Abstract
Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd 2+ for various periods of time. The H 2O 2-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd 2+ was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd 2+ concentration. Furthermore, for the same inhibition type, K i values dropped as incubation time increased. These results suggested that Cd 2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd 2+ altered the heme Soret absorption band on binding HRP and exhibited a K d which decreased as the incubation time of HRP with Cd 2+ increased. Hill plots suggested a cooperative binding of up to three Cd 2+ ions per molecule of HRP. Thus, Cd 2+ binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd 2+ ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.
Published Version
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