Inhibited reactivity of horseradish peroxidase by its conjugated proteins through redox mediated electrochemical interrogation

Abstract

In this article, a classical redox mediated metal (in this study, Au) indicator electrode approach was revisited for the direct interrogation of ‘slow’ enzymatic redox second-order kinetics of horseradish peroxidase (HRP) for oxidative conversion of the model redox substrates, ferrocenemethanol (FcMeOH) and hydroquinone (H2Q) in low HRP-concentration levels. The presented electroanalysis allowed us to recognize the inhibited enzyme reactivity of HRP for FcMeOH-oxidation by its conjugation to model proteins (streptavidin, avidin, and monoclonal antibody), whereas this effect was negligible for H2Q-oxidation by HRP. From a theoretical investigation using finite element analysis, slow kinetics of HRP can be sensitively recognized through an Au indicator electrode approach in the case where HRP bulk electrolyzes a redox substrate in a solution. Five redox substrates were electrochemically investigated, and FcMeOH and H2Q showed negligible side reactions during mediation between HRP and the indicator electrode. From the chronoamperometric approach, the apparent rate constants for oxidative conversion of FcMeOH by the non-conjugated, free HRP were estimated to be ∼104 M−1s−1 at various pH values, while that of H2Q was roughly two orders of magnitude higher at pH ∼7. The molecular docking simulation revealed the need for a significant conformational change of HRP for FcMeOH access to the heme site, whereas the structural alternation of HRP was negligible for access of the H2Q into the domain of the enzyme. The simulation result indicates that the oxidative conversion rate of FcMeOH by HRP could be more susceptible to recognize additionally induced steric hindrance for access of the redox substrate to the catalytic site of the enzyme than that of H2Q. We experimentally verified that the apparent rate constant for FcMeOH-oxidation by the HRP conjugated to streptavidin was estimated to be ∼5 times smaller than that by the free HRP, and those by other conjugates to avidin and monoclonal antibody were ∼60% of that by the non-conjugated one. In contrast to the case of FcMeOH, the estimated reactivity of H2Q by HRP was barely affected by the conjugated model proteins. The presented study demonstrated that a redox enzymatic reactivity of HRP could be significantly affected by conjugated proteins when access of a redox substrate to a catalytic site of an enzyme is susceptible to conformational changes.