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.     

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.     

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.     

Stoichiometry of the reaction between horseradish peroxidase and p-cresol.

Over a wide range of pH horseradish peroxidase compound I can be reduced quantitatively via compound II to the native enzyme by only 1 molar equivalent of p-cresol. Since 2 molar equivalents of electrons are required for the single turnover of the enzymatic cycle, p-cresol behaves as a 2-electron reductant. With p-cresol and compound I in a 1:1 ratio compound II and p-methylphenoxy radicals are obtained in the transient state. Compound II is then reduced to the native enzyme. A possible explanation for the facile reduction of compound II involves reaction with the dimerization product of these radicals, 1/2 molar equivalent of 2,2'-dihydroxy-5,5'-dimethylbiphenyl. If only 1/2 molar equivalent of p-cresol is present, than at high pH the reduction stops at compound II. The major steady state peroxidase oxidation product of p-cresol (with p-cresol in large excess compared to the enzyme concentration) is Pummerer's ketone. Pummerer's ketone is only reactive at pH values greater than about 9 where significant amounts of the enol can be formed via the enolate anion. Therefore, in alkaline solution it is reactive with compound I, but not with compound II, which is converted into an unreactive basic form. These results indicate that Pummerer's ketone cannot be the intermediate free radical product responsible for reducing compound II in the single turnover experiments. It is postulated that Pummerer's ketone is formed only in the steady state by the reaction of the p-methylphenoxy radical with excess p-cresol.     

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 formation of ES of cytochrome-c peroxidase: a comparison with lactoperoxidase and horseradish peroxidase

The activation energy for the formation of the first red compound, ES, for cytochrome-c peroxidase (ferrocytochrome-c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) by i-propyl hydroperoxide and the rate constants for the formation of ES with various hydroperoxides have been determined. Multivariate data analysis by the partial least-squares model in latent variables has been used to compare the rate constants with the corresponding rate constants for the formation of compound I from lactoperoxidase and two isoenzymes of horseradish peroxidase. The results show that the rate of formation of ES from cytochrome-c peroxidase is highly correlated with the pKa of the hydroperoxides. The activation energy for the formation of ES with i-propyl hydroperoxide is close to the corresponding value for hydrogen peroxide.     

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.     

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.     

The reaction of horseradish peroxidase with hydroperoxides derived from Triton X-100

All of the commercially available Triton X-100 examined gave Compound I upon reaction with horseradish peroxidase, followed by its gradual transition into Compound II. Titration of horseradish peroxidase with Triton X-100 to form Compound I indicated that 1% (v/v) aqueous solutions of the detergent contained 0.4 to 3.2 microM equivalent peroxide but iodometric titration revealed 1.1 to 5.0 microM peroxide, suggesting the occurrence of different types of peroxides, reactive and unreactive with the peroxidase. The rate constant for Compound I formation was 1.5 X 10(7) M-1 S-1 at pH 7.4 at 25 degrees C, and for conversion into Compound II apparent first-order rate constants were 5.2 X 10(-3) to 1.7 X 10(-2) S-1. These results indicate that the Triton peroxides are as highly reactive as hydrogen peroxide. The amount of Triton peroxides increased as aqueous solutions of the detergent were allowed to stand, but the peroxides were destroyed by treatment with sodium borohydride. Although freshly prepared aqueous solutions of sodium cholate, sodium dodecyl sulfate, Tween 20 (polyoxyethylene sorbitan monolaurate), and Emasol 1130 (an equivalent of Tween 20) did not contain any detectable amount of peroxide, aged solutions of sodium dodecyl sulfate and Emasol 1130 contained peroxides. These observations suggest the need for appropriate precautions when biologically active substances vulnerable to attack by peroxides are incubated with Triton X-100 either for their solubilization from biomembranes or for other processing.     

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.     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method.

In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.     

Titration study of guaiacol oxidation by horseradish peroxidase.

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.     

Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative study of nine methods.

Nine currently available methods for HRP neurohistochemistry have been compared with each other on matching tissue sections from four rats and four rhesus monkeys. The nine methods investigated in this report are the diaminobenzidine (DAB) procedures of LaVail JH and LaVail MM (J Comp Neurol 157:303, 1974), of Adams JC (Neuroscience 2:141, 1977) and of Streit P and Reubi JC (Brain Res 126:530, 1977); the benzidine dihydrochloride (BDHC) procedures of Mesulam M-M (J Histochem Cytochem 24:1273, 1976) and of De Olmos J and Heimer L (Neurosci Lett 6:107, 1977); the o-dianisidine (O-D) procedure of De Olmos J (Exp Brain Res 29:541, 1977); the p-phenylenediamine dihydrochloride and pyrocatechol (PPD-PC) procedure of Hanker JS et al., (Histochem J 9:789, 1977) and the tetramethyl benzidine (TMB) procedures of Mesulam M-M (J Histochem Cytochem 26:106, 1978) and of De Olmos J et al. (J Comp Neurol 181:213, 1978). Quantitative comparisons were based on counts of retrogradely labeled perikarya. The extent of anterograde transport and the size of the injection site were also compared at a more qualitative level. The results indicate that one TMB procedure (Mesulam M-M, J Histochem Cytochem 26:106, 1978) is distinctly superior to each of the other eight procedures in the number of labeled perikarya that it can demonstrate. Furthermore, these differences are statistically significant at better than the 0.05 level of confidence. Differences in sensitivity are most evident when the perikarya contain small quantities of transported HRP. The same TMB method also demonstrates more anterograde transport and a larger injection site than all the other procedures. If less sensitive procedures are employed, afferent or efferent connections that are clearly demonstrated by this TMB procedure are either underestimated or completely overlooked. It is suggested that sensitivity in HRP neurohistochemistry is determined by multiple factors which include the method of fixation, post-fixation storage, the choice of chromogen, the incubation parameters, the type of HRP enzyme that is administered, and the postreaction treatment.