Bioelectrocatalysis of Plant Peroxidases Immobilized on Graphite in Aqueous and Mixed Solvent Media

Abstract

The bioelectrocatalytic function of two plant peroxidases, horseradish peroxidase (HRP) and a newly purified royal palm tree-leaf peroxidase (RPTP), was studied on graphite electrodes in aqueous buffer solutions, over the pH range 7.0 -3.0, and in aqueous buffer solutions, at pH 6.0, containing different amounts of a polar organic cosolvent, i.e., ethanol and acetonitrile. The kinetics of direct and of mediated (using catechol as mediator) reduction of H 2 O 2 at the HRP-and RPTP-modified graphite electrodes were analyzed amperometrically at À 50 mV(vs. Ag j AgCl j 0.1 M KCl) in a flow-through wall-jet electrochemical system. Values of the apparent rate constant of the heterogeneous electron transfer between the enzyme and graphite, k s , the rate constant for enzymatic reduction of H 2 O 2 , k 1 , and the rate constant of mediated reduction of H 2 O 2 , k 3, were determined using the modified Koutecky-Levich approach. Analysis of the variation of the rate constants and the response of the peroxidase-modified electrodes to H 2 O 2 with pH and content of the organic cosolvent demonstrated that maximal peroxidase bioelectrocatalytic activity occurred at pH 5.0 -6.0 and at concentrations of ethanol of 10 -20% v/v. At a lower pH, higher concentrations of ethanol, and in aqueous solutions of acetonitrile, the bioelectrocatalytic activity of the peroxidase-modified electrodes drastically decreased. However, contrary to the data for homogeneous catalysis, both peroxidases were bioelectrocatalytically active even in 95% organic co-solvents, thus demonstrating a stabilizing effect of the enzyme immobilization on the bioelectrocatalytic performance of the peroxidases. RPTP immobilized on graphite demonstrated lower overall activity but a higher pH-and organic cosolvent-stability than HRP.

Kinetics of oxidation of hydrogen peroxide at hemin-modified electrodes in nonaqueous solvents

“…where K m app is the apparent Michaelis constant for adsorbed hemin complex into products, and Γ is the surface concentration of hemin and is the number of electrons transferred (n = 2) [18,36]. The amount of water present in the solvent should have no influence on the hemin structure, but could significantly influence the rate of the reaction catalysed by hemin.…”

“…Peroxidases (horseradish peroxidase, HRP, chloroperoxidase, soybean peroxidase [12], palm tree peroxidase [18]) have been the enzymes of choice for detection of peroxides. The need to assay hydrophobic substrates (organic hydroperoxides, cholesterol, pesticides etc.)…”

Electron transfer of peroxidase assemblies at tailored nanocarbon electrodes

“…The onset of this reduction should occur near + 0.220 V vs. Hg/Hg 2 SO 4 [2,35] but many electrochemical schemes that involve the detection of HRP immobilized at electrode surfaces report an electrocatalytic potential significantly more negative, between −0.140 and −0.500 V vs. Hg/Hg 2 SO 4 [31,34]. Such potentials are nearer to that of the HRP Fe 2+/3+ redox couple in the absence of H 2 O 2 :…”

“…The increase in A 420 due to HRP-induced oxidation of ABTS was recorded for 2 min following the addition of H 2 O 2 and used to determine HRP enzymatic activity in ABTS units (U/mg, using the mass of HRP/CNTs introduced into the assay). An ABTS extinction coefficient of 31.1 mM −1 cm −1 was used in the calculations [34]. The enzymatic activity of HRP filtrates collected from HRP immobilization at CNTs was conducted using the same protocol except that 50 L of HRP filtrate, diluted with phosphate solution to 0.5 U/mL HRP based on the original concentration of HRP used in the immobilization solution, was used rather than HRP/CNTs or HRP/N-CNTs.…”

“…Many examples of HRP-modified carbon electrodes are available in the literature [2,[31][32][33][34][35]; more specifically, HRP has been immobilized at CNTs in previous electrochemical studies, allowing us to compare the efficiency of our immobilization strategy to others reported in the literature [25][26][27][28]30]. In our study, HRP has been adsorbed at both nondoped and N-CNTs containing 5 at% N to evaluate HRP enzymatic activity upon immobilization.…”