Reactivation of inactivated horseradish peroxidase with ethyleneurea and allantoin for determination of hydrogen peroxide by micro-flow injection horseradish peroxidase-catalyzed chemiluminescence

Abstract

A method for reactivation of inactivated horseradish peroxidase (HRP) with ethyleneurea and allantoin was studied and exploited in a continuous assay of hydrogen peroxide by micro-flow injection HRP-catalyzed luminol chemiluminescence. It is necessary to maintain the activity of immobilized HRP constant during the assay of hydrogen peroxide because the HRP is used repeatedly. If no reactivating reagents are used (control), light emission from H 2O 2 (9.7 μmol l −1) decreased in the course of the assay resulting in poor reproducibility (CV=22.9%, n=3). However, if ethyleneurea (100 mmol l −1) is added to the luminol solution (0.56 mmol l −1 in Tricine buffer, pH 9.2) the reproducibility of the assay improved remarkably (CV=2.9%, n=5). Allantoin (10 mmol l −1) also improved the reproducibility of the assay (CV=4.33%, n=10). However, a side effect was the suppression of light emission from H 2O 2 in a dose-dependent manner with both ethyleneurea and allantoin. The 3 σ detection limit of H 2O 2 using ethyleneurea (100 mmol l −1) was 0.6 pmol per injection. The mechanism of the inactivation of HRP after reaction with H 2O 2 and reactivation of the inactivated HRP was postulated as follows: the active site of HRP attracts a proton from H 2O 2 resulting in protonated HRP (inactive form). Exogenous ethyleneurea or allantoin removes the proton attached at the active site of the protonated HRP to return the enzyme to the active form.