Measurement Of Catalase Activity Research Articles

An efficient protocol for quantifying catalase activity in biological samples

BackgroundCatalase is an important enzyme that helps protect cells against oxidative stress. The current protocol presents a reliable method for measuring catalase (CAT) enzyme activity in biological systems using the CUPRAC-CAT method.MethodsIn the CUPRAC-CAT method, the component of the enzymatic reaction was incubated before adding the Cu(Nc)22+ reagent. The unreacted substrates reduced the Cu(II)-the neocuproine complex, resulting in the highly colored Cu(I)-neocuproine product, which could be detected spectrophotometrically at 450 nm. The negative correlation between catalase activity and the absorbance of the Cu(I)-neocuproine complex was examined. To assess the optimization of Cu(I)-neocuproine complex production, response surface methodology (RSM) was employed, specifically utilizing the Box–Behnken design (BBD). Additionally, the reliability of the newly developed protocol was confirmed through Bland–Altman analysis of catalase activity in paired samples, employing the peroxovanadate method.ResultsThe novel method is just as accurate as the established standard; the correlation between the two methods was 0.99. The CUPRAC-CAT method is stable, sensitive, linear, reproducible, accurate, and selective and can be used for quantifying oxidative stress while measuring catalase activity in liver tissue homogenates.ConclusionsThis study has demonstrated a straightforward and dependable protocol for evaluating catalase activity. The protocol is free from interference and can be easily employed in scientific research, ensuring a high level of accuracy and precision. The CUPRAC-CAT method is an effective technique to monitor bacterial contamination. This method provides quick and reliable results that can help ensure food safety and prevent or address bacterial contamination.Graphical abstract

An improved method for measuring catalase activity in biological samples.

Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.

Tentatively Identified (UPLC/T-TOF–MS/MS) Compounds in the Extract of Saussurea costus Roots Exhibit In Vivo Hepatoprotection via Modulation of HNF-1α, Sirtuin-1, C/ebpα, miRNA-34a and miRNA-223

Saussurea costus is a plant traditionally used for the treatment of several ailments. Our study accomplished the UPLC/T-TOF–MS/MS analysis of a methanol extract of Saussurea costus roots (MESC), in addition to lipoidal matter determination and assessment of its in vivo hepatoprotective activity. In this study, we were able to identify the major metabolites in MESC rather than the previously known isolated compounds, improving our knowledge of its chemical constituents. The flavones apigenin, acacetin, baicalein, luteolin, and diosmetin, and the flavonol aglycones quercetin, kaempferol, isorhamnetin, gossypetin, and myricetin and/or their glycosides and glucuronic derivatives were the major identified compounds. The hepatoprotective activity of MESC was evaluated by measuring catalase activity using UV spectrophotometry, inflammatory cytokines and apoptotic markers using ELISA techniques, and genetic markers using PCR. Paracetamol toxicity caused a significant increase in plasma caspase 2, cytokeratin 18 (CK18), liver tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), miRNA-34a, and miRNA-223, as well as a significant decrease in liver catalase (CAT) activity and in the levels of liver nuclear factor 1α (HNF-1α), sirtuin-1, and C/ebpα. Oral pretreatment with MESC (200 mg/kg) showed a significant decrease in caspase 2, CK18, TNF-α, IL-6 and a significant increase in liver CAT activity. MESC decreased the levels of liver miRNA-34a and miRNA-223 and induced HNF-1α, sirtuin-1, and C/ebpα gene expression. The histological examination showed a significant normalization in rats pretreated with MESC. Our findings showed that Saussurea costus may exert a potent hepatoprotective activity through the modulation of the expression of cellular cytokines, miRNA-34a, and miRNA-223.

The toxicity of Aspidosperma subincanum to MCF7 cells is related to modulation of oxidative status and proinflammatory pathways

Ethnopharmacological relevanceCancer is an inflammatory disease because carcinogenesis and tumor progression depend on intrinsic and extrinsic inflammatory pathways. Although species of the genus Aspidosperma are widely used to treat tumors, and there is ethnopharmacological evidence for traditional use of the species A. subincanum as an anti-inflammatory agent, its antineoplastic potential is unknown. Aim of the studyTo evaluate toxic effects of the indole alkaloid–rich fraction (IAF) of A. subincanum on the MCF7 cell line and identify some of the anti-inflammatory mechanisms involved. Materials and methodsChromatographic analyses were performed by ultra-high-performance liquid chromatography with electrospray ionization mass spectrometry, and cytotoxic and antiproliferative effects of IAF were verified by MTT and clonogenic assays. Cell cycle alterations were analyzed by measuring DNA content, while propidium iodide and acridine orange staining was performed to determine the type of induced cell death. The expression of apoptosis markers and proteins involved in cell proliferation and survival pathways was analyzed by immunoblotting, RT-qPCR, and ELISAs. Interference with redox status was investigated using a DCFH-DA probe and by measuring catalase activity. ResultsChromatographic analyses showed that IAF is a complex mixture containing indole alkaloids. IAF selectively exerted toxic and antiproliferative effects, elevating the Bax/Bcl-xL ratio and inducing apoptosis in MCF7 cells. IAF decreased intracellular reactive oxygen species levels and increased catalase activity, while reducing the IL-8 level and suppressing COX-2 expression. ConclusionsIAF induces apoptosis in MCF7 cells by suppressing COX-2 expression while reducing IL-8 levels and intracellular content of reactive oxygen species.

A comparative proteomic analysis of Candida species in response to the oxidizing agent cumene hydroperoxide

Candida genus comprises several species that can be found in the oral cavity and the gastrointestinal and genitourinary tracts of healthy individuals. Under certain conditions, however, they behave as opportunistic pathogens that colonize these tissues, most frequently when the immune system is compromised by a disease or under certain medical treatments. To colonize the human host, these organisms require to express cell wall proteins (CWP) that allowed them to adhere and adapt to the reactive oxygen (ROS) and nitrogen (RNS) species produced in the macrophage during the respiratory burst. The aim of this study was to determine how four Candida species respond to the oxidative stress imposed by cumene hydroperoxide (CHP). To this purpose, C. albicans, C. glabrata, C. krusei and C. parapsilosis were exposed to this oxidant which is known to generate ROS in the membrane phospholipids. Accordingly, both mock and CHP-exposed cells were used to extract and analyze CWP and also to measure catalase activity and the levels of protein carbonylation. Results indicated that all four species express different CWP to neutralize ROS. Most relevant among these proteins were the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase, known as moonlight proteins because in addition to participate in glycolysis they play an important role in the cell response to ROS. In addition, a thiol-specific antioxidant enzyme (Tsa) was also found to counteract ROS.

Catalase-Loaded Silica Nanoparticles Formulated via Direct Surface Modification as Potential Oxygen Generators for Hypoxia Relief.

Enzymes are biological catalysts that have many potential industrial and biomedical applications. However, the widespread use of enzymes in the industry has been limited by their instability and poor recovery. In biomedical applications, systemic administration of enzymes has faced two main challenges: limited bioactivity mostly due to rapid degradation by proteases and immunogenic activity, since most enzymes are from nonhuman sources. Herein, we propose a robust enzyme-encapsulation strategy to mitigate these limitations. Catalase (CAT) was encapsulated in nanoporous silica nanoparticles (CAT-SiNPs) by first chemically modifying the enzyme surface with a silica precursor, followed by silica growth and finally poly(ethylene glycol) (PEG) conjugation. The formulation was carried out in mild aqueous conditions and yielded nanoparticles (NPs) with a mean diameter of 230 ± 10 nm and a concentration of 1.3 ± 0.8 × 1012 NPs/mL. CAT-SiNPs demonstrated high enzyme activity, optimal protection from proteolysis by proteinase K and trypsin, and excellent stability over time. In addition, a new electrochemical assay was developed to measure CAT activity in a rapid, simple, and accurate manner without interference from chromophore usually present in biological samples. Concentrations of 2.5 × 1010 to 80 × 1010 CAT-SiNPs/mL not only proved to be nontoxic in cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay but also conferred cell protection when cells were exposed to 1 mM hydrogen peroxide (H2O2). Finally, the ability of CAT-SiNPs to release oxygen (O2) when exposed to H2O2 was demonstrated in vivo using a rat model. Following the direct injection of CAT-SiNPs in the left kidney, partial pressure of oxygen (pO2) increased by more than 30 mmHg compared to the contralateral control kidney during the systemic infusion of safe levels of H2O2. This pilot study highlights the potential of CAT-SiNPs to generate O2 to relieve hypoxia in tissues and potentially sensitize tumors against radiation therapy.

Simple kinetic method for assessing catalase activity in biological samples

A novel kinetic method for measuring catalase activity in biological samples was evaluated. The principle of the current method is based on the oxidation effect of unreacted hydrogen peroxide (H2O2) on pyrogallol red (PGR) using the catalytic effects of molybdenum. The decrease in the absorbance of PGR in the presence of H2O2 with time from 0.5 to 4.5 min was directly proportional to the concentration of H2O2, and, in turn, directly proportional to catalase activity. Erythrocyte lysate homogenates were used to measure catalase activity and the results of the current method were significantly correlated to those of the ammonium peroxovanadate method. The 3.1% within run and 4.7% between run coefficients of variation indicated the high precision of the present novel method. The validation process confirmed that the diagnostic method is appropriate for different types of biological samples. Here, we describe a rapid, relatively easy, and reliable method for measuring catalase activity. The assay could be applied as a diagnostic tool and is suitable in research contexts.•A novel kinetic method for measuring catalase activity in biological samples was evaluated.•The validation process confirmed that the diagnostic method is appropriate for different types of biological samples.•The assay could be applied as a diagnostic tool and is suitable in research contexts.

A precise and simple method for measuring catalase activity in biological samples

The current study describes a simple, precise, and accurate protocol for measuring the activity of catalase, which controls the rate-limiting step of the dissociation of hydrogen peroxide reactions. The current protocol assesses catalase activity by incubating catalase samples with suitable concentrations of hydrogen peroxide dissolved in a phosphate buffer (pH 7.4). After the incubation period, a working solution that contained vanadate (V) and pyridine-2,6-dicarboxylic acid was added to stop the enzymatic reaction. The reaction between undissociated hydrogen peroxide and the added reagent forms a stable orange-colored chelate complex known as oxo-peroxo-pyridine-2,6-dicarboxylato-vanadate (OPDV) that demonstrates maximum absorbance at 435 nm. To optimize the formation of the method (the OPDV-CAT assay), we applied the Box–Behnken design (BBD) by utilizing the response surface methodology (RSM) as an index of precision of the assay. This novel method was validated against a Bland–Altman plot analysis of catalase activity using the carbonato-cobaltate method in matched samples. The comparison between the two methods resulted in a correlation coefficient equal to 0.9968, demonstrating that the new method is just as effective as the reference method.

Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples

Oxidative stress biomarkers are powerful endpoints in toxicological research. Cellular reductive/oxidative balance affects numerous signaling pathways involving H2O2. Detoxification and control of H2O2 levels results mainly from catalase activity.The aim of this work was to develop a precise, simple, cost-effective microassay to measure catalase activity in small tissue samples and cell extracts.We developed a protocol that quantifies H2O2 decomposition by intrinsic catalase in biological samples. Catalase activity was calculated based on rate of decomposition of H2O2, following absorbance at 240 nm. We developed a multi-well spectroscopic approach, reducing sample quantity requirements and allowing simultaneous assessment of large number of samples.The protocol is sensitive across a wide range of catalase activity (11.5–7575 U). The assay presents a 95% confidence interval with an intra-assay coefficient of variation of 3.7%, an inter-assay coefficient of variation of 6.2% and good correlation with a commercial kit. The assay was established and validated for different biological samples, including sheep hepatic tissue and human tumor and non-tumor cell lines.This high-throughput method is robust, sensitive, time-saving and cost-effective, generating highly reproducible results with precision and good correlation with a commercial kit reinforcing the method's validity for research and toxicological applications.

Dose dependent enhanced antibacterial effects and reduced biofilm activity against Bacillus subtilis in presence of ZnO nanoparticles.

Bacterial biofilms are self-produced matrix of sticky extracellular polymeric substances. They result in fouling in the food industry, water treatment plants, and possess significant environmental and industrial impacts. Nanoparticles have shown immense potential and have been effective in combating bacterial biofilm, which is the common cause of drug resistance development, biofouling in water treatment plants and the food industry. Hence, in order to explore the same, Zinc oxide nanoparticles have been synthesized by chemical synthesis method and their action against Bacillus subtilis biofilm formation was evaluated in this study by crystal violet and ROS assay. The dose-dependent reduction in biofilm biomass and density was observed as a result of nanoparticle exposure. There was considerable reduction in biofilm formation after treatment with ZnO nanoparticles. Change in surface morphology of the Bacillus subtilis cells was observed which could be due to oxidative stress induced by ZnO nanoparticles. The oxidative stress was estimated by measurement of catalase activity that also showed dose-dependent decrease.

Biologically Active Ajuga Species Extracts Modulate Supportive Processes for Cancer Cell Development.

Backround: Ajuga species have been used in traditional medicine for their diuretic, anti-inflammatory, wound-healing, and hepatoprotective properties.Purpose: The phytochemical profile and anticancer potential of three Ajuga sp. (A. genevensis, A. chamaepitys, and A. laxmannii) from Romania was investigated.Materials and Methods: The phytochemicals were extracted from the aerial parts of Ajuga sp. by using different solvents and methods. The hydroalcoholic extracts were examined for total phenolic, flavonoid and iridoid contents, and HPLC/MS was used to analyze the polyphenolic compounds and iridoids. The phytochemical profile was also evaluated by principal component analysis in connection with antitumor efficacy of extracts. The antiproliferative potential was evaluated using the ELISA BrdU-colorimetric immunoassay. Western Blot with regard to inflammatory protein NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) p65 subunit expression in cell lysates was performed. Quantification of oxidative stress marker malondialdehyde (MDA) was determined by high-performance liquid chromatography (HPLC). Enzymatic and non-enzymatic antioxidant capability was assessed by measuring catalase activity and by evaluating the total antioxidant capacity (TAC) of treated cells.Results: Ajuga laxmannii ethanol extract showed the highest total phenolic and flavonoid content, while A. genevensis ethanol extract was more abundant in iridoids. The overall cytostatic effect of the investigated plant extracts was exerted through strong inhibitory actions on NF-κB, the key molecule involved in the inflammatory response and via oxidative stress modulatory effects in both murine colon carcinoma and melanoma cell lines.Conclusion: Ajuga laxmannii showed the most significant antitumor activity and represents an important source of bioactive compounds, possibly an additional form of treatment alongside conventional anticancer drugs.

Simple spectrophotometric assay for measuring catalase activity in biological tissues

BackgroundThe details of a precise, accurate, and sensitive spectrophotometric method for measuring catalase activity are presented here. The assay was established for biological samples and depends on the rapid formation of a stable and colored carbonato-cobaltate (III) complex. Samples exhibiting catalase activity are incubated with hydrogen peroxide solution for 2 min prior to rapid mixing of the incubation enzymatic reaction mixture with cobalt-bicarbonate reagent, which assesses non-reacting hydrogen peroxide. Catalase activity is always directly proportional to the rate of dissociation of hydrogen peroxide. Hydrogen peroxide acts to oxidize cobalt (II) to cobalt (III) in the presence of bicarbonate ions; this process ends with the production of a carbonato-cobaltate (III) complex ([Co (CO3)3]Co). The formed end product has two maximum absorbance peaks: 440 nm and 640 nm. The 440-nm peak has been utilized for assessing catalase activity.ResultsThe catalase activity results of the current method for erythrocyte lysate homogenates were computationally identical to those of the dichromate method (r = 0.9950). The coefficient of variation was calculated to determine the imprecision of the current assay. The within-run and between-run results were 2.96 and 3.83%, respectively.ConclusionThis method is appropriate for analyzing bacteria, red blood cells and liver and kidney tissue homogenates.

Kinetic Parameters Analysis of Liver and Kidney Catalase Under The Influence of Cadmium and Mercury In Vitro

Cadmium (Cd) and mercury (Hg) are toxic metals that affect human organs function, including liver and kidney. This toxic activity is because the heavy metal could induce oxidative stress and interfere antioxidant activities, including catalase (CAT). The present study was aims to evaluate the effect of Cd and Hg to liver and kidney CAT kinetic parameters in vitro . In this experiment, liver and kidney were taken from male rats ( Rattus novergicus ). Sample the homogenized and divided into three groups with; T0 served as control which contains liver or kidney homogenate + H 2 O 2 , T1 which contains liver or kidney homogenate + H 2 O 2 + 0.03 mg/L CdSO 4 ; and T2 which contains liver or kidney homogenate + H 2 O 2 + 1 mg/L Hg. Solutions then incubated at 37oC for 1 hour and then was prepared for CAT activity measurement. The CAT activity was measured using spectrophotometer at 240 nm. For measuring the kinetic parameters, different concentration of H 2 O 2 were used. The kinetics parameters (Km and Vmax) were calculated using Lineweaver-Burk plot. The results shows that Cd and Hg could decrease the affinity of CAT-H 2 O 2 complex which expressed by the higher Km and Vmax values. Also from the results, Cd has better activity to decreased the affinity of CAT-H 2 O 2 complex than Hg. From this results, it can be concluded that Cd and Hg treatments could inhibit CAT activity in liver and kidney in vitro .

Effect of zinc and lead on the physiological and biochemical properties of aquatic plant Lemna minor: its potential role in phytoremediation

Plants have gained importance in situ bioremediation of heavy metals. In the present study, different concentrations of zinc (Zn2+) (0.5, 5, 10, 15, 20 mg/l) and lead (Pb2+) (1, 2, 4, 6, 8 mg/l) were used to evaluate metal tolerance level of Lemna minor. L.minor were exposed to metals for 4 days and tested for its dry to fresh weight ratio (DW/FW), photosynthetic pigments production and protein content. The oxidative damage was detected by measuring catalase activity. L.minor showed tolerance against Zn2+ and Pb2+ at a concentration of 10 and 4 mg/l, respectively. Among the metals, Pb2+ showed a significant toxicity at 8 mg/l. High concentration (20 mg/l of Zn2+ and 8 mg/l of Pb2+) of the metals displayed a considerable negative effect on soluble proteins (13 fold decrease with Zn2+ and 4 fold decrease with Pb2+) and photosynthetic pigments (twofold decrease with Zn2+ and onefold decrease with Pb2+) and lead to a consequent reduction in number of fronds. Further, the catalase was greatly increased (twofold decrease with Zn2+ and sixfold decrease with Pb2+) under metal stress. The results indicate that L.minor withstands Zn2+ and Pb2+ toxicity up to the concentration of 10 and 4 mg/l, respectively. Hence, the metal tolerant property of this plant shall be exploited for bioremediation of Zinc and Lead in polluted water. Further, the detailed and wide range of heavy metal toxicity studies should be done to reveal the possible use of this plant on large scale bioremediation purpose.

Direct measurement of catalase activity in living cells and tissue biopsies

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings.

Large-Scale Purification of Peroxisomes for Preparative Applications.

This protocol is designed for large-scale isolation of highly purified peroxisomes from Saccharomyces cerevisiae using two consecutive density gradient centrifugations. Instructions are provided for harvesting up to 60 g of oleic acid-induced yeast cells for the preparation of spheroplasts and generation of organellar pellets (OPs) enriched in peroxisomes and mitochondria. The OPs are loaded onto eight continuous 36%-68% (w/v) sucrose gradients. After centrifugation, the peak peroxisomal fractions are determined by measurement of catalase activity. These fractions are subsequently pooled and subjected to a second density gradient centrifugation using 20%-40% (w/v) Nycodenz.

PP1 - A New Protocol for the Evaluation of the Antioxidant Activity of Plant Components

A new approach is proposed to evaluate antioxidant activity. It is based on simultaneous measurement of cellular turbidity and hemoglobin. Human erythrocytes were pretreated separately with ginger oleoresin, ginger essential oil, and ascorbic acid. Untreated cells served as control. Oxidative stress was induced by H2O2. Samples were then evaluated by simultaneous measurement of cellular turbidity and the released hemoglobin. Additionally, morphological changes of erythrocytes, catalase activity, and lipid peroxydation were investigated. The results showed that the hemoglobin concentrations were significantly higher in samples treated with ginger extracts compared to the control. Surprisingly, cell concentrations were also higher in these same samples. This indicates that in the presence of antioxidants the measurement of hemoglobin levels alone is not a sufficient indicator of hemolysis. These findings were supported by the measurement of catalase activity and lipid peroxidation. We show that a concurrent measurement of erythrocyte concentration and hemoglobin levels is essential in such assays and provide a new protocol that is based on simultaneous measurement of cellular turbidity and hemoglobin levels.

Prebiotics prevent the appearance of aberrant crypt foci (ACF) in the colon of BALB/c mice for increasing the gene expression of p16 protein.

Studies have shown fibers to be effective in reducing the appearance of aberrant crypt foci (ACF) in rodents. The goal of this study was to investigate the preventive effects of fructooligosaccharide (FOS) and inulin prebiotics on the appearance of ACF in mice. The techniques used were: RT-PCR to evaluate the gene expression of p16, p21, p54, cyclin D1 and cyclin E in the distal colon; the quantification of Number of aberrant crypt foci (ACF) and measurement of catalase activity in the liver and distal colon. The animals were divided into five treatments (n=8); C-: AIN93M diet without fibers + DMH (1,2-dimethylhydrazine); INL: AIN93M diet with inulin; INLCA: AIN93M diet with inulin + DMH; FOS: AIN93M diet with FOS; FOSCA: AIN93M diet with FOS + DMH, during 15 weeks. Inulin prevented the appearance of ACF in the proximal, middle and distal colon, compared to the control without fibers. In the middle and distal colon, FOS was also effective in preventing the incidence of ACF. This effectiveness may be attributed to the increased gene expression of p16 following FOS treatment. Both prebiotics also decreased catalase activity in the distal colon, thus suggesting an antioxidant effect. These results suggesting an antioxidant effect prebiotics that may be attributed to the increased gene expression of p16.

Catalase Activity Assay in Candida glabrata

Commensal and pathogenic fungi are exposed to hydrogen peroxide (H2O2) produced by macrophages of the host. Pathogenic fungi counteract the harmful effects of H2O2 with the enzyme catalase (EC 1.11.1.6), which decomposes two molecules of H2O2 to two molecules of H2O and O2. Contribution of antioxidant systems on fungal virulence is actively studied. Measurement of catalase activity can contribute to the elucidation of the factors that influence the regulation of this pivotal enzyme. Here we describe a simple spectrophotometric method in which the activity of catalase is measured in total yeast extracts. Decomposition of H2O2 by the yeast extract is followed by the decrease in absorbance at 240 nm. The difference in absorbance through time (ΔA240) is inferred as the measure of catalase activity.

Biochemical and Biophysical Characterization of the Two Isoforms of cbb3-Type Cytochrome c Oxidase from Pseudomonas stutzeri

The cbb3-type cytochrome c oxidases (cbb3-CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb3-CcOs are present only in bacteria and play a primary role in microaerobic respiration, being essential for nitrogen-fixing endosymbionts and for some human pathogens. As frequently observed in Pseudomonads, Pseudomonas stutzeri contains two independent ccoNO(Q)P operons encoding the two cbb3 isoforms, Cbb3-1 and Cbb3-2. While the crystal structure of Cbb3-1 from P. stutzeri was determined recently and cbb3-CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb3-CcOs. In this work, both isoforms were homologously expressed in P. stutzeri strains from which the genomic version of the respective operon was deleted. We purified both cbb3 isoforms separately by affinity chromatography and increased the yield of Cbb3-2 to a similar level as Cbb3-1 by replacing its native promoter. Mass spectrometry, UV-visible (UV-Vis) spectroscopy, differential scanning calorimetry, as well as oxygen reductase and catalase activity measurements were employed to characterize both cbb3 isoforms. Differences were found concerning the thermal stability and the presence of subunit CcoQ. However, no significant differences between the two isoforms were observed otherwise. Interestingly, a surprisingly high turnover of at least 2,000 electrons s(-1) and a high Michaelis-Menten constant (Km ~ 3.6 mM) using ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb3-CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb3 isoforms specifically.