Oxidation of p-cresol by horseradish peroxidase compound I.

Rate constants for the reaction between horseradish peroxidase compound I and p-cresol have been determined at several values of pH between 2.98 and 10.81. These rate constants were used to construct a log (rate) versus pH profile from which it is readily seen that the most reactive form of the enzyme is its most basic form within this pH range so that base catalysis is occurring. At the maximum rate a second order rate constant of (5.1 +/- 0.3) x 10(-7) M-1 s-1 at 25 degrees is obtained. The activation energy of the reaction at the maximum rate was determined from an Arrhenius plot to be 5.0 +/- 0.5 kcal/mol. Evidence for an exception to the generally accepted enzymatic cycle of horseradish peroxidase is presented. One-half molar equivalent of p-cresol can convert compound I quantitatively to compound II at high pH, whereas usually this step requires 1 molar equivalent of reductant. The stoichiometry of this reaction is pH-dependent.     

A kinetic study of the reaction between glutaraldehyde and horseradish peroxidase.

Horseradish peroxidase was reacted with glutaraldehyde under various reaction conditions. The reaction product was, in a second step, bound covalently to aminohexyl groups attached to Sepharose particles. The influence of pH, time and the concentration ratio of enzyme:glutaraldehyde on the reaction was evaluated. A first step reaction with 100-fold molar excess of glutaraldehyde to horseradish peroxidase at pH 9.5 for 2 hr at room temperature results in a high yield of conjugated enzyme with well preserved enzymatic activity.     

The reaction between indole 3-acetic acid and horseradish peroxidase

Three distinct phases of the reaction between indole 3-acetic acid (IAA) and horse-radish peroxidase (isoenzymes B and C) were observed. When 100 μm IAA was added to an aerobic solution of the 7μm enzyme at pH 5.0 the oxidation of IAA occurred after a lag time of several seconds, during which the enzyme was partially converted into peroxide Compound II. At a time when the lag time was over the conversion of the enzyme into a green hemoprotein, called P-670 suddenly occurred at a considerable speed. The oxidation of IAA was almost over at the end of the second phase. The last phase was the restoration of the free enzyme from the remaining Compound II.Ascorbate and cytochrome c peroxidase elongated the lag phase of IAA oxidation. From these inhibition experiments it was suggested that a peroxide form of IAA would react with peroxidase to form its peroxide compounds as does hydrogen peroxide and cause the oxidation of IAA. A reaction path that the enzyme is directly reduced by IAA might be involved as an initiation step but appeared to play no essential role in the oxidation of IAA at steady state.Contrary to the cases with dihydroxyfumarate and NADH, Superoxide dismutase did not inhibit the aerobic oxidation of IAA by peroxidase. IAA peroxide radical instead of superoxide anion radical was suggested to be an intermediate in the oxidation of IAA.On the basis of stoichiometric relation of reactions between IAA and peroxidase peroxide compounds a tentative scheme of P-670 formation during the oxidation of IAA was presented.     

The reaction of coumarins with horseradish peroxidase

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.     

Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

A kinetic study of the reaction of horseradish peroxidase with hydrogen peroxide.

A kinetic study has been carried out over the pH range of 2.63-9.37 for the reaction of horseradish peroxidase with hydrogen peroxide to form compound I of th;e enzyme. Analysis of the results, indicates that there are two kinetic influencing, ionizable groups on the enzyme with pKa values of 3.2 and 3.9. Protonation of these groups results in a decrease in the rate of reaction of the enzyme with H2O2. A previous study of the kinetics of cyanide binding to horseradish peroxidase (Ellis, W.D. & Dunford, H.B.: Biochemistry 7, 2054-2062 (1968)) has been extended to down to pH 2.55, and analysis of these results also indicates the presence of two kinetically important ionizable groups on the enzyme with pKa values of 2.9 and 3.9.     

Kinetics of the reaction of superoxide anion with ferric horseradish peroxidase

Reaction of horseradish peroxidase A2 and C with superoxide anion (O2-) has been studied using pulse radiolysis technique. Peroxidase C formed Compound I and an oxy form of the enzyme due to reaction of ferric enzyme with hydrogen peroxide (H2O2) and O2-, respectively. At low concentrations of O2- (less than 1 mM), O2- reacted with ferric peroxidase C nearly quantitatively and formation of H2O2 was negligible. The rate constant for the reaction was found to be increased below pH 6 and this phenomenon can be explained by assuming that HO2 reacts with peroxidase C more rapidly than O2-. In contrast the formation of oxyperoxidase could not be detected in the case of peroxidase A2 after the pulse, and only Compound I of the enzyme was formed. Peroxidase A2, however, produced the oxy form upon aerobic addition of NADH, suggesting that O2- can also react with peroxidase A2 to form the oxy form. The results at present indicate that the rate constant for the reaction of O2- with peroxidase A2 is smaller than 103 M-1.s-1.     

A tetramethylbenzidine/tungstate reaction for horseradish peroxidase histochemistry

Tracing of neuroanatomical pathways commonly involves the histochemical demonstration of horseradish peroxidase, using the chromogen tetramethylbenzidine. A new modification of this reaction using ammonium paratungstate stabilizer retains high sensitivity while permitting the reaction to be performed at pH 6.0 in isotonic solutions. The reaction product resists solvents, allowing Nissl-stained sections to retain their peroxidase labeling. With subsequent stabilization by diaminobenzidine, the tissue is suitable for electron microscopic study and is compatible with post-embedding immunocytochemistry.     

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k(1)) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k(2)) and compound II (k(3)) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 x 10(5)M(-1)s(-1) (k(1)), 2.25 x 10(5)M(-1)s(-1) (k(2)), and 2.4 x 10(4)M(-1)s(-1) (k(3)), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 x 10(7)M(-1)s(-1) and 2.7 x 10(4)M(-1)s(-1), respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.     

Calcium-related properties of horseradish peroxidase.

Horseradish peroxidase has been shown to be a metalloprotein in which calcium contributes to the structural stability of the protein. Isoenzyme C and A contain 2.0 and 1.4 moles calcium/mole enzyme, respectively, which can be removed by treatment with guanidine hydrochloride and EDTA. Calcium-free isoenzyme C, but not isoenzyme A, reconstitutes upon addition of calcium and regains enzymatic activity. Free calcium readily exchanges with isoenzyme C, but only to a small extent with isoenzyme A. In addition the role of calcium in maintaining molecular conformation is evidenced by the effects of calcium removal from the isoenzyme C on the thermal stability of the protein.     

The blue reaction product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility.

A blue reaction product is formed at sites that contain horseradish peroxidase (HRP) activity when benzidene is used as the chromogen. With neutral red as a counter stain, this method affords excellent visualization of both retrograde and orthograde axonal transport of intracerebrally injected HRP. The visibility of this blue reaction-product is better than the visibility of the brown reaction-product obtained in the commonly used diaminobenzidene procedures. Variations in incubation times and reagent concentrations resulted in significant differences in the extent to which transported HRP could be demonstrated with benzidene. One of these benzidene procedures demonstrated a wider extent of HRP transport than a representative diaminobenzidene procedure. The substantia nigra and the nucleus locus ceruleus did not display artifactual deposition of the blue reaction-product.     

The dual oxygenase and peroxidase activities of porphobilinogen oxygenase and horseradish peroxidase: a study using the reaction with phenylhydrazine.

Porphobilinogen oxygenase and horseradish peroxidase show dual oxygenase and peroxidase activities. By treating porphobilinogen oxygenase with phenylhydrazine in the presence of H2O2 both activities were inhibited. When horseradish peroxidase was treated in the same manner only the peroxidase activity was lost while its oxygenase activity toward porphobilinogen remained unchanged. The phenylhydrazine treatment alkylated the prosthetic heme group of porphobilinogen oxygenase and N-phenylheme as well as N-phenylprotoporphyrin IX were isolated from the treated hemoprotein. In horseradish peroxidase the modified heme was mainly 8-hydroxymethylheme. The apoproteins of the alkylated enzymes were isolated and recombined with hemin IX. The oxygenase and peroxidase activities of porphobilinogen oxygenase were entirely recovered in the reconstituted enzyme, while the reconstituted horseradish peroxidase regained 75% of its peroxidase activity.     

Preparation and properties of matrix-supported horseradish peroxidase.

A new method of enzyme immobilization has been described using poly(4-methacryloxybenzoic acid) as the carrier. Activation of the polymer, prior to enzyme attachment, was achieved with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The enzyme coupling step proceeded through nucleophilic attack by the protein on a mixed carbonic anhydride. The degree of polymer activation was determined by analysis for quinoline, a by-product of the reaction. The polymer-enzyme complex was compared to the enzyme in solution in terms of pH optimum, substrate kinetics, and thermal denaturation. Potential uses of the polymerenzyme system in chemical synthesis of benzoquinone derivatives are discussed.