Additional factors influencing sensitivity in the tetramethyl benzidine method for horseradish peroxidase neurohistochemistry

In experiments that use horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) for tracing neural connections, the activity of tissue-bound enzyme as well as the stability of the resultant reaction product are influenced by the duration of storage, the composition of the storage medium, the type of counterstaining and even the details of histological dehydration. Furthermore, the conditions for preserving HRP activity are very different from those necessary for preserving the stability of the tetramethyl benzidine (TMB) reaction product. Thus, tissue-bound HRP activity is stable at a neutral pH, while a much lower pH, around 3.3, is required for preserving the stability of the TMB reaction product. Recent evidence indicates that the stabilization bath in sodium nitroferricyanide that was previously recommended is not necessary. However, gradual dehydration of mounted sections is essential for long-term stability. Excessive counterstaining and excessive dehydration interfere with the detection of reaction product. These considerations are pertinent to experiments using free HRP as well as to those where the enzyme has been conjugated to wheat germ agglutinin.     

Peroxidase-catalyzed benzidine binding to DNA and other macromolecules

[14C]Benzidine is rapidly oxidized by a peroxidase/H2O2 system to products which bind irreversibly to DNA. The presence of exogenous DNA also prevented benzidine polymerization to 'benzidine brown' and azobenzidine. Two molar equivalents of H2O2 were required to oxidize the benzidine and achieve maximal DNA binding. Furthermore, 95% of the benzidine was trapped and 36 nmol benzidine was bound per mg DNA. Polyriboguanylic acid was as effective as DNA in binding benzidine, but polyriboadenylic acid, polyribouridylic acid and polyribocytidylic acid were much less effective. Binding of [14C]benzidine correlated well with the absorbance at 295 nm and 390 nm of the modified DNA or various synthetic homopolymers of ribonucleotides isolated from the reaction mixture. The peroxidase/H2O2 system also catalyzed the binding of dichlorobenzidine, o-tolidine and o-dianisidine to DNA but 3,5,3',5'-tetramethylbenzidine, a non-carcinogen, did not bind. The binding could be prevented by various biological hydrogen donors, thiols, or phenolic antioxidants. The mechanisms for DNA protection were investigated; the oxidized benzidine species involved in binding can be reduced with ascorbate, NADPH, or thiols, and trapped by thiols or phenolic antioxidants to form conjugates or adducts.     

Peroxidative metabolism of benzidine derivatives by Salmonella typhimurium

Benzidine and several derivatives are activated to mutagenic species in an H2O2-dependent Ames test system. Optical and electron paramagnetic resonance (EPR) spectroscopy are employed in studies of the H2O2-dependent oxidation of benzidine and 3,5,3',5'-tetramethylbenzidine (TMB) catalyzed by intact bacteria, and provide direct evidence for peroxidase activity in Salmonella typhimurium. The acetylase-proficient Ames tester strain TA98 and its acetylase-deficient derivative TA98/1,8-DNP6 are equally responsive to H2O2-dependent mutagenicity; enzymatic acetylation appears not to be involved in activation of benzidine, in this system. The H2O2-dependent mutagenicity of benzidine and oxidation of TMB are observed when the assays are carried out in acetate buffer (pH 5.5), but not in 2-[N-morpholino]ethane sulfonic acid (MES) buffer, at the same pH. This difference is interpreted in terms of the effects of these buffers on the intracellular pH of the bacteria. The H2O2-dependent mutagenicity of several benzidine congeners is also described.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes

We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis.     

Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O₂ evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II_f and one Signal II_s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z⁺ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.     

Studies on the Energy-coupling Sites of Photophosphorylation: V. Phosphorylation Efficiencies (P/e(2)) Associated with Aerobic Photooxidation of Artificial Electron Donors

The rate of Hill reaction can be measured accurately as O₂ uptake (the Mehler reaction) if a rapidly autoxidizable electron acceptor (e.g., methylviologen) is used. However, when an artificial electron donor-ascorbate couple (or ascorbate alone) replaces the natural donor, water, the rate of O₂ consumption is no longer a reliable measure of the electron flux, because superoxide radical reactions contribute to O₂ uptake. Such radical reactions, however, can be suppressed by adding enough superoxide dismutase to the reaction mixture. Indeed in all of the photosystem I- and photosystem II-donor reactions tested (except with benzidine which was tested without ascorbate added), the O₂ uptake was inhibited by 30 to 50% by the addition of superoxide dismutase. The rate of phosphorylation was totally unaffected by the enzyme. The reasessment of the phosphorylation efficiencies thus made by the use of superoxide dismutase led us to the following conclusions. The phosphorylation efficiency associated with the transfer of electrons from a donor to methlylviologen (than to O₂) through both photosystems II and I is practically independent of the donor used—catechol, benzidine, p-aminophenol, dicyanohydroquinone, or water. The P/e₂ ratio is 1.0 ± 0.1. Only ascorbate gives a slightly lower value (P/e₂ = 0.9). (NH₂OH-treated, non-water-splitting chloroplasts were used for reactions with these artificial donors.) The phosphorylation efficiency associated with DCMU-insensitive, photosystem I-mediated transfer of electrons from a donor to methylviologen (then to O₂) is again largely independent of the donor used, such as diaminodurene, diaminotoluene, and reduced 2,6-dichlorphenol-indophenol. The P/e₂ ratio is 0.6 ± 0.08.     

Quantification of small amounts of hemoglobin in polyacrylamide gels with benzidine.

Benzidine-hydrogen peroxide is a sensitive blood stain. Since benzidine is carcinogenic, less hazardous substitutes have been sought which retain the specificity and sensitivity of benzidine. We have tested two benzidine derivatives, 3,3′-dimethoxybenzidine and 3,3′, 5,5′-tetramethylbenzidine, reported to be satisfactory substitutes, but have found both to be unsatisfactory for quantifying small amounts of hemoglobin (Hb) on polyacrylamide gels. However, our data show that benzidine stains gels with an intensity proportional to the amount of Hb present when the staining time and temperature are controlled. The stain is fast, sensitive, and specific for Hb, gives very little background stain, and is stable if the gels are thoroughly rinsed and stored in distilled-deionized water. The relative amounts of Hb in different bands or different gels may be quantified later by densitometry. Procedures are suggested for using benzidine while giving adequate attention to governing regulations and personnel safety.     

Amine functional monodisperse microbeads via precipitation polymerization of N-vinyl formamide: immobilized laccase for benzidine based dyes degradation

Densely cross-linked poly(vinylamine) microbeads (∼2 μm) were prepared by precipitation copolymerization of N-vinyl formamide and ethylene glycoldimethacrylate in acetonitrile. The formamido groups of the microbeads were hydrolyzed into amino groups. Then, amino-functionalized microbeads were used for covalent immobilization of laccase via glutaraldehyde coupling. The average amount of immobilized enzyme was 18.7 mg/g microbeads. Kinetic parameters, V_max and K_m values were determined as 20.7 U/mg protein and 2.76 × 10⁻² mmol/L for free enzyme and 15.8 U/mg protein and 4.65 mmol/L for the immobilized laccase, respectively. The immobilized laccase was operated in a batch reactor for the degradation of two different benzidine based dyes (i.e., Direct Blue 1 and Direct Red 128). The laccase immobilized on the microbeads was very effective for removal of these dyes which interfere with the hormonal system.     

Cytochemical and electrophoretic studies of haemoglobin synthesis in the fat body of a midge, Chironomus thummi

The fat body of developing mid- and late fourth instar larvae of a midge, Chironomus thummi, has been investigated by means of the benzidine reaction for the localization of haemoglobin within cells. In the subepidermal fat body the reaction deposits of the haemoglobin pseudo-peroxidase activity appear predominantly in the intracisternal cavities of ER and the Golgi, and later, in the pharate pupal stage, in small dense granules (0.5–1 μm in. diameter).All the major protein bands of fat body extracts, which are resolved in electrophoresis, give the benzidine reaction and show incorporation of ¹⁴C-amino levulinic acids, in this case a specific marker for haemoglobin synthesis. In addition, labelled proteins show identical electrophoretic mobility as the haemoglobins of the haemolymph, suggesting that haemoglobins are synthesized in the fat body. Two types of fat body cells seem to differ with respect to their rôle in haemoglobin metabolism.     

Photooxidation of ferrocyanide and iodide ions and associated phosphorylation in NH2OH-treated chloroplasts

NH₂OH-treated, non-water oxidizing chloroplasts are shown to be capable of oxidizing ferrocyanide and I^? via Photosystem II at appreciable rates (? 200 μequiv/h per mg chlorophyll). Using methylviologen as electron acceptor, ferrocyanide oxidation can be measured as O₂ uptake, as ferricyanide formation, or as H⁺ consumption (2 Fe²⁺ + 2H⁺ + O₂ → 2 Fe³⁺ + H₂O₂). I^? oxidation can be measured as methylviologen-mediated O₂ uptake, or spectrophotometrically, using ferricyanide as electron acceptor. The oxidation product I₂ is re-reduced, as it is formed, by unknown reducing substances in the reaction system.The rate-saturating concentrations of these donors are very high: 30 mM with ferricyanide and 15 mM with I^?. Relatively lipophilic Photosystem II donors such as catechol, benzidine and p-aminophenol saturate the photooxidation rate at much lower concentrations (< 0.5 mM). It thus seems that the oxidation of hydrophilic reductants such as ferricyanide and I^? is limited by permeability barriers. Very likely the site of Photosystem II oxidation is embedded in the thylakoid membrane or is situated on the inner surface of the membrane.The efficiency of phosphorylation (P/e₂) is 0.5 to 0.6 with ferrocyanide and about 0.5 with I^?. In contrast the P/e₂ ratio is 1.0 to 1.2 when water, catechol, p-aminophenol or benzidine serves as electron donor. These differences imply that only one of two phosphorylation sites operate when ferrocyanide and I^? are oxidized. Ferrocyanide and I^? are also chemically distinct from other Photosystem II donors in that their oxidation does not involve proton release. It is suggested that the mechanism of energy conservation associated with Photosystem II may be only operative when the removal of electrons from the donor results in release of protons (i.e. with water, hydroquinones, phenylamines, etc.).     

A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride

Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3'-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels.     

Pitfalls in the light microscopical detection of NADH oxidase

Summary NADH oxidase activity has been detected at the ultrastructural level using cerium ions to trap H₂O₂ generated by the enzyme (via intermediate reactive oxygen species). In an attempt to localize NADH oxidase activity at the light microscope level using the cerium-diaminobenzidine (DAB)-nickel-H₂O₂, the cerium-DAB-cobalt-H₂O₂ or the cerium-alkaline lead procedures, the distribution patterns of the revealed enzyme were found to be identical to those for non-specific alkaline phosphatase and especially 5-nucleotidase activity. With the cerium-DAB-cobalt-H₂O₂ visualization procedure, the distribution pattern of the inial reaction product was similar to that obtained with the other two techniques but much less final reaction product was formed. Incubations for NADH oxidase activity performed in the presence of exogenous catalase or in the absence of catalase or peroxidase inhibitors did not affect the staining intensity, whereas inhibitors of 5-nucleotidase (EDTA) and non-specific alkaline phosphatase (levamisole) always did. Therefore, phosphatases contribute to the formation of the final reaction product. Since NADH initially cannot be hydrolysed by either of these two phosphatases, then presumably nucleotide prophosphatase (E.C.3.6.1.9) cleaves NADH into 5-AMP and nicotinamide mononucleotide in a first step. Both nucleotides can be hydrolysed further by the two monophosphatases. These then generate cerium phosphate which is detected by the DAB-nickel-H₂O₂, DAB-cobalt-H₂O₂ or lead visualization methods.     

Haemoglobin adducts of aromatic amines: diamines and polyaromatic amines

Aromatic amines and nitroarenes are important antioxidants and intermediates in the synthesis of dyes, pesticides and plastics. In the present paper we introduce methods for the synthesis of deuterated standards: 3-[²H₈]aminofluoranthene, 3,3′-dimethyl-[²H₄]benzidine, [²H₄]benzidine, N′-acetyl-[²H₄]benzidine, 2,4-[²H₆]toluenediamine, 2,6-[²H₆]toluenediamine. These standards have been used for the quantification of haemoglobin adducts of diamines and polyaromatic amines. Haemoglobin was hydrolysed in 0.1 M sodium hydroxide and the hydrolysate extracted with dichloromethane. The extracts were derivatised with heptafluorobutyric anhydride and analysed by GC–MS with negative chemical ionisation. In one run up to 15 aromatic amines can be determined: 6-aminochrysene, 3-aminofluoranthene, 2-aminofluorene, 1-aminopyrene, benzidine, 3,3′-dichlorobenzidine, 3,3′-dimethoxybenzidine, 3,3′-dimethylbenzidine, 3,3′-methylenedianiline, 4,4′-methylenedianiline, N′-acetyl-benzidine, N′-acetyl-4,4′-methylenedianiline, 4,4′-methylene bis(2-chloroaniline), 2,4-toluenediamine and 2,6-toluenediamine.     

A rapid assay for peroxidase activity

1. Peroxidase has been assayed by a chronometric method involving the coupled reaction of ascorbic acid with the product of the enzymic action on benzidine. 2. Measurements of the activities of horseradish and tea peroxidase by this and two other methods, involving respectively pyrogallol and o-dianisidine, are compared. 3. It is claimed that the chronometric method is relatively simple, rapid and accurate. 4. The method can be used in the presence of polyphenol oxidases.     

Changes in proton-magnetic-resonance spectra during aminolysis and enzymic hydrolysis of cephalosporins

1. Changes in proton-magnetic-resonance spectra were followed during the reaction of cephalosporins, deacetylcephalosporins, deacetoxycephalosporins and a Δ²-cephalosporin with ND₃ in D₂O. 2. Changes in proton-magnetic-resonance spectra were also followed during the hydrolysis of a cephalosporin and a deacetylcephalosporin in D₂O with a β-lactamase. 3. Structures for the reaction products are proposed. 4. The signals obtained after aminolysis of the β-lactam ring of a cephalosporin indicate that the reaction is accompanied by expulsion of the acetoxy group as acetate, formation of a double bond in the Δ⁴-position and the appearance of an exocyclic methylene group. 5. Aminolysis of deacetyl- and deacetoxycephalosporins can occur without immediate structural changes in the dihydrothiazine ring. 6. In contrast, the ring structure of the first product of enzymic hydrolysis of a deacetylcephalosporin is apparently identical with that of the product of aminolysis of the cephalosporin itself.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.     

Inactivation of Escherichia coli by superoxide radicals and their dismutation products.

Escherichia coli cells are inactivated by the products of the reaction between dialuric acid and oxygen, of which the primary product is Superoxide. The rate of inactivation is decreased by Superoxide dismutase, by catalase, and by EDTA, whereas it is increased by addition of cupric ions or hydrogen peroxide. It is concluded that a toxic product is formed in a reaction involving Superoxide, hydrogen peroxide, and metal ions, which might be the Haber-Weiss reaction, O₂^? + H₂O₂ → OH + OH^? + O₂. In radiation chemical experiments it is shown that this reaction does not occur in the absence of metal ions.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.