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Reactive oxygen species (ROS) at low concentrations effectively regulates intracellular pathways such as gene expression. Whereas their high concentrations are involved in the pathogenesis of many diseases by causing oxidative stress and damaging vital macromolecules. Each cell is equipped with an antioxidant defense system to neutralize high levels of ROS. Peroxidase, as an essential antioxidant enzyme, catalyzes the oxidation of various substrates using hydrogen peroxide which is a reactive oxygen species. Since, the caffeine and theobromine are widely consumed daily in the world, and their concentrations affect the activity of many enzymes. Therefore, in the present study, the inhibitory effect of these methylxanthines on peroxidase activity has been examined. The peroxidase activity is measured by a spectrophotometer at 510 nm for 3 minutes with following absorption due to the oxidation of 4-aminoantipyrine in the presence and absence of caffeine and theobromine. In this study, it was observed that both compounds had an inhibitory effect on peroxidase activity. The values of IC₅₀ for theobromine and caffeine were obtained as 0.5 and 0.6 mmol, respectively. Moreover, the values of K_m and V_max showed that both inhibitors acted by an un-competitive mechanism of inhibition. Also, K_i values for theobromine and caffeine were calculated 0.03 and 0.08 mM, respectively. The values of K_i and IC₅₀ for theobromine was lower than those of caffeine indicating that theobromine has a higher inhibition strength and binding affinity to the enzyme-substrate complex. Therefore, it can be concluded that theobromine has a stronger inhibitory effect on peroxidase activity.

Keywords: Peroxidase, Caffeine, Xanthine stimulant, Theobromine, ROS

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