Quantitative determination of hemoglobin and cytochemical staining for peroxidase using 3,3',5,5'-tetramethylbenzidine dihydrochloride, a safe substitute for benzidine.

The quantitative determination of hemoglobin employing 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB-d) is described. This agent can also be used for staining myeloperoxidase-containing granules in granulocytes. TMB-d is at least as sensitive a reagent as benzidine but, unlike benzidine and diaminobenzidine, it is not a carcinogen.     

Peroxidase activities in bull spermatozoa

The present study was carried out to determine the localization of peroxidase activity in bull spermatozoa. 3,3′-Diaminobenzidine (DAB) was used as a substrate for revealing peroxidase activity, and light and electron microscopic analysis of the results obtained was performed. Peroxidase activity was detected in the mitochondria of the middle piece and the outer acrosomal membrane. Catalase was excluded as an enzyme, catalyzing the detected peroxidase activity. Concerning the biochemical properties of bull sperm peroxidases, peroxidase activity was found to be manifested in a large pH range, 4–10.5. Bull sperm peroxidase activity appeared to be temperature sensitive and azide sensitive and could be readily inhibited by phenylhydrazine. Electrophoretic analysis of the proteins from bull sperm extracts separated in a Davis-Ornstein system of 7% polyacrylamide gel, followed by the determination of peroxidase activity on the polyacrylamide gels, revealed that all 14 sperm protein fractions available on the gel possessed peroxidase when benzidine was used as a substrate. The possible reasons for the electrophoretic heterogeneity of bull sperm peroxidases are discussed. © 1994 Wiley-Liss, Inc.     

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-H₂O₂ as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.     

The effect of acetyl-CoA supplementation on the mutagenicity of benzidines in the Ames assay

The conventional Ames assay metabolising system was confirmed to be deficient in its ability to N-acetylate. This may render the test less sensitive to compounds which normally have an acetylation step during their in vivo activation to carcinogens. The addition of acetyl-coenzyme A to the S9 mix in the Ames assay increased the mutagenicity of benzidine in Salmonella typhimurium strains TA98 and TA1538 4-5-fold. This was consistent with the observation that benzidine is N-acetylated prior to DNA binding in vivo in rat liver. Two 3,3'-disubstituted benzidines, o-tolidine and o-dianisidine, were also tested. A smaller increase in o-tolidine mutagenicity, compared to that observed with benzidine, occurred with the addition of acetyl-coenzyme A. However, the production of acetylated metabolites from o-tolidine was only 37% of that from benzidine. The mutagenicity of o-dianisidine was unaffected by acetyl-coenzyme A. Acetylation of o-dianisidine was only 16% of that observed with benzidine, and the N-acetyl derivatives of o-dianisidine showed lower mutagenicity than the parent amine. The differing responses of benzidine, o-tolidine and o-dianisidine to addition of acetyl-coenzyme A suggests it may not be possible to simply infer the metabolism of 3,3'-disubstituted benzidines to DNA binding species from data on benzidine itself.     

GelStar nucleic acid gel stain: high sensitivity detection in gels.

GelStar nucleic acid gel stain can be used for sensitive fluorescent detection of both double-stranded (ds) and single-stranded (ss) DNAs, oligonucleotides and RNA in gels. The stain can be added to agarose gels at casting for immediate imaging after electrophoresis or can be used after electrophoresis with both agarose and acrylamide gels. GelStar stain is highly fluorescent only when bound to nucleic acids thus giving superior signal-to-noise ratios and obviating the need to destain the gel. The detection limits of GelStar strain are 20 pg for dsDNA, 25 pg for ssDNA and 10 ng for native or glyoxal-treated RNA.     

Specific indication of hemoproteins in polyacrylamide gels using a double-staining process

Hemoproteins were revealed in polyacrylamide gels in the presence of sodium dodecyl sulfate by staining with different benzidine derivatives. When the protein samples were treated with either beta-mercaptoethanol or dithiothreitol, a significant decrease in peroxidase activity of the proteins possessing noncovalently bound heme led to diminished staining. However, when Coomassie blue R-250 staining followed the hemespecific stain it was observed that the hemoprotein bands stained more intensely than duplicate sample bands that had been stained only with the Coomassie blue R-250. This staining property allows the indication of hemoproteins in gels even after the peroxidase yield has been significantly depleted by reducing agents.     

Limitations of the hemoglobin method for detecting lipid hydroperoxides

The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides, trilinoleoylglycerol hydroperoxides and phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were different. Some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. Front these results, the Hb method needs to be carefully applied to biological materials that contain mixtures of different typos of lipid classes.     

Binding of chlorinated benzidines to the rat hepatic aromatic hydrocarbon receptor

The binding of benzidine, 3,3'-dichlorobenzidine (3,3'-Cl2BZ), and the asymmetrically-substituted chlorinated benzidines 3,5-dichlorobenzidine (3,5-Cl2BZ) and 3,5,3'-trichlorobenzidine (Cl3BZ) to the rat hepatic cytosolic aromatic hydrocarbon (Ah) receptor was measured, in order to assess the mechanism of P-450I induction by 3,3'-Cl2BZ. Cl3BZ is the most mutagenic benzidine derivative in the Ames assay. Binding affinity to the Ah receptor protein was determined by displacement of labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the receptor, measured with the sucrose density gradient centrifugation technique. The rank order of affinities and the apparent inhibitor constants were: Cl3BZ (4 microM) greater than 3,5-Cl2BZ (8.4 microM) greater than 3,3'-Cl2BZ (10 microM). Benzidine did not displace TCDD from the receptor protein. 4-Aminobiphenyl a structural link between the benzidine and biphenyl series competed weakly with TCDD. The 50% inhibition concentration was about 150 microM. The results are consistent with the hypothesis that the induction of P-450 enzymes by 3,3'-Cl2BZ in vivo is mediated by the Ah receptor.     

A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels

Mycobacteria produce two classes of catalase, designated T and M. Only the T-catalase also has a peroxidase-like function. When a 3,3'-diaminobenzidine (DAB) peroxidase stain was applied to polyacrylamide gel electrophoresis gels, followed by a ferricyanide negative stain for catalase, isoenzymes of T-catalase appeared as dark bands within a zone of clearing in the green background; the M-catalase appeared only as a clear zone. Heated and unheated preparations could be used to demonstrate the presence of comigrating bands of M and T. The application of the ferricyanide stain after the DAB stain of T-catalase resulted in marked intensification of the positive bands of T-catalase due to nonenzymatic, peroxide-independent reduction of the ferricyanide by the DAB product.     

Polyphenols of Eucalyptus sideroxylon wood

Twenty-three major components were detected in the methanol extractives of the heartwood of Eucalyptus sideroxylon. The components identified include resveratrol, resveratrol-β-glucoside, 3,3′-di- and 3,3′,4-tri-o-methylellagic acids and their glucosides. The 3,3′-di-o-methylellagic acid 4′-glucoside isolated had properties significantly different from those previously reported for this compound. Also present were gailic acid, catechin, ellagic acid, an unidentified stilbene, the ellagitannins D-6 and D-13, polymerized leucocyanidin and an oily material. The sapwood contained gailic acid, small amounts of ellagitannins and ellagic acids and traces of other components. The heartwood extractives of related eucalypt species were also examined.     

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.     

Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.     

Study on the enzymatic 5′-deiodination of 3′,5′-diiodothyronine using a radioimmunoassay for 3′-iodothyronine

A radioimmunoassay for 3′-iodothyronine has been developed. All iodothyronine analogues (except 3,3′-diiodothyronine) showed very little (0.02% at most) cross-reactivity, and the assay was sensitive to 1 pg 3′-iodothyronine/ tube. We have studied the 5′-deiodination of 3′,5′-diiodothyronine by rat liver microsomal fraction in the presence of dithiothreitol. Production of 3′-iodothyronine at 37°C was found to be linear with time of incubation up to 30 min and with concentration of microsomal protein up to 100 μg/ml. The reaction rate reached a limit on increasing 3′,5′-diiodothyronine concentration to 10 μM. The effect of pH on 3′-iodothyronine production was found to depend on 3′,5′-diiodothyronine concentration. Increasing 3′,5′-diiodothyronine concentration from 0.1 to 10 μM resulted in a shift of the pH optimum from 6–6.5 to 7.5. Similar effects on the 5′-deiodination of 3,3′,5′-triiodothyronine were observed, supporting the hypothesis that these reactions are catalysed by a single enzyme (iodothyronine 5′-deiodinase).     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

Oxidation of Benzidine and Its Derivatives by Thyroid Peroxidase

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.     

微量蛋白检测中不同银染方法的比较与分析

随着生物化学技术的不断发展,作为检测SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)中微量蛋白的银染方法也在不断改进和发展.采用4种不同的银染方法检测不同含量的牛血清白蛋白,结果显示单纯的银染过程中如果使用戊二醛固定会使蛋白检出更快速灵敏,而结合考马斯亮蓝的复合银染则较单纯银染灵敏度提高了5～7个数量级.     

Studies on the effect of reagent and protein charges on reactivity of the β93 sulfhydryl group of human hemoglobin using selected mutations

The reactivity of the β93 sulfhydryl (SH) group of human oxyhemoglobins with the negatively charged 5,5′-dithiobis(2,2′-nitrobenzoate) and the uncharged 2,2′-dithiodipyridine was determined as a function of pH. Selected mutant hemoglobins having increased oxygen affinity and having residue substitutions altering charge near the SH group (Wood, Malmö, Yakima, Kempsey, Andrew-Minneapolis, Osler, and Chesapeake) were compared to hemoglobin (Hb) A. Although both reagents reacted with GSH at the same rate and with the same enthalpies of activation, the rates with Hb were different and the difference showed a pronounced pH dependence. The charged reagent was sensitive to charges near the SH group; a positive charge increased the rate and a negative one decreased the rate. The uncharged reagent which reacted with Hb A with activation enthalpies similar to those for GSH was insensitive to neighboring charges, but was sensitive to tertiary and quaternary structural changes. The rates obtained with the latter reagent did not correlate with oxygen affinity. The evolutionary aspects of the β93 cysteine in relation to structure and function are reviewed.     

Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic

We describe here the use of Alta, a pre-existing scarlet-red stain of cosmetic use, for staining proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels, as well as for a single step staining of gels and nitrocellulose membranes during Western blot analysis. This stain, which is composed of 0.8% Crocein scarlet (brilliant crocein) and 0.2% Rhodamine B, is inexpensive, easy to use and nearly as sensitive as Coomassie Brilliant Blue (CBB) R-250. The gels can be stained in 10% Alta (2 h) and can be destained effectively only with 7% acetic acid as opposed to the conventional destainer (methanol/acetic acid/water) required for CBB-stained gels. In an alternative procedure, the proteins can be stained on the gel while electrophoresis by simply using 5% Alta in the top tank buffer and the stain can be viewed under UV-transilluminator. This procedure can also be used for Western blot analysis, as a single step procedure for staining of proteins on the gel as well as on the nitrocellulose membrane, as the stain is retained on the membrane after protein transfer. Thus, this staining procedure allows monitoring of proteins after each step in the Western blot, thereby eliminating the need to run separate gels for staining and Western blot analysis, and also the need for Ponceau Red S staining of the nitrocellulose membrane during Western blot analysis.     

A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.     

Evaluation of new 2,2'-dimethyl-5,5'-dipropoxybenzidine- and 3,3'-dipropoxybenzidine-based direct dye analogs for mutagenic activity by use of the Salmonella/mammalian mutagenicity assay

As part of a continuing study aimed at establishing structure-activity relationships and heuristic principles useful for the design of non-genotoxic azo dyes, a series of new direct dyes based on two non-mutagenic benzidine analogs, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 3,3'-dipropoxybenzidine, were evaluated for mutagenic activity in Salmonella typhimurium strains TA98 and TA100. These strains are widely used for mutagenicity screening and have been shown to detect the mutagenic activity of benzidine analogs. While some toxicity was seen with some dyes at high doses, all of the dyes examined were judged non-mutagenic with and without metabolic activation in the standard Salmonella plate-incorporation assay. The results in the standard test are consistent with the properties of the diamines themselves. However, only one of the dyes was non-mutagenic when a reductive-metabolism pre-incubation assay was used. The results of this study suggest that although benzidine analogs are potential replacements for benzidine, there is a need to understand which mutagenic products are produced when reductive metabolism is present. There is also a need to know whether or not metal complexes of these dyes are mutagenic. Such information will allow the development of new non-mutagenic azo dyes.