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

摘要

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 HPR is used repeatedly.If no reactivating reagents are used(control),light emission from H_2O_2(9.7 mu mol 1~-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 mmoll~-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 sigma detection limit of H_2O_2 using ethyleneurea(100 mmoll~-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.