Substrates POase

The rate of an enzyme-catalyzed reaction as a function of pH generally yields a bell-shaped curve. Some enzymes are very sensitive to small changes in pH (lysozyme), whereas others (POase) are relatively insensitive (within 1-2 pH units near their optima). Enzymes with similar activities but from different origin may have very different optima. APase from Escherichia coli is optimally active at a pH of about 8, whereas APase from calf intestine is most active around pH 10 and the activity of these enzymes decreases strongly outside their optima. Nevertheless, the E. coli enzyme is often assayed at the pH optimum of the intestinal enzyme. The optimum substrate concentrations may also be pH dependent for some enzymes (Chapter 10). 

Immobilization of the enzyme may also have direct effects on its catalytic ability in that conformational changes may lead to partial inactivation which affects the Michaelis-Menten parameters. Allosteric enzymes may, moreover, loose their ability to undergo allosteric activation. Steric restrictions may also be responsible for lower activities of immobilized enzymes by preventing or hindering the access of the substrate or effectors. On the other hand the stability or activity of enzymes on a solid phase is often better than in the fluid phase, probably due to the local high concentration of enzyme. Certain solid phases may, however, directly inactivate the enzyme, such as polystyrene for horseradish POase (Berkowitz and Webert, 1981). 

The purity of the enzyme is reflected by its specificity for a panel of substrates. Many preparations may contain contaminating activities and the number of crystallizations indicated for commercial preparations is a poor criterion for purity. The absence of proteases is important to maintain maximum stability of the preparations. Contaminating microorganisms very often produce proteases however, the addition of preservatives may interfere with enzyme activity. For example, POase is extremely sensitive to both contaminating bacteria and NaNs, Sometimes, enzymes can be stabilized by the addition of substrate homologues. 

The six co-ordination positions of iron are occupied by the nitrogen atoms of porphyrin, by the protein (probably through histidine Haschke and Friedhoff, 1978) and by the substrate. The complete amino acid sequence of POase-C has been established (Welinder, 1979). The apoprotein specifies and amplifies the inherent properties of the prosthetic group (Saunders et al., 1964). 

Fig. 10.2. Scheme of catalysis by POase. POase combines with H2O2 (substrate) to form component 1 and is reduced in two successive steps by a H-donor to the original state. Generally, the appearance of oxidized donor is measured. An excess of H,0, will inactivate enzyme (compound HI and IV). The activation energy for 1 is 9 kJ-mole (Marklund et al., 1974). 

An excess of substrate inactivates the enzyme by forming compound III (red) or IV (emerald green). This substrate inhibition is very substantial in some of the current EIA procedures using POase (Tijssen et al., 1982). 

A sensitive and versatile chromogenic assay for POase is based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB) (Ngo and Lenhoff, 1980) in the presence of substrate. MBTH is the donor which, after oxidation, reacts with DMAB to form a cationic in-... 

Like POase, MPOase is also inhibited by high concentrations of substrate (H2O2). MPOases are also sensitive to cyanide (67% inhibition at 10 M) and, importantly, very sensitive to glutaralde-hyde used in fixation or conjugation procedures. In contrast, MPOases are very heat-stable, e.g., resistant to boiling for 1 h. 

The preparation of substrate solutions and measurement of activity of horseradish peroxidase The usefulness of POase... 

Fig. 14.1. Determination of the optimum dilution of H202as substrate with horseradish POase C with DMAB/MBTH as H-donor. High dilutions as well as high concentrations of the substrate decrease the detectability of the enzyme. The optimum is around 0.0025-0.003% H2O2 (reproduced from Tijssen et al., 1982 courtesy of Archives...

The ideal enzyme label for EIH should convert a soluble substrate to an insoluble product so that an identifiable precipitate forms immediately at the site of enzyme action, whereas the substrate should not contribute to background staining. The precipitate in the cell can then be seen with a light microscope and may be rendered, in some systems, electron opaque for studies by electron microscopy. In particular POase (Avrameas and Uriel, 1966 Nakane and Pierce, 1966) and GOase are useful for this purpose. In EIH an amplification of the signal is possible to a degree seldom attained in enzyme cytochemistry and often much less tedious. Recent developments in EIH made routine applications of these techniques possible (reviews Bullock and Petrusz, 1982 Van Noorden and Polak, 1983). 

POase (Section 10.1.1) in EIH is so universal that the techniques described here have been widely known as immunoperoxidase. The popularity of POase is due to the fact that several H-donors fulfill the requirements of suitable substrates, the enzyme is stable, of moderate size, and affordable. 

Endogenous enzyme activity is readily recognizable in some cases (e.g. staining of peroxisomes with POase substrates) and can be inhibited specifically (Section 17.3.4.3.2.1). 

Chromogenic detection of horseradish peroxidase POase is widely used in enzyme immunoassays (EIA) and many suitable chromogens (which are oxidized by the enzyme in the presence of the peroxide or urea peroxide substrates) have been developed. Peroxide is the usual substrate, particularly on solid phases since its reduction results in the formation of inert water near the solid phase (Fig. 7.9). It should be realized that POase has a very pronounced optimum concentration of H2O2 substrate (Tijssen et al., 1982). Activity is low at low substrate concentrations, but inhibition is considerable at high substrate concentrations. The universally used POase, C isozyme, has an optimum in solution of 0.003% peroxide but higher concentrations are usually required on a solid phase. 

Fig. 7.9. General catalytic reaction mechanism of POase. Note that excess substrate leads to enzyme inactivation. Hydrogen donors such as 3,3 -diaminobenzidine (DAB), 4-chloro-l-naphthol, 3,3, 5,5 -tetramethylbenzidine (TMB) and o-dianisidine (ODA) are often used, but the color reaction (III) proposed by Conyers and Kidwell (1991) yields superior results.

J. J.5.2. Luminol substrates for peroxidase Cyclic diacylhydrazides, such as luminol (Fig. 7.11), can be oxidized by POase in the presence of H2O2 (Matthews et al., 1985). Luminol is then converted via an... 

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