Role of Amino Thiols as Radical Quenchers in Delayed Chemiluminescence of Luminol Catalyzed by Horseradish Peroxidaset

Abstract

A horseradish peroxidase (HRP)-catalyzed luminol chemiluminescence (CL) reaction was applied to the detection of hydrogen peroxide (H2O2) formed during the Cu(II)-catalyzed oxidation of amino thiol with oxygen . Cysteamine, Lcysteine, L-cysteine methyl ester and L-cysteine ethyl ester were used as amino thiols. A CL flash suddenly appeared after a certain dark period from the start of the reaction in all thiols used. The appearance of the delayed CL could be explained in terms of quenching function of the thiol for luminol radical formed through the enzymatic cycle of HRP. The quenching rate of luminol radicals with the cysteine esters was remarkably higher than those with cysteamine and cysteine. KeywordsChemiluminescence, luminol, peroxidase, thiolHorseradish peroxidase (HRP: EC 1.11.1.7) has been widely used as a catalyst in luminol chemiluminescence (CL). The HRP-catalyzed luminol CL provides a sound basis for an assay of hydrogen peroxide (H202) and enzymatically generated H2O2.' On the other hand, the Cu(II)-catalyzed oxidation of amino thiol with oxygen is focused on a model reaction for elucidating the mechanism of the biological oxidation with copperbearing enzymes such as galactose oxidase and diamine oxidase.2In the catalytic oxidation, H2O2 is shown to be produced as an intermediate from oxygen.3In the course of our studies on the application of the HRP-catalyzed luminol CL to the detection of H2O2 formed during the catalytic oxidation of cysteamine, we have found that a CL flash suddenly appeared after a certain dark period from the initiation of the reaction.4 The delay time and the CL intensity were dependent on the concentrations of Cu(II). Based on this finding, we proposed a delayed luminol CL method for the determination of Cu(II).4 The purpose of the present study is to investigate the effects of amino thiols on CL intensity-time profiles and to clarify the function of the thiols in the appearance of the delayed CL of luminol. ExperimentalReagents HRP (type VI), L-cysteine methyl ester (CME), I.-cysteine ethyl ester (CEE) and L-cystine dimethyl ester (CDME) were obtained from Sigma Chemical Co. Luminol (5-amino-2,3-dihydrophthalazine-l ,4-dione), cysteamine and L-cysteine were purchased from Kanto Chemical Co. All thiols obtained were hydroclorides. All chemicals used were guaranteed-grade reagents and were used without further purification. HRP solution was prepared with Carmody's buffer (pH 8.0) involving 0.2 M (1 M=1 mol dm 3) boric acid, 0.05 M citric acid and 0.1 M tertiary sodium phosphate. The concentration of HRP was determined spectrophotometrically with an 6403 value of 1.02X105 M-1 cm 1.5 A 1.0X102 M stock solution of luminol was prepared by dissolving the compound with 0.1 M NaOH solution. Working solutions of luminol were prepared by serial dilution with Carmody's buffer solution. Standard solutions of thiols were made daily. All solutions used were prepared with water from a Millipore Milli-Q water purification system. CL detectionAll CL measurements were made using a luminomete...

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