A precise and simple method for measuring catalase activity in biological samples
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ORIGINAL PAPER
A precise and simple method for measuring catalase activity in biological samples Mohammed A. Kadhum1 · Mahmoud H. Hadwan1 Received: 16 July 2020 / Accepted: 20 October 2020 © Institute of Chemistry, Slovak Academy of Sciences 2020
Abstract 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.
* Mahmoud H. Hadwan [email protected] 1
Chemistry Department., College of Science, University of Babylon, p.o. 51002, Hilla City, Babylon Governorate, Iraq
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Vol.:(0123456789)
Chemical Papers
Graphic abstract
Keywords Box–Behnken design · Catalase activity · Pyridine-2,6-dicarboxylic acid · Response surface methodology · Spectrophotometry · Vanadate (v)
Introduction Antioxidant enzymes such as catalase and glutathione peroxidase scavenge free radical and reactive oxygen species, thereby preventing oxidative modification of proteins, lipids, and DNA (Ceballos-Picot et al. 1992). Although these enzymes compete with peroxiredoxin when scavenging hydrogen peroxide ( H2O2), their specific contributions to the detoxification of hydrogen peroxide remain extensively regulated via posttranslational modification (Rhee et al. 2005). Catalase is expressed in all biological tissues and hydrolyzes H 2O2 into water and oxygen (Loewen et al. 1985). Additionally, catalase protects biological tissues against H 2O 2 that is formed from host immune cells to attack pathogens (Day et al. 2000). Catalase comprises
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four hemoprotein groups and has a molecular weight equal to 250 kDa (Van Lente and Pepoy 1990). Catalase and other antioxidant enzymes are highly expressed in plant and animal cells, including hepatic cells, renal cells, and erythrocytes (Kodydková et al 2014; Jenkins 1981). On the other hand, although catalase is produced in all aerobic bacteria and most facultative anaerobes, it is not found in ob
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