Electrochemical studies on reconstituted horseradish peroxidase modified carbon paste electrodes

Horseradish peroxidase (HRP) is a heme protein that acts specifically on H2O2 as the electron acceptor. Hemin (Ferriprotoporhyrin-IX) is the prosthetic group of the enzyme. A direct molecular wire to the redox center of the enzyme is expected to enhance the electrochemical response of the enzyme. Native HRP was immobilized onto the surface of glassy carbon (GC) matrix using a 16-atom spacer arm. We have also immobilized the redox center of the enzyme (hemin) through one of the propionate groups onto the surface of glassy carbon matrix using an 11-atom spacer arm with amino terminus. Apoperoxidase was isolated according to the Tealeʹs method and was allowed to reconstitute with the hemin-bound matrix for enzyme reconstitution. The HRP paste and reconstituted-HRP (rec-HRP) paste electrodes were used to study the electrochemical response to substrate H2O2 using electrochemical techniques like cyclic voltammetry (CV) and flow injection (FI) studies. Flow injection studies using HRP paste electrode showed a linearity from 25 to 200 μM H2O2. The rec-HRP paste showed ∼100 times increase in the electron transfer rates compared to native HRP paste, and substrate linearity from 25 to 100 μM was observed.