Spectrophotometric assay for superoxide dismutase based on the nitroblue tetrazolium reduction by glucose-glucose oxidase.
ABSTRACT A new spectrophotometric assay of superoxide dismutase (SOD) is described. The assay is based on the SOD-mediated inhibition in the rate of nitroblue tetrazolium reduction to the blue formazan at alkaline pH. The optimized assay of SOD is performed in 50 mM glycine-NaOH buffer, pH 9.5, at 25 degrees C. The SOD concentration is determined from the V/v ratio of rates measured in the absence (V) or the presence (v) of SOD. One unit of SOD has been defined as the concentration that decrease the rate to 50% (V/v = 2). The assay is simple, sensitive, uses commercially available reagents, rapid and easy to perform and could be used routinely for monitoring superoxide dismutase levels in purified protein fractions.
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ABSTRACT: Nitroblue tetrazolium (NBT) reduction to formazan has been used as a marker for nitric oxide synthase (NOS). Since inducible NOS activity is elevated in urine from patients with urinary tract infections (UTIs), we investigated the accuracy of NBT reduction as an early predictor of UTIs and quantified the relationship between inducible NOS and NBT. Urine samples from 434 patients were screened for the presence of UTIs with leukocyte-esterase and nitrite dipsticks and with NBT reduction. The rapid screening results from each test were compared to urine culture results. In addition, NBT reduction parameters were measured in urine pellet at 595 nm after incubation with one of four factors: NOS cofactors, NOS inhibitors, NADH, or superoxide dismutase/catalase. As a urine screening test for UTIs, NBT reduction was more sensitive with a higher negative predictive accuracy than the nitrite dipstick. NBT reduction also was more specific with a higher positive predictive accuracy and negative predictive accuracy than the leukocyte-esterase dipstick. In infected urine pellet, both NADPH, a NOS cofactor, and NADH increased NBT reduction. Superoxide dismutase/catalase decreased NBT reduction. Although NOS may not be the only NBT reducing enzyme, rapid, visible reduction of NBT is induced in urine from patients with UTIs.Kidney International 11/1998; 54(4):1331-6. DOI:10.1046/j.1523-1755.1998.00102.x · 8.52 Impact Factor
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ABSTRACT: Aerobic organisms have devised several enzymatic and non-enzymatic antioxidant defenses to deal with reactive oxygen species (ROS) produced by cellular metabolism. To combat such stress, cells induce ROS scavenging enzymes such as catalase, peroxidase, superoxide dismutase (SOD) and glutathione reductase. In the present research, we have used a double staining technique of SOD and catalase enzymes in the same polyacrylamide gel to analyze the different antioxidant enzymatic activities and protein isoforms present in Saccharomyces and non-Saccharomyces yeast species. Moreover, we used a technique to differentially detect Sod1p and Sod2p on gel by immersion in NaCN, which specifically inhibits the Sod1p isoform. We observed unique SOD and catalase zymogram profiles for all the analyzed yeasts and we propose this technique as a new approach for Saccharomyces and non-Saccharomyces yeast strains differentiation. In addition, we observed functional correlations between SOD and catalase enzyme activities, accumulation of essential metabolites, such as glutathione and trehalose, and the fermentative performance of different yeasts strains with industrial relevance.Applied Microbiology and Biotechnology 01/2013; 97(10). DOI:10.1007/s00253-012-4672-1 · 3.81 Impact Factor
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ABSTRACT: Background Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Recently it has been characterized in this human pathogen a flavodiiron protein and a rubrerythrin, but no physiological reductants have been identified. Methods The present work deals with biochemical studies aimed to reach a better understanding of the kinetic and structural properties of an amebic rubredoxin reductase and two ferredoxins from E. histolytica. Results We have complemented the characterization in E. histolytica of two different metabolic pathways for O2 and H2O2 detoxification. We characterized a novel amebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, this enzyme exhibited NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. Additionally, we described how different ferredoxins were also efficient as reductant substrate for flavodiiron protein and rubrerythrin. Conclusions and General Significance These proteins could contribute to O2 and H2O2 detoxification as two alternative systems in vivo, playing an important role in the parasite defense against reactive oxidant species. So far we know this is the first characterization of eukaryotic NROR protein (from E. histolytica) and the first kinetic study of ferredoxin-dependent reduction of flavodiiron protein and rubrerythrin.Biochimica et Biophysica Acta (BBA) - General Subjects 02/2015; 1850(6). DOI:10.1016/j.bbagen.2015.02.010 · 3.83 Impact Factor