The Mechanism of Inhibition of Human Erythrocyte Catalase by Azide

Yasemin AKSOY, Mevhibe BALK, İ. Hamdi ÖĞÜŞ, Nazmi ÖZER
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Abstract


In order to elucidate the kinetic mechanism of human erythrocyte catalase a well-known substrate, H2O2 and inhibitor azide were used. The catalase-mediated conversion of H2O2 to H2O and O2, in the presence and absence of azide, was studied in 50 mM phosphate buffer pH 7.0 at 37 ºC under conditions where the peroxidation side reaction (RH2 + H2O2 &#61614; R + 2H2O) is negligible ([H2O2] =< 25 mM; assay time, 10 s). The kinetics conformed to the Michaelis-Menten model. Lineweaver-Burk plots for H2O2 at different fixed concentrations of azide were linear and intersected on the abscissa indicating a noncompetitive or irreversible type of inhibition. To identify the inhibition type Vm vs. [E] plots were constructed at different [N3-]. The plots were linear and converged at the origin, indicating that N3- is a noncompetitive inhibitor. Using a non-linear curve-fitting program, the kinetic parameters were calculated. The Km value for H2O2, and the Ki value for azide were found to be 10.97 ± 1.46 mM and 1.107 ± 0.093 µM, respectively.

Keywords


Human erythrocytes, catalase, substrate kinetics, azide inhibition

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