Substrate For Horseradish Peroxidase Research Articles

Comparative study of some synthesised and commercial fluorogenic substrates for horseradish peroxidase and its mimetic enzyme hemin by a flow injection method

Four 3,4-dihydroquinoxalin-2(1H)-one derivatives, i.e., 3,4-dihydroquinoxalin-2(lH)-one (DHQ), 3-methyl-3,4-dihydroquinoxalin-2(1H)-one (MDHQ), 3,4-dihydroquinoxalin-2(1H)-one-6-acid (DHQ-6-A) and 3-methyl-3,4-dihydroquinoxalin-2(1H)-one-6-acid (MDHQ-6-A), and N, N′-dicyanomethyl- o-phenylenediamine (DCM-OPA) were synthesised as potential substrates for horseradish peroxidase (HRP). Of these compounds DCM-OPA, DHQ and MDHQ can be prepared by very simple methods in a pure form in large quantities. Their properties for use as fluorogenic substrates for HRP and its mimetic enzyme hemin were compared with commercially available substrates, i.e., p-hydroxyphenylacetic acid ( p-HPA), p-hydroxyphenylpropionic acid ( p-HPPA), homovanillic acid (HVA) and tyramine, by a flow injection method. The results showed that DCM-OPA and MDHQ were the best among the five synthesised substrates and p-HPPA and p-HPA are better than HVA and tyramine. Substrates p-HPPA, p-HPA, DCM-OPA and MDHQ showed comparable ability for H 2O 2 detection in HRP and hemin catalysed reaction systems, with detection limits in the nmol l −1 region. The stability of DCM-OPA is better than MDHQ, but both are stable for at least a month in a refrigerator.

Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels.

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses.

Kinetic investigations of horseradish peroxidase in AOT/n-heptane reverse micelles

The oxidation of typical horseradish peroxidase substrate, 2,2'-azino-bis[3-ethyl-benzothiazoline-(6)-sulfonic acid] (ABTS), has been studied in AOT/n-heptane reverse micelles. The rate of product formation has been found to be over an order of magnitude higher in reverse micelles than in homogeneous aqueous solution. The rate constants of all three elementary steps of horseradish peroxidase reaction with ABTS are higher in reverse micelles than in homogeneous aqueous solution. The rate limiting step of compound I formation is the exchange between enzyme filled and hydrogen peroxide filled micelles. The exchange step most probably also influences the rate of ABTS reactions with compound I and compound II, although the changes in their rate constants due to a different water and enzyme structure in reverse micelles cannot be excluded.

A Novel Fluorogenic Substrate for the Use of Nucleic Acid Hybridization.

The phosphates derived from newly synthesized 3′-O-aralkyl-5- (4-biphenylcarboxamido) fluorescein derivatives were examined for the alkaline phosphatase-linked fluorescence assay of the membrane-bound DNA. λDNA was detectable to the amount of 5 fg in the assay using phosphorylated 5- (4-biphenylcarboxamido) -3′-O- (1-naphthyl) methylfluorescein (BNFP). The spots gave distinguishably clear fluorescence without diffusion and nonspecific adsorption. In the Southern blot hybridization, 0.1 pg of DNA could be detected. Detection of two different DNAs on a single blot was successfully performed by using two different fluorogenic phosphates, Phosphrorilated N- (2-biphenyl) -3-hydroxy-2-naphthalenecarboxamide (HNPP) and BNFP. The fluorescein derivatives were also examined as substrates for horse radish peroxidase.

A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E 2 using enhanced luminol as substrate

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E 2 (PGE 2) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE 2 antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE 2 conjugate. Then, after preincubating, the anti-PGE 2 antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE 2 conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated antimouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10–50,000 pglml. The lower limit of quantification is 100 pglml (2 pglwell) and 150 pglml (3 pglwell) for buffer and plasma, respectively. The intea-day coefficients of variation (CV) for the range of 100–50,000 pglml are 3.2–8.9% (buffer) and 4.2–17.7% (plasma) and inter-day CV are 2.9–19.8% (buffer) and 3.6–21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.

2,4,5-TRIHYDROXYBUTYROPHENONE (2,4,5-THBP) AS A SUBSTRATE FOR HORSERADISH PEROXIDASE

2,4,5-trihydroxybutyrophenone (2,4,5-THBP), in the presence of hydrogen peroxide (H 2 O 2 ), is a substrate for horseradish peroxidase (HRP) with a Km value of 2.5 mM. An intermediate red product(s), probably 2,4,5-THBP quinone, characterized by a peak at 490-500 nm, was detected and the time course of its conversion to the final red product(s) was studied. The relationships between the rate of 2,4,5-THBP oxidation to pigmented product(s) as a function of various concentrations of H 2 O 2 , 2,4,5-THBP and HPR are described.

Trans-2-Phenylcyclopropylamine is a substrate for and inactivator of horseradish peroxidase

Horseradish peroxidase (HRP) is well known for mediating the electron-transfer oxidation of electron-rich aromatic ‘donors’ such as phenols and anilines, but has not been described to oxidize aliphatic amines. We here confirm the inability of HRP to oxidize typical aliphatic amines, even those which would exist significantly as free bases at the operative pH. In contrast, trans-2-phenylcyclopropylamine (2-PCPA) is both a substrate (turnover product is cinnamaldehyde) and a time-dependent inactivator of HRP. These activities of 2-PCPA are consistent with either a concerted or rapid sequential one-electron-oxidation/ring-opening to give an intermediate capable of covalent binding to the enzyme. 2-PCPA is the first known example of a simple aliphatic amine which serves as a substrate for HRP under turnover conditions.

Nonsteroidal antiinflammatory drugs interact with horseradish peroxidase in an in vitro assay system for hydrogen peroxide scavenging

The established horseradish peroxidase/guaiacol in an in vitro assay system was used for investigation of the reactivity of nonsteroidal antiinflammatory drugs with hydrogen peroxide. Although the drugs rapidly seemed to react in the selected conditions, difficulties were encountered in attempts to quantify the reaction and an interaction with horseradish peroxidase was suspected. A more specific assay system based on the absolute specificity of the enzyme glutathione peroxidase for glutathione was subsequently used which demonstrated that none of the investigated nonsteroidal antiinflammatory drugs was able to scavenge hydrogen peroxide. An original procedure to further evidence the interaction was developed thereafter, based on the reaction of 5-aminosalicylic acid with similar hemoproteins. This led to the demonstration that nonsteroidal antiinflammatory drugs were substrates for horseradish peroxidase and explained their reactivity in the horseradish peroxidase/guaiacol assay system. The compound 5-aminosalicylic acid showed an unusual behaviour that was attributed to its ability to both scavenge hydrogen peroxide and interact with horseradish peroxidase. It was concluded that the lack of specificity of horseradish peroxidase for its donor substrate may lead to erroneous results in assays for hydrogen peroxide scavenging of some drugs. An alternative method is however available and a simple spectroscopic assay can evidence the interaction with horseradish peroxidase.

Comparison of Three Chemiluminescent Horseradish Peroxidase Substrates for Immunoblotting

Comparison of Three Chemiluminescent Horseradish Peroxidase Substrates for Immunoblotting

Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.

Role of the reductant substrates on the inactivation of horseradish peroxidase by m-chloroperoxybenzoic acid.

Horseradish peroxidase reacts with H2O2 and other hydroperoxides to form Compound I, the first active enzymatic form m-Chloroperoxybenzoic acid, a xenobiotic hydroperoxide, acts as an oxidant substrate of horseradish peroxidase. However, this hydroperoxide is also a powerful inactivator of the enzyme and in this sense is more effective than H2O2. The coupled reductant substrates used in the peroxidatic reaction protect the enzyme from the inactivating process. A reaction mechanism is proposed with two competitive routes: one catalytic and one inactivating. Using a kinetic approach, the ratio between the hydroperoxide and the reductant substrate appears to be a decisive factor in the catalytic turnover of the enzyme. The role of the reductant substrates in protecting the enzyme, and the physiological and biotechnological implications of this process are discussed.

Synthesis and Characteristics of a New Fluorogenic Substrate for Horseradish Peroxidase

N,N′-Dicyanomethyl-o-phenylenediamine was synthesized with a 90% yield by a reaction ofo-phenylenediamine with chloroacetonitrile in triethylamine. Our experimental results showed that it was the effective fluorogenic substrate for horseradish peroxidase (HRP) and hemin. TheKmfor the HRP system was 48 μM,and that for hemin was 1.3 μM.Properties of the substrate were evaluated from the detection limits of enzymes and H2O2. The linear ranges for the determination of HRP and hemin were 21–150 pMand 2–20 nM,respectively. The linear range for the determination of H2O2using HRP or hemin was 18–140 and 60–1000 nM,respectively. The structural elucidation of the fluorescent product using NMR and mass spectral techniques was proposed to be 1,2-dihydro-2-imido-imidazo[1,2-a] quinoxaline. Based on the product structure and earlier reports, the possible reaction mechanism of HRP and the substrate was also proposed, i.e., two steps for ring closures, one step of isomerization, and a final step of oxidative dehydrogenation.

Amperometric determination of butanone peroxide and hydroxylamine via direct electron transfer at a horseradish peroxidase-modified platinum electrode

An amperometric organic-phase peroxide biosensor was constructed by immobilizing horseradish peroxidase (HRP) entrapped within poly(ester-sulfonic acid) Eastman AQ-55D polymer matrix on a platinum electrode surface. The enzyme electrode functioned by direct electron transfer between the HRP redox centre and the platinum surface. This mediator-free biosensor was applied to the determination of butanone peroxide and hydroxylamine, which are HRP substrate and inhibitor, respectively. Fast sensor responses were obtained in 30 s for both the substrate and the inhibitor. The biosensor showed higher catalytic efficiency in acetonitrile (1.1 µA l mmol–1) than in methanol (0.9 µA l mmol–1). However, the sensor exhibited greater percentage inhibition of HRP activity in the less catalytically efficient methanol medium (516% inhibition 1 mmol–1) than in acetonitrile (345% inhibition l mmol–1). This reversal in the performance of the sensor towards hydroxylamine in the organic solvents is attributed to the reaction media polarity combined with the butanone peroxide-induced conformational change of the enzyme active site.

A chemiluminescent immunoassay for the diagnosis of grapevine closteroviruses on nitrocellulose membrane

A dot-immunobinding assay was adapted on enhanced chemiluminescence (DIBA-ECL), which employs luminol, a cyclic diacylhydrazide, as substrate for horseradish peroxidase conjugated with a secondary antibody, for the diagnosis of grapevine closteroviruses I and III. The sensitivity of DIBA-ECL was also compared to other immunoenzymatic methods. DIBA-ECL proved to be at least 16 times more sensitive than the dot-immunobinding assay using chloronaphthol/diaminobenzidine mixture as a substrate, which was at least twice as sensitive as DAS-ELISA, DAS-indirect avidin-biotin complex ELISA, and dot-immunobinding assay, using alkaline phosphatase as enzyme. Optimisation of all parameters involved in the DIBA-ECL procedure and its advantages are discussed.

New screening system for simultaneous determination of two marker proteins by homogeneous enzyme immunoassay.

To save time and labour in mass screening, by detecting two marker proteins on one specimen using only one test. alpha Fetoprotein and ferritin were chosen to demonstrate the principal of this system. The assay reagents were horseradish peroxidase (HRP)-labelled anti-alpha fetoprotein and HRP-labelled anti-ferritin antibodies. After the serum sample had been incubated with these reagents the substrate for HRP was added and the absorbance measured. An absorbance value below the cutoff point indicated that both parameters were within normal limits; a value above the cutoff point indicated that at least one of the two parameters was abnormally high. Fifty sera from healthy Japanese subjects were assayed by the simultaneous assay method. All samples gave absorbancy values below the cutoff point. Fifty serum samples from patients with high alpha fetoprotein concentrations (over 20 ng/ml) and 50 samples with high ferritin concentrations (over 200 ng/ml) were also assayed. The absorbancy values of all samples with high alpha fetoprotein concentrations, and all but one sample with high ferritin concentrations gave values above the cutoff point. Although this homogeneous enzyme assay method was applied to the combination of alpha fetoprotein and ferritin, it could be used in mass screening for any other combination of two markers.

Fluorometric determination of hydrogen peroxide with peroxidase and 1-methyl-1,2,3,4-tetrahydro-?-carboline-3-carboxylic acid

The possibility of the fluorometric determination of hydrogen peroxide using various tetrahydro-β-carbolines as hydrogen-donating substrates for horseradish peroxidase was examined. A simple and sensitive method has been developed by using 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid as a substrate. The fluorescence intensity was proportional to the hydrogen peroxide concentration in the range of 0.05–1 μmol/l (30–600 pmol/tube) in the sample solution with a relative standard deviation of 3.8% (0.1 μmol/l).

Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).

Hydrogen peroxide, peroxidase and low rank coal

Horseradish peroxidase was tested to determine if it catalysed any reaction between coal and hydrogen peroxide. Experiments were performed in aqueous buffers of pH 5.0, 6.5 or 8.0 and used either Wyodak, Beulah Zap, Texas lignite, Mississippi Wilcox lignite or solubilized Mississippi Wilcox lignite. Reactions were monitored by determining the amounts of hydrogen peroxide consumed at various points in time. All coals reacted rapidly with hydrogen peroxide in the absence of peroxidase and these reaction rates increased as pH increased. Horseradish peroxidase did not measurably increase the reaction rates even when present in large concentrations. These data suggest that coal is not a substrate for horseradish peroxidase.

Evaluation of anionic histological dyes as co-injectable cell markers in pre-implantation mouse embryos.

The enzyme horseradish peroxidase (HRP) is a widely used microinjectable cell marker for studying cell position, lineage, and migration in many kinds of animal embryos. Marked cells are easily identified because they darken when exposed to a chromophore and an HRP substrate such as hydrogen peroxide. This assay, however, requires cytochemical fixation. Thus, when HRP-marked cells need to be identified prior to fixation, visible co-injectants such as dyes and fluorescent substances have been used with HRP. Fluorescent substances have limitations because their excitation could be harmful to the marked cells. Visible but non-fluorescent co-injectants, however, would permit visualization of HRP-marked cells without inflicting such damage. We tested the compatibility of several histological dyes and electrolytic carriers with HRP iontophoresed as a cell marker in 2-cell mouse embryos. The dyes tested were Evans Blue, Cibacron Blue F3GA, Fast Green FCF, and Patent Blue Violet; the electrolytic carriers were KCl, K2SO4, CH3CO2K, and KH2PO4. The combination found most useful was Patent Blue Violet in K2SO4. Survival of embryos incubated to the blastocyst stage following injection with HRP + Patent Blue Violet in K2SO4 at the 2-cell stage was significantly greater than that of embryos injected with any other dye. Although the proportion of embryos undergoing the 8-cell-to-morula transition was somewhat decreased by this treatment, the proportion of embryos reaching the blastocyst stage was comparable to that in the uninjected (control) group. Our results indicate that Patent Blue Violet is a useful, HRP-co-injectable dye for short-term cell marking in preimplantation mouse embryos.

3-p-Hydroxyphenylpropionic Acid—A Sensitive Fluorogenic Substrate for Automated Fluorometric Enzyme Immunoassays

The application of 3-p-hydroxyphenylpropionic acid (HPPA), a fluorogenic substrate of horseradish peroxidase (HRP) to an automated microplate fluorometric enzyme immunoassay is described. Fluorescence intensity of the end product was highly dependent on the pH of the buffer and on the concentrations of the substrate mixture ingredients. The determination of human thyrotropin (TSH) and recombinant hepatitis B surface antigen (rHBsAg) were performed using a fluorometric enzyme immunoassay (FEIA) with HPPA as the substrate, and a colorimetric one with tetramethylbenzidine (TMB) as the chromogenic substrate. The sensitivity of both types of assays proved comparable. The distinct advantage of a fluorometric assay is the possibility to perform a quantitative detection of analyte over a very wide dynamic range. Clinical evaluation of both assays showed good correlation between the FEIA and conventional methods.