A novel method for the fluorometric assay of horseradish peroxidase and microperoxidase activities is described based on their catalytic reactions between 1,2-diarylethylenediamines and catechol compounds as fluorogenic substrates in the presence of hydrogen peroxide. meso-l,2-Diphenylethylenediamine and epinephrine as a couple of substrates are most recommendable in practical use. This method is highly sensitive: the detection limits (S/ N=2) are 10 µU tube-1 for horseradish peroxidase and 500 fmol tube-1 for microperoxidase.Keywords Horseradish peroxidase, microperoxidase, activity assay, fluorometry, 1,2-diarylethylenediamine, meso-1,2-diphenylethylenediamine, epinephrinePeroxidase has been used as a marker enzyme in immunoassay and DNA hybridization assay because of its high stability. Activity is commonly assayed by colorimetric or fluorometric methods: the former uses 4-aminoantipyrine-phenols, 2,2'-azino-di(3-ethylbenzothiazoline-6-sulfonic acid)2, benzidine3 or di-o-anisidine;4 the latter uses p-substituted phenolic compounds, such as homovanillic acids, tyramine6, p-cresol' and phydroxyphenylpropionic acids as substrates. Although the fluorometric methods are more sensitive than the spectrophotometric methods, the reaction products from the substrates fluoresce in too short wavelength region to observe with the naked eye as in DNA hybridization assay.In our previous papers, it was shown that 1,2-diarylethylenediamines (DAEs) react with catechol compounds in the presence of potassium hexacyanoferrate(III) to provide intense fluorescence (emission maxima, longer than 480 nm).9-12 We have found that this reaction can be mediated by peroxidase (HRP) and microperoxidase (MP) in the presence of hydrogen peroxide.This paper describes the enzymatic reactivities of various DAEs with catechol compounds and the characteristics of the yielding fluorescence, and optimal conditions of the assay for HRP and MP using meso-l,2-diphenylethylenediamine (DPE) and epinephrine as a selected pair of substrates.
Experimental Reagents, solutions and apparatusThe following 29 DAEs were synthesized as described previously: s s DPE, 1,2-bis(2-hydroxyphenyl)ethylenediamine, 1,2-bis(2-, 3-and 4-methoxyphenyl)ethylenediamines, 1,2-bis(2-and 4-ethoxyphenyl)ethylenediamines, 1,2-bis(2-, 3-and 4-methylphenyl)ethylenediamines, 1,2-bis(4-ethylphenyl)ethylenediamine, 1,2-bis(3,4-dimethoxyphenyl)ethylenediamine, 1,2-bis(3,4-methylenedioxyphenyl)ethylenediamine, 1,2-bis(4-fluorophenyl)ethylenediamine, 1,2-bis(2-, 3-and 4-chlorophenyl)ethylenediamines, 1,2-bis(2,6-and 3,4-dichlorophenyl)ethylenediamines, 1,2-bis(4-cyanophenyl)ethylenediamine, 1,2-bis(4-dimethylaminophenyl)-ethylenediamine, 1,2-bis(4-biphenylyl)ethylenediamine, 1,2-bis(1-and 2-naphthyl)ethylenediamines, 1,2-bis(2-furyl)ethylenediamine and l,2-bis(3-pyridyl)ethylenediamine. Although these were all synthesized in the meso form, 1,2-diphenylethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis(4-methylphenyl)-ethylenediamine, were all in the DL form. Each DAE solut...