PH-activated

Table 9. Use of indicator dyes to detect pH-active substances.

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

Magnesium is a relatively reactive metal, and can be chromated in nearly neutral solutions as well as in acid solutions. The range of treatments possible illustrates well the role of pH, activating anion, temperature and duration of treatment in promoting the breakdown of passivity in the chromate solution and the consequent formation of a chromate him. 

Krieger, N., and Hastings, J. W. (1968). Bioluminescence pH activity profiles of related luciferase fractions. Science 161 586-589. 

Figure 5. pH activity profile of PGE on polygalacturonate. Mcllvaine buffers were used in the pH range from 2.5 to 5,5, Reducing sugars were detected by the neocuproine method. PGE (2 mg/ml) was 80-times diluted and incubated with 500 pi of a 0.25% (w/v) polygalacturonate solution, at SO C. 

There are two catalytically active residues in pepsin Asp-32 and Asp-215. Their ionizations are seen in the pH-activity profile, which has an optimum at pH 2 to 3, and which depends upon the acidic form of a group of pKa 4.5 and the basic form of a group of pKa 1.1.160,161 The pKa values have been assigned from the reactions of irreversible inhibitors that are designed to react specifically with ionized or un-ionized carboxyl groups. Diazo compounds—such as A-diazoacetyl-L-phenylalanine methyl ester, which reacts with un-ionized carboxyls—react specifically with Asp-215 up to pH 5 or so (equation 16.28).162-164 Epoxides, which react specifically with ionized carboxyls, modify Asp-32 (equation 16.29). 

Enzymes require an optimum pH at which their catalytic activity is maximal. The pH-activity profiles of enzymes indicate the pH at which the catalytic sites are in their necessary state of ionization. The optimal pH of an enzyme may be different from that of its normal environment. The action of enzymes in cells may be regulated by variation in the pH of the surrounding medium. 

Three cyanide-degrading nitrilases were recently cloned and purified and their kinetic profiles were evaluated in order to better understand their applicability to cyanide bioremediation. CynD from Bacilluspumilus Cl and DyngD from Pseudomonas stutzeri exhibit fairly broad pH profiles with >50% activity retained across pH 5.2 to pH 8.0 while the CHT (NHase) from Gloeocercospora sorghi exhibited a more alkaline pH activity profile with almost all of its activity retained at pH 8.5, slightly lower thermal tolerance, and quite different metal tolerance compared with the two bacterial enzymes [46]. 

The sensor for the measurement of high levels of CO2 in gas phase was developed, as well90. It was based on fluorescence resonance energy transfer between 0 long-lifetime ruthenium polypyridyl complex and the pH-active disazo dye Sudan III. The donor luminophore and the acceptor dye were both immobilized in a hydrophobic silica sol-gel/ethyl cellulose hybrid matrix. The sensor exhibited a fast and reversible response to carbon dioxide over a wide range of concentrations. 

An extensive study of the effect of salts on the pH-activity curves of PM was made on alfalfa49 and on orange21 and tomatoes.44 In general, the effect of salts is to lower the pH at which maximum activity is attained and to extend the activity into lower pH regions. At the higher pH values (7-8), salts have practically no activating effect. The main usefulness of salt activation of PM seems to lie in counteracting adverse pH conditions. 

Due to shifts of the pH-activity profiles, pH values often must be adjusted to obtain the optimal activity of the enzyme under investigation (Maurel and Douzou, 1975). When the above requirements are fulfilled, there is always a residual effect of the cosolvent on enzyme activity. In most cases, such an effect is small compared to the effect of lowering temperature. It must be checked that the effect is instantaneous upon addition of the solvent, independent of time, and fully reversible by infinite dilution or dialysis. If these conditions are not met, one should suspect denaturation. 

In the current study we report the investigation of the thermal denaturation behavior of glutaraldehyde modified -D-glucosidase by differential scanning microcalorimetry (DSC), along with the effect of glutaraldehyde crosslinking on the pH/activity-stability of the enzyme at elevated temperatures. 

The protein(s) is relatively unstable at its true pHopt, and this lack of stability has not been corrected in the pH-activity plot. Thus, the observed pHopt is a compromise of the effect of pH on both catalytic activity (under the assay conditions) and protein denaturation and/or conformation. 

An interesting observation is that an enzyme may exhibit different pH activity profiles for various neutral substrates. The explanation of this is that the enzyme binds or transforms such various substrates differently. For example. Taka amylase has different pH optima for long chain amyloses and for low molecular mass substrates. Some specific chemical modifications of the side chains of the enzyme may also alter the pH activity profiles. Kobayashi, Miura and Ichisima (1992) modified the lysine amino groups using bifimctional reagent o-phtalaldehyde, and observed a pronounced shift in the pH-dependence of ohgomaltoside hydrolysis. 

With the exception of the enzyme from the limpet, P. vulgata, a-D-mannosidase from most of the important sources shows optimal activity at pH values lying between 4 and 5. For the enzyme from jack-bean meal39 and that from rat epididymis,80 we employed a pH of 5 for routine assays. If this is not the actual optimum, it is close to it on the broad pH-activity curves, and, at this pH, the addition of Zn2+ and other cations has relatively little effect in the assay, thus simplifying the study of the various metal complexes that can be formed by the enzyme protein. 

The dissociation of the active protein-Zn2+ complex at low pH can be seen from the pH-activity curves shown in Fig. 1. This Figure also shows the effect of Cl-. Chloride ion not only accelerates hy-... 

Yes, we have made some pH activity studies. One example is shown in the article (Fig. 20) for the decarboxylation of oxalacetate. Interestingly, the bell shape is similar to that found by others in the catalysis of this reaction by small-molecule amines. 

A coating bearing one enzyme (papain) is produced on the surface of a glass pH electrode by the method previously introduced (co-crosslinking). The papain reaction decreases the pH, and the pH-activity variation gives an autocatalytic effect for pH values greater than the optimum under zero-order kinetics for the substrate (benzoyl arginine ethyl ester) the pH inside the membrane is studied as a function of the pH in the bulk solution in which the electrode is immersed. A hysteresis effect is observed and the enzyme reaction rate depends not only on the metabolite concentrations, but also on the history of the system. 

Dennehy PH Active immunization in the United States Developments over the past decade. Clin Micro Rev 2001 14 872. 

Lyophilized enzymes have a pH memory, meaning that the activity of the enzyme in organic solvent parallels its pH-activity profile of the aqueous solution from which it was lyophilized [36, 79-81]. However, very often acidic or basic mixtures within a nonaqueous reaction mixture such as reactant, products, or impurities, can disrupt this delicate protonation state, leading to changes in catalytic activity. To counteract this potential problem, solid-state buffers have been developed to protect the enzyme s protonation state in the nonaqueous environment [53, 82]. These solid-state buffers contain pairs of crystalline solids that can be intercon-... 

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