ANALYTICAL SCIENCES JUNE 1991, VOL. 7 437 Assay for Peroxidase Using 1,2-Diarylethylenediamines and Catechol Compounds as Fluorogenic Substrates Hitoshi NoHTA, Tomohiro WATANABE,Hiroaki NAGAOKAand YOsuke OHKURAt Faculty of Pharmaceutical Sciences, Kyushu University62, Maidashi, Fukuoka 812, Japan 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, epinephrine Peroxidase has been used as a marker enzyme in previously: ss DPE, 1,2-bis(2-hydroxyphenyl)ethylene- immunoassay and DNA hybridization assay because of diamine, 1,2-bis(2-, 3- and 4-methoxyphenyl)ethylene- its high stability. Activity is commonly assayed by diamines, 1,2-bis(2- and 4-ethoxyphenyl)ethylenedi- colorimetric or fluorometric methods: the former uses amines, 1,2-bis(2-, 3- and 4-methylphenyl)ethylenedi- 4-aminoantipyrine-phenols, 2,2'-azino-di(3-ethylbenzo- amines, 1,2-bis(4-ethylphenyl)ethylenediamine, 1,2- thiazoline-6-sulfonic acid)2, benzidine3 or di-o-anisi- bis(3,4-dimethoxyphenyl)ethylenediamine, 1,2-bis(3,4- dine;4 the latter uses p-substituted phenolic compounds, methylenedioxyphenyl)ethylenediamine, 1,2-bis(4- such as homovanillic acids, tyramine6, p-cresol' and p- fluorophenyl)ethylenediamine, 1,2-bis(2-, 3- and 4- hydroxyphenylpropionic acids as substrates. Although chlorophenyl)ethylenediamines, 1,2-bis(2,6- and 3,4- the fluorometric methods are more sensitive than the dichlorophenyl)ethylenediamines, 1,2-bis(4-cyanophe- spectrophotometric methods, the reaction products from nyl)ethylenediamine, 1,2-bis(4-dimethylaminophenyl)- the substrates fluoresce in too short wavelength region to ethylenediamine, 1,2-bis(4-biphenylyl)ethylenediamine, observe with the naked eye as in DNA hybridization 1,2-bis(1- and 2-naphthyl)ethylenediamines, 1,2-bis(2- assay. furyl)ethylenediamine and l,2-bis(3-pyridyl)ethylenedi- In our previous papers, it was shown that 1,2- amine. Although these were all synthesized in the meso diarylethylenediamines (DAEs) react with catechol form, 1,2-diphenylethylenediamine, 1,2-bis(4-methoxy- compounds in the presence of potassium hexacyanofer- phenyl)ethylenediamine and 1,2-bis(4-methylphenyl)- rate(III) to provide intense fluorescence (emission ethylenediamine, were all in the DL form. Each DAE maxima, longer than 480 nm).9-12 We have found that solution (0.1 M; for the screening of DAEs) was prepared this reaction can be mediated by peroxidase (HRP) and in a mixture of methanol-0.2 M hydrochloric acid (1:1, microperoxidase (MP) in the presence of hydrogen v/v). DPE solutions (0.1 M and 80 mM; for the peroxide. This paper describes the enzymatic re- screening of catechol compounds and the assay of the activities of various DAEs with catechol compounds and enzyme activities) were prepared in 0.1 M hydrochloric the characteristics of the yielding fluorescence, and acid. The solutions were usable for at least one week optimal conditions of the assay for HRP and MP using when stored at 4°C. meso-l,2-diphenylethylenediamine (DPE) and epineph- The following catechol compounds were purchased rine as a selected pair of substrates. from the companies given in parentheses and used without further purification: norepinephrine hydrogen- tartrate and dopamine hydrochloride (Wako, Tokyo, Experimental Japan); isoproterenol hydrochloride, 4-methylcatechol, protocatechualdehyde, protocatechuic acid (Nacalai Reagents, solutions and apparatus Tesque, Kyoto, Japan); 3,4-dihydroxyphenylacetic acid, The following 29 DAEs were synthesized as described a-propyldopacetamide and Tiron (Aldrich, Milwaukee, WI, USA); epinephrine hydrogentartrate, 3,4- t To whom correspondence should be addressed. dihydroxybenzylamine, 3,4-dihydroxycinnamic acid, 438 ANALYTICAL SCIENCES JUNE 1991, VOL. 7 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphen- 0.1 M DAE solution, 1.0 ml of 50 mM phosphate buffer ylalanine methyl ester, 3,4-dihydroxynorephedrine, DL- (pH 7.0), 1.0 ml of hydrogen peroxide (1 mM for HRP, threo-3,4-dihydroxyphenylserine, 3,4-dihydroxyphenyl- 40 mM for MP) and 0.1 ml of 0.5 U mL1 HRP or 9 µM ethyleneglycol, D-DOPA and L-DOPA (Sigma, St. MP, and the mixture was incubated at 37° C for 10 min in Louis, MO, USA); adrenalone hydrochloride (Fluka, HRP reaction and for 1 h in MP reaction. The reaction Buchs, Switzerland). Dobutamine hydrochloride and mixture was chilled in ice-water to stop the enzyme carbidopa (both, catecholic drugs) were supplied by reaction. The reagent blank was prepared in the same Shionogi (Osaka, Japan) and Sankyo (Tokyo, Japan), way, except that 0.1 ml of the enzyme solution was respectively. Their solutions (0.1 M) were prepared in replaced with 0.1 ml of the phosphate buffer. The water and stored at -20° C; they were then diluted with fluorescence spectra and intensities of the test and blank water to the desired concentrations when required for were measured at the excitation and emission maxima use. (see Table 1). A hydrogen peroxide solution (Mitsubishi Gas Chemical, Tokyo, Japan) was standardized by acid Procedure for the screening of catechol compounds using permanganate titration. HRP (type VI; 200 purprogal- DPE lin U mg 1protein, Sigma) and MP (microperoxidase-11, The same procedure as that used in the screening of Sigma) solutions were prepared in 50 mM phosphate DAEs was carried out, except that 10 mM solutions of buffers (pH 7.0 for HRP; pH 6.3 for MP); these solutions each catechol compound and DPE solution (0.1 M for were stable for at least one month when stored at 4° C. HRP, 80 mM for MP) were used in place of a 10 mM Catalase (from bovine liver, 24000 U mg-1 protein) and epinephrine and 0.1 M DAE solution, respectively (for superoxide dismutase (from bovine erythrocytes, the fluorescence excitation and emission maxima, see 3100 U mg 1 protein) were purchased from Sigma. All Table 2). other chemicals were of reagent grade. Deionized and distilled water was used. Procedure for the assay of HRP and MP activities Uncorrected fluorescence spectra and intensities were To 1.0 ml of 10 mM epinephrine were added 0.2 ml of measured with a Hitachi MPF-4 spectrofluorometer in DPE solution (0.1 M for HRP, 80 mM for MP), 1.0 ml of 1OX 10 mm quartz cells; spectral bandwidths of 5 nm were 50 mM phosphate buffer (pH 7.0 for HRP, pH 6.3 for used in both the excitation and emission monochroma- MP), 1.0 ml of hydrogen peroxide (0.6 mM in HRP tors. reaction for 10 min; 2 mM in HRP reaction for 2 h; 40 mM in MP reaction) and 0.1 ml of sample solution of Procedure for the screening of DAEs using epinephrine HRP or MP. The mixture was incubated at 37° C for To 1.0 ml of 10 mM epinephrine were added 0.2 ml of 10 min or 2 h in the HRP reaction, and for 1 h in the MP Table 1 Excitation and emission maxima of the fluorescence produced by HRP- and MP-mediated reactions of epinephr with various DAEs, and their relative fluorescence intensities (RFI)a a. A portion (100 µl) of 0.5 U ml-' HRP or 1 mM MP was treated according to the procedure. b. The intensity obtained with DPE was taken as 100 in each reaction. ANALYTICAL SCIENCES JUNE 1991, VOL. 7 439 Table 2 Excitation and emission maxima of the fluorescence produced by HRP- and MP-mediated reactions of DPE with various catechol compounds, and their relative fluorescence intensities (RFI)a a. A portion (100 µl) of 0.3 U ml-' HRP or 1 mM MP was treated according to the procedure. b. The intensity obtained with epinephrine was taken as 100 in each reaction. reaction. To stop the enzyme reaction, the reaction method). The chlorination of the phenyl groups of mixture was chilled in ice-water. To prepare the blank, DAEs caused the excitation maxima to be shifted to the same procedure was carried out, except that the longer wavelengths, and the introduction of electron- 0.1 ml of the sample solution was replaced with 0.1 ml of donating groups (methyl, methoxy, ethoxy and methyl- the phosphate buffer. The fluorescence intensities were enedioxy) produced more intense fluorescence: the measured at an emission wavelength of 500 nm with observations were identical with those obtained using the irradiation at 360 nm. chemical oxidation method. i 1 HRP reaction was much susceptible to aryl groups of DAEs: bulky groups (dichlorophenyl and naphthyl) seemed to sterically Results and Discussion hinder the enzyme reaction. DL-DAEs gave the same fluorescence spectra as those obtained with the Screening of DA Es corresponding meso-DAEs, though they were less soluble For screening of the DAEs, epinephrine was used as a in water. Of the DAEs in the meso form, 1,2-bis(4- model catechol compound (selected as described later). methoxyphenyl)ethylenediamine is the most sensitive in Table 1 shows the characteristics of the fluorescence both HRP and MP reactions, but DPE is more readily from the DAEs that responded intensely to epinephrine available, and still sensitive. DPE was therefore of the tested DAEs. The relative fluorescence inten- selected as a practical substrate. sities of the blanks were less than 0.1 % of that given by 0.5 U ml-1 HRP or 1 mM MP with DPE.