Peroxidase complexes

Bronckers, A.J.J., Gay, S., Finkelman, R.D., and Butler, W.T. (1987) Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs Comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement./. Histochem. Cytochem. 35, 825-830. 

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... 

Whereas antigen-retrieval technique serves to amplifying the immunocytochemical signal at the predetection phase, conventional methods of signal amplification, such as avidin biotin complex (ABC) and soluble enzyme-anti-enzyme immune complex techniques (peroxidase-anti-peroxidase complex and alkaline phosphatase-anti-alkaline phosphatase complex PAP and APAAP respectively), are applied in the phase of detection. For many years, the PAP and APAAP procedures represented the most sensitive and reliable and hence most popular techniques in many pathology laboratories. However, today these techniques are only rarely used, being substituted by modem more sensitive methods. 

A wide spectrum of hepatic lesions has been reported in AIDS (H4), but it is not known whether the changes are related to the presence of HIV-1. Therefore, sections from livers of autopsied patients with AIDS were examined for the presence of HIV-1 antigen p 24 (core) and gp 41 (envelope) by the avidin-biotin-peroxidase complex methods using monoclonal antibodies. The most common histologic abnormalities were steatosis, portal inflammation, Kupffer cell hyperplasia, and focal hepatocellular and bile duct damage. Immunoreactivity for HIV-1 antigens was demonstrated in 80% of cases. 

As part of a subsequent study concerning primarily second-site revertant yeast iso-l-cytochrome c variants, Hazzard et al. evaluated the effect of converting Lys-72 to an aspartyl residue by site-directed mutagenesis on the electron transfer kinetics of the cytochrome c-cytochrome c peroxidase complex [136]. Lys-72 was of interest for this purpose, because it is involved in the hypothetical model for the complex formed by these two proteins that was proposed by Poulos and Kraut on the basis of molecular graphics docking [106]. In these... 

Hsu, S., Raine, L., and Eanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577. 

Fig. 1. Diagram illustrating the molecular interactions of the ABC procedure. The primary antibody against the antigen of interest is linked to the avidin-biotinylated peroxidase complex via a biotinylated secondary antibody raised against immunoglobulin of the animal species used to generated the primary antibody (A, immunoglobulin , biotin A, avidin , peroxidase).

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. 

A test was conducted to determine if the coverplate plastic had any deleterious effect on the AEG chromogen. Coverplates were finely ground, and the resulting plastic powder was added to AEG. Various concentrations of plastic were used. After 10 min of incubation with the plastic, streptavidin-peroxidase complex was added. After another 5 min, the tubes were photographed. Over a range of added plastic from 0.01 to 0.04 g, there was no detectable decrease in the chromogenic reaction as compared to a tube with no added plastic. 

Fig. 11.3 Nitrogen configuration example Indoline/cytochrome C peroxidase complex (PDB laek). Alternative hydrogen positions indicated by dotted lines.

The last example (Figure 11.3) underlines the importance of the correct nitrogen configuration. In the indoline/cytochrome C peroxidase complex, only one of the three sketched hydrogen positions allows for an interaction with an active site aspartate. 

The crystal structures of two ferulic acid complexes of HRP C have been solved, one with resting state enzyme (to 2.0 A resolution) and the other with the cyanide-ligated enzyme (to 1.45 A resolution) 195). These represent a major achievement for the crystallography of peroxidase complexes. The binary complex is heterogenous, according to the 2Fo-Fc omit difference electron density map of the active site. The disordered density observed has been interpreted in terms of three... 

Fig. 10 X-ray co-crystal structure of praziquantel 211 and glutathione-S-peroxidase complex (PDB ID IGTB). Praziquantel is shown as yellow sticks and the receptor binding site is shown as hydrophobic surface...

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)... 

This intermediate could then halogenate substrate AH (Eq. 16-13, step a), lose HOC1 (Eq. 16-13, step b), or generate Cl2 by reaction with CL (Eq. 16-13, step c). These are all well-established reactions for the enzyme. In each case the chlorine in the peroxidase complex can be viewed as an electrophile which is transferred to an attacking nucleophile. A fourth reaction that can go... 

The sections are incubated for 72 hr at 4°C with primary antiserum in PBS containing 2% normal serum and 0.25% gum arabic (Sigma). The sections are then incubated overnight at 4°C with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), diluted 1 200. They are next incubated overnight at4°C with avidin-biotinylated peroxidase complex (ABC) diluted 1 100 in PBS containing 0.25% gum arabic. Note that the peroxidase-antiperoxidase procedure can also be used. 

Before being removed from the autoclave, thejars are allowed to cool in the autoclave until the temperature has reached 50°C. After thejars have reached a temperature of 30°C, the sections are incubated overnight at 4°C with MIB-1 antibody at a concentration of 2.67 p,g/ml. The sections are treated successively with biotinylated antimouse IgG antibody diluted 1 300 for 30 min, streptavidin-biotinylated peroxidase complex, diluted 1 50... 

Cattoretti G, Berti E, Schiro R, D Amato L, Valeggio C, Rilke F. Improved avidin-biotin-peroxidase complex (ABC) staining. Histochem J 1988 20(2) 75-80. 

Figure 6.2 The mechanism of cytochrome-c-peroxidase complex formation, (a) Native enzyme, (b) Activated complex with the acid-base catalytic function of distal histidine (His) and stabilization of negative charge by arginine (Arg) residue of the active site, (c) Hypothetic intermediate oxene complex, (d) Complex I after intramolecular electron regrouping of oxene complex with Fe4+ and free radical X fragment formation.

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