Horse radish peroxidase reaction

Experiments on Efficiency in the Forced Oscillatory Horse-Radish Peroxidase Reaction... 

In the reaction of luminol, hydrogen peroxide, and horse radish peroxidase 122> the chemiluminescence intensity is proportional to the square of luminol radical concentration. The lifetime of these luminol radicals was found by ESR techniques to be about 10 sec. Titration studies revealed that luminol acts as two-electron donor during the reduction of a hydrogen peroxide-horseradish peroxidase complex. The enzyme is not involved in the reaction step leading directly to light emission. This step is formulated as... 

The enzymes commonly used as labels in ELISA and other immunochemical reactions include horse radish peroxidase (HRP) and alkaline phosphatase (AP). The enzyme can be covalently coupled to the antibody using glutaraldehyde conjugation to reactive amino groups on the enzyme (lysines) in a phosphate buffered aqueous solution at neutral pH, as shown in Fig. 19 (103). Alternatively, carbohydrates present in the immunoglobulin structure can be cleaved by periodate treatment (see Fig. 20) and bound to free amino groups on the enzyme through a Schiff base reaction (103). 

The oxidation of DOPA and adrenaline to dopachrome and adrenochrome, respectively, by a horse radish peroxidase-H202 system has been reported by Herzmann.29,30 The oxidation process was activated by trace quantities of caffeic acid, its esters, and related compounds.30 Ascorbic acid inhibited the oxidation of adrenaline by this enzyme in the initial stages of the reaction, but later had a stimulatory effect.30... 

If we consider the fate of substrate AH2 during the action of a peroxidase, we see that donation of an electron to compound I to convert it into II (Fig. 16-14, step c) will generate a free radical AH as well as a proton. The radical may then donate a second electron to II to form the free enzyme. Alternatively, a second molecule of AH2 may react (Fig. 16-14, step c) to form a second radical AH. The two AH radicals may then disproportionate to form A and AH2 or they may leave the enzyme and react with other molecules in their environment. Compound II of horse radish peroxidase is able to exchange the oxygen atom of its Fe(IV)=0 center with water rapidly at pH 7, presumably by donation of a proton from the nearby histidine side chain (corresponding to His 52 of Fig. 16-13).227/230a/b This histidine presumably also functions in proton transfer during reactions with substrates (see Fig. 16-11B).224... 

The sections are incubated for 1 hr with the primary monoclonal antibody, mouse antihuman mast cell tryptase antibody (DAKO), diluted 1 200 with 1% BSA/PBS. They are washed for 10 min in PBS using magnetic stirring, incubated with biotinylated antimouse antibody for 15 min, and washed in PBS. This is followed by adding avidin-biotin-horse-radish peroxidase for 15 min. A Vector DAB Substrate kit is applied to develop the reaction product by using nickel-DAB (5 min developing time) according to the manufacturer s instructions. This step yields a black reaction product at sites of mast cell tryptase. 

Ternaux and Chamoin described an enhanced chemiluminescence assay method for the determination of acetylcholine [48]. Reaction medium was prepared by mixing 250iu/mL of choline oxidase (100 pL), 2mg/mL of horse-radish peroxidase (50 pL) and 10-120 pM luminol in 100 pL of 0.1 M Tris buffer (pH 8.6), or 100 pL of 10-100 pM 7-dimethylamino-naphthalene-l,2-dicarbonic acid hydrazide, for 10min in 5mL of 0.1 M sodium phosphate buffer (pH 8.6). Aqueous 0.325-80 pmol of acetylcho-lineesterase (50 pL) purified on a Sephadex G50 coarse column was mixed with 450 pL of reaction mixture, and the chemiluminescence was measured at 21° C. 

Pascual et al. studied the effect of antioxidants on the luminal chemiluminescence produced by dipyridamole [54], The method is based on the use of the reaction between luminal and horse radish peroxidase, and is proposed for the determination of dipyridamole. 

In spite of a long-time paradigm that enzymes can be active only in their natural aqueous media and other solvents cause deactivation and denaturation of proteins, at present a growing number of investigations are devoted to enzymatic reactions in organic solvents (Klibanov, 2001 Ke et al., 1996 Koskinen and Klibanov, 1996 and references therein). Such enzymes as a-chymotrypsin, subtilisin ribonuclease, pancreatuc lipase, and horse radish peroxidase have been found to be markedly active in organic solvents (alcohols, amines, tiols,anhydrous alkanes, acetonitril, dichloromethane, methyl acetate, etc.). 

In this test, oxygen from air is used for the enzymatic oxidation of (3-D-glucose in the presence of immobilized GOD to gluconic acid and hydrogen peroxide. Hydrogen peroxide can be determined by a second enzyme reaction. With horse radish peroxidase as catalyst o-phenylendiamine is oxidized by H2O2 to 2,3-diaminophenazine, which can be photometrically determined at 490 nm, thus establishing a quantitative relationship between active GOD sites and the intensity of the absorption band. 

Electrochemical biosensors have been divided into two basic types enzyme-based sensor and electrochemical probe-based sensor. Alkaline phosphatase (ALP) and horse radish peroxidase (HRP) have been often employed for enzyme-based biosensors using p-nitrophenyl phosphate (PNP), a-naphtyl phosphate, 3-3, 5,5 -tetramethylbenzidine (TMB) and 2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrates of electrochemically active species, and ferrocene (Fc) and methylene blue as the electrochemical mediators. In general, enzymatic amplification of electrochemical signals enables highly sensitive detection of analytes. On the other hand, a direct detection of analytes by using electrochemical probes allows a more rapid time-response onto the detector surface and needs no enzymatic reaction. Based on the reason, a direct detection of analytes by using electrochemical probes has been... 

A C-60 linked oligodeoxynucleotide has been demonstrated to facilitate sequence-specific modification of guanosine residues in a complementary oligonucleotide. The hybridisation of an oligonucleotide conjugated to Horse Radish Peroxidase can be directly measured as an electrical current which results from detection of the peroxide reaction upon hybridisation. ... 

Another endogenous HNO source relies on the oxidation of hydroxylamine (HA), or other alcohol amine, such as hydroxyurea or A -hydroxy arginine. In vivo, such a process is postulated to depend on the activity of several heme proteins, which are able to stabilize oxo ferryl species (compound I and compound II), such as peroxidases. Recently, Donzelli et al. evaluated HNO production by this mechanism (22), with a newly developed selective assay in which the reaction products, GS(0)NH, in the presence of reduced glutathione (GSH) are quantified by HPLC. Their results showed that metmyoglobin, horse radish peroxidase, and myeloperoxidase were efficient HNO producers using hydroxylamine as substrate. However, there are several remaining unresolved questions concerning the proposed mechanism (which is outlined below, Eq. (2)). 

PiETTE et al. (1964) measured the dismutation reaction constant, at pH 4.8 to be 15.0 M xs b This extreme stabihty of the free radical allowed further oxidation of the free radical by horse radish peroxidase in the substrate-limited case the rate constant for this reaction Piette et al. (1964) foimd to be 3.9x10 M" xs", approximately 10 times slower than ki, the first oxidation. 

The chemiluminescent reaction between Horse Radish Peroxidase (HRP)/ Alkaline Phosphatase (AP) and the luminol/CSPD/hydrogen peroxide substrate is used in a multianalytical ELISA approach to simultaneous analysis of different pesticides. The pesticides included in the present study were 2,4-D, Atrazine and Simazine. A novel variant of peroxidase (from transgenic tobacco, TOP) has also been investigated. 

In their studies on peroxide reaction, Ohnishi et al. (1969) further showed that certain phenols like p-cresol were very fast substrates for horse radish peroxidase. 

In this section, we turn to experiments on the horse radish peroxidase (HRP) reaction [14,15], which is the oxidation of nicotinamide adenine dinucleotide (NADH) catalyzed by HRP... 

The structure of horse metHb has been determined at 2.0 A resolution. The increased resolution (c/. 5.5 and 2.8 A) is sufficient to show up a number of bound water molecules in the contact regions between the subunits which had been overlooked previously, but it is not, unfortunately, sufficient to decide whether the porphyrin rings are fiat or slightly domed, puckered or ruffled. The effects of pressure on the visible spectra of metHb and metMb have been reported (together with those of cytochrome c and horse radish peroxidase) the effect is to shift the equilibrium between open and closed crevice structures in favour of the latter. The spectrum of the short-lived intermediate formed in the reaction of eaq" with metHb is consistent with a low-spin iron(ii) species the rate of its subsequent transition to the stable high-spin derivative is solvent dependent. The results of a resonance Raman study on inositol hexaphosphate binding to metHb fluoride are consistent... 

Figure 1.2 Repetitive spectoa at 1.25 ms interval during the reaction of a mutant of horse-radish peroxidase (Arg38 - Lys) compound 1 (1.25 iM) with /vaminobenzoic acid (200 pM) at pH 7 and 25 C. Experiments were carried out with a HiTech Scientific Ltd SF-61 stopped-flow spectrophotometer with MG-6000 rapid scanning system by Drs A. T. Smith and R. N. F. Thomley (unpublished observations).

Table XII, based partly on Table III of Millikan s review (137), summarizes the various constants, reduced to 20 and pH 7.4. In the case of sheep hemoglobin we have retained the values given by Roughton as nearly as possible rather than attempt to reconcile the equilibrium constant and the velocity constants on the assumption of independent hemes. This table includes rough, indirect estimates of constants for the oxygen reaction of the respiratory ferment (232) and the hydrogen peroxide reaction of catalase (67) as well as the recent elegant results of Chance (29) on the hydrogen peroxide reaction of horse-radish peroxidase.

Peroxidase can be activated by a thermostable factor in pea seedlings to be a direct oxidase toward phenylacetaldehyde (413). Purified horse-radish peroxidase plus manganous ions behaves similarly toward this substrate. In either case, benzaldehyde and formic acid are the primary products, but the oxygen stoichiometry of the reaction is obscured by side reactions. Catalase has an inhibiting effect, which suggests that peroxide is an obligatory intermediate or activator. 

Conn and Seki have reported that mitochondrial preparations from higher plants catalyze the oxidation of phenylpyruvic acid to benz-aldchyde, CO2 and other products, with the consumption of one molecule of oxygen per molecule of substrate (159a). The reaction is heat labile, and is inhibited by catalase as well as by cyanide and by catechol and hydroquinone, but all attempts to demonstrate hydrogen peroxide in the reaction mixture during the course of the reaction have been unsuccessful. A mixture of horse-radish peroxidase and manganous chloride also catalyzes the reaction. 

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