Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro
Ouardia Bendou, Ismael Gutiérrez-Fernández, Emilio L. Marcos-Barbero, Nara Bueno-Ramos, Ana Isabel González-Hernández, Rosa Morcuende, Juan B. Arellano
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.