Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

Abstract

A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis–Menten model and is inactivated by H2O2. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H2O2] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H2O2 decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H2O2 decomposition by catalase in 32 samples in triplicates if kept below 8 mM min−1 in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost.

Diaporthe atlantica enhances tomato drought tolerance by improving photosynthesis, nutrient uptake and enzymatic antioxidant response

“…Samples of fresh leaves previously stored at −80°C were ground with liquid nitrogen and kept at −80°C until the measurement of the antioxidant enzyme activities. The antioxidant activities of catalase (CAT), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR) were measured in leaf samples of four plant replicates per treatment following the methods described below by Bendou et al. (2022) and Pérez-López et al.…”

“…Samples of fresh leaves previously stored at −80°C were ground with liquid nitrogen and kept at −80°C until the measurement of the antioxidant enzyme activities. The antioxidant activities of catalase (CAT), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR) were measured in leaf samples of four plant replicates per treatment following the methods described below by Bendou et al (2022) and Peŕez-López et al (2009). APX was selected as a representative peroxidase activity enzyme because it belongs to the ascorbate-glutathione cycle, it is very sensitive to stress conditions, and it is well established that APX also regulates redox signaling pathways in normal plant development (Caverzan et al, 2012).…”

The Effect of Lemon Juice (Citrus limon L.) Treated with Melatonin on the Health Status and Treatment of K14HPV16 Mice

“…For quantitative analysis, a calibration standard curve constructed by SOD from bovine erythrocytes was used (0-3.75 U mL −1 ). The activity of catalase (CAT) was determined at 240 nm in accordance with a previously published method [25] and was calculated using bovine catalase as a standard (0-5 U mL −1 ).…”

One-Step Suicide Substrate Inactivation Kinetics of a Ping-Pong Reaction with One Substrate Undergoing Disproportionation: A Theoretical Approach with Approximate Solutions

“…Now, the respective numerically and analytically integrated solutions for [A], [E], [P], and [I] are merged, and their overlap remains regardless of the values for [A] 0 and [E] 0 , and for k a and k i . These approximate analytically integrated solutions are equivalent to those shown in [34,39,41] for the one-step suicide inactivation of catalase by H 2 O 2 , in which the kinetic mechanism analysis does not consider the formation of the intermediate active state of the enzyme. Therefore, when there is one-step suicide substrate inactivation in an irreversible uni-uni Michaelis-Menten model, it can be established that the ODE system of the reaction scheme can be analytically solved if the approximation [EA] ≈ 0 is accepted in any time domain in which the enzyme reaction takes place (Figure 2).…”

“…This simplified reaction scheme has been extensively used with success to determine the overall inactivation rate constant of catalase when the enzyme was exposed to prolonged incubation with H 2 O 2 under different experimental conditions such as temperature and pH [38][39][40]. More recently, a theoretical analysis of the inactivation reaction was conducted to design a rapid and high-throughput measurement of catalase in vitro [41].…”