Horseradish peroxidase complexes

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 technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

Make streptavidin-biotin/horseradish peroxidase complex from 20 pi streptavidin, 20 pi biotinylated horseradish peroxidase, 1 ml TBS (shake well and wait 30 min)... 

Let the fluid drain from the sections and incubate with the streptavidin-biotin/horseradish peroxidase complex for 30-60 min Wash with TBS (20 min, 3 times)... 

Karniya N, Okazaki S-y, Goto M (1997) Surfactant-horseradish peroxidase complex catalyt-ically active in anhydrous benzene. Biotechnol Techniques 11 375-378... 

Kiamiya, N., Okazaki, S. Y, and Goto, M., Surfactant-horseradish peroxidase complex catalytically active in anhydrous benzene, Biotechnol. Tech., 11, 375-378, 1997. 

Dietrichs E, Walberg F, Haines DE (1985b) Cerebellar nuclear afferents from feline hypothalamus demonstrated by retrograde transport after implantation of crystalline wheat germ agglutinin-horseradish peroxidase complex. Neurosci. Lett., 54, 129-133. 

Biotinylated secondary antibodies and streptavidin-biotm-horseradish peroxidase complex (SABHRP Amersham, UK). 

If a rabbit primary antibody has been used, then a suitable secondary antibody directed against rabbit immunoglobulins should be selected if a mouse pnmary has been used, select antimouse, and so forth. Secondary antibodies are commonly available with a fluorophore attached for use in immunofluorescence studies. Alternatively, they may be biotinylated, which permits subsequent binding of streptavidin-biotin-horseradish peroxidase complex, or they may have enzymes, such as horseradish peroxidase, attached directly. 

Streptavidin is available in the form of a streptavidin-biotm-horseradish peroxidase complex. This is available either as a preformed complex (e.g, from Amersham) or as kits containing two separate components that must be mixed immediately prior to their use (e.g., kits from Vector). We use the preformed complex because in our hands it is simple to use, gives excellent results, and is considerably cheaper. When diluting the stock solution, make sure that no sodium azide is present in the dilution buffer because azide inhibits the HRP enzyme. 

Chance et al. concluded that the valence states for the central iron of horseradish peroxidase Compounds I and II were both more- highly oxidized than Fe [144]. The identical conclusion had been reached in the earlier study of high-valent horseradish peroxidase complexes [143]. Furthermore, an Fe=0 bond distance of 1.64 + 0.02 A was determined by Chance et al. for horseradish peroxidase Compound I [144]. This finding was essentially identical to that of Penner-Hahn et al. [143],... 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

It should be pointed out that the addition of substances, which could improve the biocompatibility of sol-gel processing and the functional characteristics of the silica matrix, is practiced rather widely. Polyethylene glycol) is one of such additives [110— 113]. Enzyme stabilization was favored by formation of polyelectrolyte complexes with polymers. For example, an increase in the lactate oxidase and glycolate oxidase activity and lifetime took place when they were combined with poly(N-vinylimida-zole) and poly(ethyleneimine), respectively, prior to their immobilization [87,114]. To improve the functional efficiency of entrapped horseradish peroxidase, a graft copolymer of polyvinylimidazole and polyvinylpyridine was added [115,116]. As shown in Refs. [117,118], the denaturation of calcium-binding proteins, cod III parvalbumin and oncomodulin, in the course of sol-gel processing could be decreased by complexation with calcium cations. 

Extensive studies have established that the catalytic cycle for the reduction of hydroperoxides by horseradish peroxidase is the one depicted in Figure 6 (38). The resting enzyme interacts with the peroxide to form an enzyme-substrate complex that decomposes to alcohol and an iron-oxo complex that is two oxidizing equivalents above the resting state of the enzyme. For catalytic turnover to occur the iron-oxo complex must be reduced. The two electrons are furnished by reducing substrates either by electron transfer from substrate to enzyme or by oxygen transfer from enzyme to substrate. Substrate oxidation by the iron-oxo complex supports continuous hydroperoxide reduction. When either reducing substrate or hydroperoxide is exhausted, the catalytic cycle stops. 

Answers to these questions were initiated over a decade ago during our studies on catalase (CAT) and horseradish peroxidase (HRP) (30). Both native enzymes are ferric hemoproteins and both are oxidized by hydrogen peroxide. These oxidations cause the loss of two electrons and generate active enzymatic intermediates that can be formally considered as Fe + complexes. 

Deciphering a Complex Electroenzymatic Response Horseradish Peroxidase... 

Enzymatic catalysis mechanisms may well be considerably more complex than the basic ping-pong mechanism, involving inhibition and hysteresis phenomena. Horseradish peroxidase offers a remarkable example... 

The enzyme horseradish peroxidase is a hemoprotein and the region of the Soret band exhibits large differences between the position and extinction coefficients of the uncombined and combined forms. Both forms were first studied by spectrophotometry, but the E—S complexes were 0 labile that they could not be examined extensively by any other spectroscopic method. Using rapid-scanning spectrophotometry and rapid mixing, Chance was able to distinguish the spectra of compound I and II and determine the various rate constants of the multistep reaction with rather poor precision. 

The PAP method was pioneered by Sternberger in 1979 (1). The method uses an immunological sandwich amplification and the enzyme peroxidase to effect a signal. The unique feature of this procedure is the enzyme/antibody solution, the PAP immune complex. The horseradish peroxidase enzyme, itself an immunogenic protein, is used to inoculate a given species, and a polyclonal immune response is generated against the enzyme. This antiserum is harvested and placed in solution with the enzyme so that immune complexes form that... 

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