fumago was analysed for its ability to oxidize ten different monoterpenes with hydrogen peroxide as oxidant. In the absence of halide ions geraniol and, to a lesser extent, citronellol and nerol were converted into the corresponding aldehydes, whereas terpene hydrocarbons did not serve as substrates under these conditions. In the presence of chloride, bromide and iodide ions, every terpene tested was converted into one or more products. (1S)-(+)-3-carene was chosen as a model substrate for the CPO-catalysed conversion of terpenes in the presence of sodium halides. With chloride, bromide and iodide, the reaction products were the respective (1S,3R,4R,6R)-4-halo-3,7,7-trimethyl-bicyclo[4.1.0]-heptane-3-ols, as identified by 1H and 13C nuclear magnetic resonance. These product formations turned out to be strictly regio- and stereoselective and proceeded very rapidly and almost quantitatively. Initial specific activities of halohydrin formation increased from 4.22 U mg-1 with chloride to 12.22 U mg-1 with bromide and 37.11 U mg-1 with iodide as the respective halide ion. These results represent the first examples of the application of CPO as a highly efficient biocatalyst for monoterpene functionalization. This is a promising strategy for 'green' terpene chemistry overcoming drawbacks usually associated with cofactor-dependent oxygenases, whole-cell biocatalysts and conventional chemical methods used for terpene conversions.

Ascomycota↗
PubMed1988

Determination of peroxidative halogenation in mixtures of chloride and bromide.

A method for the differentiation of chlorinated and brominated products from peroxidative oxidation of mixtures of the halides is presented. Chlorination or bromination of monochlorodimedone (MCD) by fungal chloroperoxidase (CPO) was measured by loss of MCD absorbance. Although the Vmax was similar for both halides [approximately 0.08 mM (2 min)-1], the apparent Km for chlorination was 10 times greater than that for bromination (5.88 vs 0.67 mM). Chlorination was also quantitated as I3- produced from N-chlorotaurine and I-. The Vmax [0.076 mM (2 min)-1] and apparent Km (6.31 mM) determined by this method agreed with those determined with MCD. Selective reduction by H2O2 of the I-oxidizing potential of N-bromotaurine allowed determination of the brominated product from the difference between the amounts of halogenated MCD and N-chlorotaurine. The brominated product predominated at saturating and at physiologic halide levels. Hence, it is suggested that Br- plays a significant role in halogenation even though in vivo levels of Cl- are equal to or greater than 1000 times those Br-.

Bromides↗