Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 1. Spectral properties of the products of dianisidine oxidation.
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Benzidines↗
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Determination of histaminase (diamine oxidase) activity by o-dianisidine test: interference of ceruloplasmin.
Until now o-dianisidine was used as an indicator substance in a test system for the determination of diamine oxidase. More recently, however, this substance was also used to measure ceruloplasmin activity. A study of the test principles revealed that o-dianisidine was the one denominator for both enzymes. As it was found for diamine oxidase the indicator was oxidized via peroxidase mediated H2O2 cleavage. Ceruloplasmin, however, oxidized o-dianisidine directly with resulting free radical formation. An addition of histamine dihydrochloride or putrescine dihydrochloride to an incubation mixture, containing ceruloplasmin as enzyme and o-dianisidine or p-phenylene-diamine as substrates, produced an activation of the enzyme, being more than 10-fold in the presence of 1 X 10(-2) M putrescine at pH 7.0. It was assumed that an allosteric effect of the dihydrochloride component might be responsible for this activation. When the activity of purified diamine oxidase was determined by the o-dianisidine test and by the isotope assay, a very good correlation between both methods was found. But, in a mixture of diamine oxidase and ceruloplasmin, no differentiation between the two enzymic activities by the o-dianisidine test was possible. This observation demonstrated an interference of ceruloplasmin when the o-dianisidine method was used for the determination of diamine oxidase activity. To apply our findings also in vivo the amine oxidase activity increasing in guinea-pig plasma during inflammation, was determined by the o-dianisidine test and by specific methods for some amine oxidase. Despite an enhanced oxidation of the o-dianisidine observed, only an increase of ceruloplasmin activity was found. It was concluded that ceruloplasmin had no 'histaminase activity' as has been assumed by other authors using the o-dianisidine test.
Amine Oxidase (Copper-Containing)↗
DNA sensor for o-dianisidine.
o-Dianisidine (3,3'-dimethoxybenzidine) is applied in the production of some dyes and also used in analytical tests. However, this compound is anticipated to be a human carcinogen. An analytical strategy utilizing square wave voltammetry for the determination of o-dianisidine is presented. An electrochemical system was consisted of three electrodes: carbon paste working electrode, platinum wire counter electrode and silver-silver chloride (Ag/AgCl) reference electrode. However, square wave voltammograms of direct measurements of o-dianisidine were found to be hardly reproducible, exhibiting few peaks due to some labile short-lived intermediates with the only exception of a quite stable peak at +0.7 V vs. Ag/AgCl. Quantitative determination of o-dianisidine gave satisfactory results only when the carbon paste working electrode was replaced by deoxyribonucleic acids (DNA) electrode obtained by immobilization of double-stranded (ds) DNA on carbon electrode. Square wave voltammogram of DNA showed two peaks attributed to adenine and guanine and the latter was used as analytical signal. After interaction with o-dianisidine, guanine oxidation peak was reduced to the extent related to the concentration of the analyte. Initial reduction of guanine peak took place already at the concentration of o-dianisidine equal to 0.4 microM; high concentrations (above 100 microM) of the analyte quenched completely a guanine response. The presented electrochemical system enables a specific detection of o-dianisidine by the presence of an oxidation peak at +0.7 V and its quantitative determination by measuring a reduction of guanine peak by means of a DNA sensor.
Adenine↗
[Kinetics and mechanism of individual and combined oxidation of o-dianisidine and bismuthol I by hydrogen peroxide catalyzed by horseradish peroxidase].
The individual and combined oxidation of 5-mercapto-1,3,4-thiadiazolthione-2 (bismuthol I) and 3,3'-dimethoxybenzidine (o-dianisidine) by hydrogen peroxide catalyzed by horseradish peroxidase (pH 5,0) was studied. It was shown that bismuthol I is a substrate for peroxidase, which is competitive towards o-dianisidine. In the presence of bismuthol I the Km value for o-dianisidine is increased, while kappa cat remains unchanged. The competitive inhibition constant for bismuthol is equal to 19,4 mkM. An addition of bismuthol I causes the appearance of the induction period (tau) on the kinetic curves of o-dianisidine oxidation, whose duration is proportional to bismuthol concentration. The kinetic patterns allowing interpretation of the experimental results are proposed. A kinetic analysis permitted to determine the rate constants for the individual steps of omicron-dianisidine oxidation catalyzed by peroxidase.
Benzidines↗
Kinetics of oxidation of o-dianisidine by hydrogen peroxide in the presence of antibody complexes of iron(III) coproporphyrin.
The complex of iron(III) coproporphyrinI (FeCPI) with antibody D5E3 was studied as an artificial peroxidase, using o-dianisidine as a substrate. At saturation with respect to antibody, the initial rates of o-dianisidine oxidation are practically the same for free and bound FeCPI at a concentration 5 x 10(-9)M, but the catalytic rate constant (kc) for bound FeCPI exceed (kc) for free FeCPI by two- to three-fold. This difference can be explained by a real enhancement of (kc) at the antibody-active site. The dependence of initial rates of the reaction on substrate concentrations obeyed Michaelis-Menten kinetics and revealed substrate activation at high concentrations of o-dianisidine. A comparison of the Stern-Volmer constants for o-dianisidine-induced quenching of the porphyrin fluorescence proves that antibody-bound coproporphyrin is equivalently accessible to the substrate as protoporphyrin bound to apoperoxidase from horseradish peroxidase (HRP). Based on analysis of the (kc) dependence on H2O2 concentrations in the FeCPI-antibody system, we suggest that interaction with hydrogen peroxide is the rate-limiting step for the oxidation reaction.
Antibodies, Catalytic↗
[Cooxidation of potassium ferrocyanide and o-dianisidine by hydrogen peroxide catalyzed by horseradish peroxidase. Substrate-substrate activation].
The kinetics of cooxidation of potassium ferrocyanide and o-dianisidine by hydrogen peroxide catalyzed by horseradish peroxidase were studied. The peroxidation of potassium ferrocyanide is activated by o-dianisidine. A scheme illustrating the direct involvement of the enzyme in substrate-substrate activation is proposed. A method for determination of the rate constants of the first electron transfer from o-dianisidine to peroxidase (i. e. reduction of peroxidase E1 to E2) and of the constants for o-dianisidine binding by E1 was developed.
Benzidines↗
Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 2. Evidence for a substrate radical--enzyme complex and its reaction with nucleophiles.
Changes in the optical absorption spectrum of horseradish peroxidase, during the oxidation of o-dianisidine at pH 7.5, reveal an intermediate distinct from the previously described compounds I and II. The rate of decay of this new complex appeared to be rate limiting for the catalytic cycle, in this pH range, since imidazole, which augments the catalytic reaction, also enhanced the rate of decay of this complex. Nitrogenous compounds reportedly unable to ligate to hemes, such as 2-methylimidazole and benzimidazole, were nevertheless capable of augmenting the HRP-catalyzed rate of oxidation of o-dianisidine. The activity of nitrogenous compounds, in this regard, appeared to be a function of their nucleophilicity and was sensitive to steric factors but relatively free of a deuterium solvent isotope effect. The data presented in this and in the preceding paper [Claiborne, A., & Fridovich, I. (1979) Biochemistry 18 (preceding paper in this issue)] lead to the suggestion that the nucleophile-responsive intermediate is an enzyme--dianisidine radical complex and that abstraction of the second electron from the bound radical is facilitated by binding of nitrogenous nucleophiles.
Benzidines↗
[Kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase].
A kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase within the pH range of 3.7-9.0 has been carried out. It was shown that the reaction of o-dianisidine peroxidase oxidation obeys the Michaelis--Menten kinetics; the kcat and Km values within the pH range used were determined. The optimum of peroxidase catalytic activity during o-dianisidine oxidation was observed at pH 5.0-6.0. The kinetic pattern of the reaction is discussed. It was demonstrated that deprotonation of the group at pK 6.5 decreases the kcat value 60 times. At pH greater than 8.0 an additional ionogenic group controls the enzyme activity.
Benzidines↗
Purification of the o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities.
Extracts of aerobically grown Escherichia coli B exhibit both catalase and dianisidine peroxidase activities. Polyacrylamide gel electrophoresis demonstrates two distinct catalases which have been designated hydroperoxidases I and II (HP-I and HP-II) in order of increasing anodic mobility. HP-I has been purified to essential homogeneity and found to be composed of four subunits of equal size. Its molecular weight is 337,000, and it contains two molecules of protoheme IX per tetramer. Its amino acid composition is unusual, for so large a protein, in lacking half-cystine. HP-I is a very efficient catalase with an activity optimum at pH 7.5, a Km for H2O2 of 3.9 mM, and a turnover number of 9.8 x 10(5) per min. It is also a broad specificity peroxidase capable of acting upon dianisidine, guaiacol, p-phenylenediamine, and pyrogallol. Dianisidine acted as a powerful reversible inhibitor of the catalatic activity of HP-I and as a suicide substrate when HP-I functioned in its peroxidatic mode.
Amino Acids↗
Steady-state kinetics of combined oxidation of hydroquinone and o-dianisidine by hydrogen peroxide in the presence of horseradish peroxidase
The steady-state kinetics of horseradish peroxidase-catalyzed oxidation of hydroquinone was studied. Hydroquinone was shown to be a rapidly oxidizable substrate of the peroxidase. Values of kcat and Km for this substrate were determined in the pH range 4-7. The oxidation of hydroquinone and o-dianisidine was distinguished when both were present in the reaction mixture. o-Dianisidine was not oxidized until hydroquinone was completely converted. The rate of hydroquinone oxidation by peroxidase in the presence of o-dianisidine was 3-10 times higher than the rate of its individual oxidation. The activator decreased the Km for hydroquinone oxidation.
Journal Article↗
Automated assay of ceruloplasmin by kinetic analysis of o-dianisidine oxidation.
Automated procedures for the kinetic assay of serum ceruloplasmin activity using a bichromatic and a centrifugal analyzer are described. The method is based on the oxidase activity of ceruloplasmin at pH 5.0 with o-dianisidine as substrate. Enzyme activity is reported in I.U./l, based on the molar absorption coefficient of o-dianisidine consumed. The substrate is stable and is not subject to non-enzymatic oxidation. Comparison with a manual reference end-point assay using the same substrate indicates good correlation of the bichromatic and centrifugal methods. The analytical precision is comparable to the manual assay for both methods.
Autoanalysis↗
The use of o-dianisidine for serum haptoglobin electrophoresis using cellulose acetate.
1. Due to the carcinogenicity of benzidine, a method by which o-dianisidine is used to stain serum haptoglobin is described. Serum haptoglobin is determined by electrophoresis using cellulose acetate as the medium. 2. A comparison of the two staining systems demonstrates good agreement. 0-Dianisidine can be substituted for benzidine without loss of specificity.
Dianisidine↗
Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin.
Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H2O2 was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type 1b Cu(II).
Animals↗
Pyrocatechol as a stabilizing agent for o-tolidine and o-dianisidine: a sensitive new method for HRP neurohistochemistry.
A new procedure for detecting HRP in nerve tissue is described which is based on the use of pyrocatechol to stabilize the oxidation products of o-tolidine and o-dianisidine in citric acid/ammonium acetate buffer of pH 4.85. In both cases the precipitate obtained is insoluble, stable and more visible than when any variant of the diaminobenzidine method is employed, and the morphological image of neurons and nerve fibres labelled with HRP is superior to that produced by the tetramethylbenzidine/sodium nitroprusside method. There is no non-specific precipitation, and no retraction of nerve tissue has been observed. The performance of the method is improved further using either o-tolidine/pyrocatechol or o-dianisidine/pyrocatechol in conjunction with glucose oxidase, which may be useful if it is desired to obtain Golgi-like images of HRP-bearing cells or to display weakly HRP-labelled nerve fibres.
Animals↗
Health hazard alert--benzidine-, o-tolidine-, and o-dianisidine-based dyes DHHS (NIOSH) publication No. 81-106.
Recent data from animal tests, case reports, and other sources about the carcinogenic effects and metabolism of benzidine-, o-tolidine-, and o-dianisidine-based dyes have come to the attention of OSHA and NIOSH. Both agencies have reviewed the data and concluded that the findings establish the potential of these dyes to cause cancer in humans. OSHA and NIOSH conclude that persons working with these dyes should be aware of the potential health hazards that could result from excessive exposure to them. The intent of this document is to summarize the information available on the carcinogenic effects and metabolism of benzidine-, o-tolidine-, and 0-dianisidine-based dyes and to provide guidance so that employers, employees, and physicians may work together to reduce potential health hazards that could result from excessive exposure to these dyes.
Animals↗
Measurement of constitutive L-pyrrolidonyl peptidase activity from Streptococcus and Enterococcus using tetrazotized 0-dianisidine.
The detection of L-pyrrolidonyl peptidase activity is extremely useful for the differentiation of Enterococcus species and Streptococcus pyogenes from other members of the family Streptococaceae. This analysis has generally been performed utilizing the hydrolyzable substrate L-pyrrolidonyl beta-naphthylamine. After the substrate was hydrolyzed, free beta-naphthylamine has been detected utilizing the reagent para-dimethylaminocinnamaldehyde. The cinnamaldehyde and naphthylamine reagents combined to form an orange color, much like the indole reaction. The use of the cinnamaldehyde reagent had several drawbacks however: color development was not sharp, and the reagent was difficult to produce, and it was not stable. A new indicator system employing tetrazotized 0-dianisidine was developed. An extremely deep burgundy colored complex resulted from the reaction between the new indicator and B-Naphthylamine. This diazo reagent showed excellent correlation with results obtained with para-dimethylaminocinnamaldehyde and yielded more objective, distinct endpoints. This inexpensive reagent can be utilized either in a liquid form or dried on paper discs.
Aminopeptidases↗
o-Dianisidine: a new reagent for selective spectrophotometric, flow injection determination of chlorine.
A flow injection analysis (FIA) procedure for the determination of free chlorine in industrial formulations and water samples is proposed. The manifold is provided with a gas-diffusion unit which permits the removal of interfering species and also the preconcentration of chlorine. The determination of chlorine is performed on the basis of the oxidation by o-dianisidine as a chromogenic reagent to a coloured product which can be monitored at 445 nm. The method (for a preconcentration step of 60 s) is linear over the range 0.04-1.00 mg l(-1) of chlorine, the limit of detection is 0.04 mg l(-1), the reproducibility of the procedure (as RSD of the slope) is 3.7% for a series of four independent calibrations, the precision (as RSD of a series of 30 continuous FIA peaks of 0.56 mg l(-1) of chlorine) is 1.4% and the sample throughput is 40 h(-1). A detailed comparative study of the analytical characteristics of a single mono-channel reverse FIA assembly and the same system but provided with a Fluoropore membrane filter of 0.5 microm pore size was performed to check the advantages of the new approach in terms of sensitivity, selectivity and limit of detection.
Chlorine↗
Prostaglandin H synthase oxidation of benzidine and o-dianisidine: reduction and conjugation of activated amines by thiols.
Prostaglandin H synthase oxidized the carcinogens benzidine and o-dianisidine to their respective quinonediimines. Analysis of the reaction media by u.v./visible spectroscopy and liquid chromatography with electrochemical and radiochemical detection revealed that these quinonediimines can be both conjugated and reduced by glutathione, cysteine and N-acetylcysteine. Analysis of the purified conjugate formed between synthetic benzidinediimine and glutathione by proton magnetic resonance spectroscopy demonstrated the product to be 3-(glutathion-S-yl)-benzidine. This metabolite was also formed during peroxidation of benzidine by prostaglandin H synthase in the presence of excess glutathione. These conjugates may be useful markers of peroxidatic activation of aromatic amines in vivo.
Amines↗