University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Enzyme, horseradish peroxidase

@ ChemSpider@ NIST@ PubChem

Citations 7

"Kinetic Studies On The Oxidation Of Nitrite By Horseradish Peroxidase And Lactoperoxidase"
Acta Biochim. Pol. 1999 Volume 46, Issue 4 Pages 919-927
Lidia Gebicka

Abstract: The reaction of nitrite (NO2-) with horseradish peroxidase and lactoperoxidase was studied. Sequential mixing stopped-flow measurements gave the following values for the rate constants of the reaction of nitrite with compounds II (oxoferryl heme intermediates) of horseradish peroxidase and lactoperoxidase at pH 7.0, 13.3±0.07 mol-1 dm(3) s-1 and 3.5±0.05 . 10(4) mol-1 dm(3) s-1, respectively. Nitrite, at neutral pH, influenced measurements of activity of lactoperoxidase with typical substrates like 2,2-azino-bis[ethyl-benzothiazoline-(6)-sulphonic acid] (ABTS), guaiacol or thiocyanate (SCN-). The rate of ABTS and guaiacol oxidation increased linearly with nitrite concentration up to 2.5-5 mmol L-1. On the other hand, two-electron SCN oxidation was inhibited in the presence of nitrite. Thus, nitrite competed with the investigated substrates of lactoperoxidase. The intermediate, most probably nitrogen dioxide ((NO2)-N-.), reacted more rapidly with ABTS or guaiacol than did lactoperoxidase compound II. It did not, however, effectively oxidize SCN- to OSCN-. NO2- did not influence the activity measurements of horseradish peroxidase by ABTS or guaiacol method.
Stopped-flow Kinetic

"Continuous-flow Potentiometric Determination Of Horseradish Peroxidase With A Fluoride-selective Electrode"
Anal. Chim. Acta 1988 Volume 208, Issue 1-2 Pages 173-181
Peter W. Alexander and Carmelita Maitra

Abstract: The horse-radish peroxidase(I)-catalyzed oxidation of 0.104 M 4-fluoroaniline in 0.16 M acetate buffer (pH 4.6) by 4.45 mM H2O2 was monitored at 45°C with a F--selective electrode in a continuous-flow system (described with diagram; flow-rate 6.8 mL min-1). The detection limit was 1 miu mL-1 of I. Interference by I inhibitors was studied at 1 µM to 1 mM levels; Cu(II), Fe(III) and Co had little effect, whereas CN- and S2- caused considerable interference. The method was applied in the determination of I in turnip extract and milk.
Vegetable Milk Electrode Sample preparation Potentiometry Heated reaction Interferences

"Comparative Study Of Some Synthesized And Commercial Fluorogenic Substrates For Horseradish Peroxidase And Its Mimetic Enzyme Haemin By A Flow Injection Method"
Anal. Chim. Acta 1997 Volume 340, Issue 1-3 Pages 159-168
Yuan-Zong Li and Alan Townshend*

Abstract: Four 3,4-dihydroquinoxalin-2-(1H)-one derivatives (details given) were evaluated as fluorogenic substrates for horseradish peroxidase and its mimetic enzyme haemin. The performances of these substrates were compared to those of commercially available substrates, namely, p-hydroxyphenylacetic acid (p-HPA), p-hydroxyphenylpropionic acid (p-HPPA), homovanillic acid (HVA) and tyramine. The evaluations were performed by a FIA method in which a mixture, formed by merging substrate and buffered enzyme streams, was subsequently merged with a water carrier stream containing 150 µL H2O2. The reaction was monitored by fluorimetry at the optimum wavelength for each substrate. The substrates p-HPPA, p-HPA, NN'-dicyanomethyl-o-phenylenediamine (DCM-OPA) and 3-methyl-3,4-dihydroquinoxalin-2-(1H)-one (MDHQ) exhibited comparable performances; detection limits were at the nM level for H2O2. DCM-OPA had a better stability than MDHQ but both were stable for at least one month in a refrigerator.
Fluorescence Buffer Optimization

"3,3',5,5'-Tetramethylbenzidine As Electrochemical Substrate For Horseradish Peroxidase Based Enzyme Immunoassays. A Comparative Study"
Analyst 1998 Volume 123, Issue 6 Pages 1303-1307
G. Volpe, D. Compagnone, R. Draisci and G. Palleschi

Abstract: The use of 3,3',5,5'-tetramethylbenzidine (TMB) as an electrochemical substrate for horseradish peroxidase (HRP) was investigated. HRP activity has been detected using flow injection analysis at a glassy carbon working electrode polarized at +100 mV vs. Ag/AgCl in 0.1 mol L-1 citrate-phosphate buffer (pH 5.0). The optimum concentrations were 2 x 10^-4 mol L-1 TMB and 10^-3 mol L-1 H2O2. The detection limit obtained after 15 min of incubation was 8.5 x 10^-14 mol L-1 HRP with the amperometric method. This limit was lower than that obtained using hydroquinone as HRP substrate and comparable to that with the p-aminophenyl phosphate-alkaline phosphatase system. Better performance was achieved with amperometric than spectrophotometric detection using TMB in a competitive ELISA for rabbit IgG as a model analyte.
Electrode Amperometry Method comparison Optimization

"Studies On Flow Injection Chemiluminescence Immunoassay. 2. Hetermination Of HRP And Its Conjugates With Coupled Reaction"
Acta Chim. Sin. 1997 Volume 55, Issue 6 Pages 590-594
Shao Qian; Ma Wangbai; Feng Manliang; Zhang Zhujun

Abstract: In this paper chitoson, controlled pore glass and silica - gel have been investigated with respect to its potentiality of being used as solid support of flow - injection immunoassay. A new flow - injection immunoassay end - point detection method has also been developed on the basis of coupling the reaction of H2O2 and K4Fe(CN)(6), catalyzed by HRP, with H2O2 and K3Fe(CN)(6) co - oxidized chemiluminescence reaction of luminol. As enzymatic and chemiluminescent reaction were generated at different sites of the detection system. The general shortcomings of such as unable to select a optimum pH for both enzymatic and chemiluminescent reaction, incomplete contact between enzyme and substrate, and scattering of support have been eliminated It has the advantages of high sensitivity and good accuracy. HRP and its conjugates at f mol level can be detected within 1 similar to 2 min. and the relative standard deviation is 3.9%. 2 References
Chemiluminescence Immunoassay Controlled pore glass Silica gel

"Highly Sensitive Determination Of Hydrogen Peroxide And Peroxidase With Tetrathiafulvalene-based Electrodes And The Application In Immunosensing"
Biosens. Bioelectron. 1997 Volume 12, Issue 1 Pages 43-52
Frank Wendzinski, Bernd Gründig, Reinhard Renneberg and Friedrich Spener*

Abstract: A tetrathiafulvalene modified graphite electrode coupled with horseradish peroxidase is described, capable of monitoring H2O2 by FIA with a detection limit of 7 nM at a potential of +20 mV vs. a SCE. Holding the H2O2 constant, the detection limit of horseradish peroxidase was 150 pM. The system was evaluated and applied to the determination of 5-100 ng/ml of rabbit IgG as a model analyte by sandwich immunoassay.
Biological Immunoassay Electrode Electrode

"Amperometric Detection In TMB/HRP-based Assays"
Anal. Bioanal. Chem. 2005 Volume 382, Issue 2 Pages 297-302
Pablo Fanjul-Bolado, María Begoña González-García and Agustín Costa-García

Abstract: 3,3',5,5'-Tetramethylbenzidine (TMB) is the most commonly used chromogen for horseradish peroxidase (HRP) and so its performance as an electrochemical substrate was evaluated. Measurements of HRP activity in solution were carried out by using an amperometric detector coupled to a flow injection analysis (FIA) system. The enzymatic product was easily detected at a potential of +0.1 V (vs. Ag-pseudoreference electrode) at a bare screen-printed electrode placed in a homemade electrochemical flow cell. A high flow rate (4.3 mL min-1) of 0.5 M H2SO4 was used to obtain repeatable signals and a short analysis time. The detection limit achieved after 15 min of incubation was 2 x 10^-14 M of HRP. The applicability of the amperometric detector to ELISAs was demonstrated by using a commercially available kit for the quantification of interleukin-6 (IL-6) without modifying the kit manufacturer's protocol or the reagents for this test.
Immunoassay Electrode Electrode Amperometry Method comparison