An efficient protocol for quantifying catalase activity in biological samples

Abstract

Abstract Background Catalase 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. Methods In the CUPRAC-CAT method, the component of the enzymatic reaction was incubated before adding the Cu(Nc)2 2+ 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. Results The 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. Conclusions This 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

Keywords

• Catalase activity
• Enzymology
• Spectrophotometry
• CUPRAC
• Bland Altman