Effect of pH on activity

Fig. 7. Effect of pH on activity of soluble invertase ( ) and Dowex-lx4-200/inver-tase complex (O).

Figure 4 36 Effect of pH on activity and stability of an enzyme. Curve A v versus pH plot. Curve J3 v at pH 6.8 after preincubating the enzyme at the indicated pH values. The decline in activity between pH 6.8 and 5.0 and between 6.8 and 8.0 can be ascribed to the effect of pH on ionizable groups of the active site or substrate, The decline in activity above pH 8.0 and below 5.0 can be ascribed to irreversible denaturation of the enzyme.

First of all the pH value and the temperature of the assay have to be chosen. The pH-optimum of the enzyme is determined by measuring the effect of pH on activity. It has to be recognized that the location of the pH-optimum depends on... 

The inhibitory activity of sorbates is attributed to the undissociated acid molecule. The activity, therefore, depends on the pH of the substrate. The upper limit for activity is approximately pH 6.5 in moist appHcations the degree of activity increases as the pH decreases. The upper pH limit can be increased in low water activity systems. The following indicates the effect of pH on the dissociation of sorbic acid, ie, percentage of undissociated sorbic acid at various pH levels (76,77). 

Since hypohalous acid is a much more active disinfectant than the hypohalite ion, the effect of pH on ionization becomes important. Hypobromous acid has a lower ionization value than hypochlorous acid and this contributes to the higher disinfectant activity of BrCl compared with chlorine. 

Fig. 1.30 Corrosion of a metal in an acid in which both metal dissolution and hydrogen evolution are under activation control so that the .log i curves are linear, (a) Effect of pH on and I o Hi increase in pH (decrease in an + ) lowers E and decreases / o (b) Effect of...

Fig. 3.1.6 Effects of pH on the activity and stability of Cypridina luciferase (solid lines) and the quantum yield of Cypridina luciferin (dashed line). In the measurements of activity and quantum yield, luciferin (1 pg/ml) was luminesced in the presence of luciferase (a trace amount for the activity measurement 20 pg/ml for the quantum yield) in 20 mM buffer solutions of various pH containing 0.1M NaCl, at 20°C. In the stability measurement, luciferase (a trace amount) was left standing in 0.1 ml of the buffer solutions of various pH for 30 min at 20°C, then the activity was measured by adding 1 ml of 50 mM sodium phosphate buffer, pH 6.5, containing 0.1 M NaCl and 1 pg of luciferin, at 20°C. The activity and stability data are taken from Shimomura et al., 1961, with permission from John Wiley 8c Sons Ltd.

Fig. 4.3.1 Effect of pH on the total light emission of phialidin (A), and the temperature stability profiles of phialidin (minute open circles) and aequorin (solid line) (B). In A, each buffer contained 0.1 M CaCl2 plus 0.1 M Tris, glycine or sodium acetate, the pH being adjusted with NaOH or HC1. In B, the photoprotein samples in 10 mM Tris-EDTA buffer solution, pH 8.0, were maintained at a test temperature for 10 min, and immediately cooled in an ice water bath. Then total luminescence activity was measured by injecting 1ml of 0.1 M CaCl2/Tris-HCl, pH 7.0, to 10 pd of the test solution. From Levine and Ward (1982), with permission from Elsevier.

Fig. 4.5.5 Effect of pH on the luminescence of coelenterazine catalyzed by Periphylla luciferases A, B and C, and on the stability of the luciferases. The effect on light intensity (solid lines) was measured in 3 ml of 50 mM phosphate buffers, pH 4.1-7.25, and 50 mM Tris-HCl buffers, pH 7.1-9.7, all containing 1 M NaCl, 0.025% BSA, and 0.3 pM coelenterazine. To measure the stability (dotted lines), a luciferase sample (5 pi) was left standing for 30 min at room temperature in 0.1 ml of a buffer solution containing 1 M NaCl and 0.025% BSA and having a pH to be tested, and then luciferase activity in 10 pi of the solution was measured in 3 ml of 20 mM Tris-HCl, pH 7.8, containing 1M NaCl, 0.05% BSA, and 0.3 pM coelenterazine at 24°C. The amounts of luciferases used for measuring each point were luciferase A, 150 LU luciferases B and C, 170 LU. One LU = 5.5 x 108 quanta/s. From Shimomura etal., 2001.

Robertson, S.P. Kerrick, W.G.L. (1979). The effects of pH on Ca -activated force in frog skeletal muscle fibers. Pfluegers Arch. 380,41 5. 

Figure 8-2. Effect of pH on enzyme activity. Consider, for example, a negatively charged enzyme (EH ) that binds a positively charged substrate (SH ). Shown is the proportion (%) of SH+ [ ] and of EH [///] as a function of pH. Only in the cross-hatched area do both the enzyme and the substrate bear an appropriate charge.

It has been shown that in some compounds the active species is the non-ionized molecule while the ion is inactive (benzoic acid, phenols, nitrophenols, salicylic acid, acetic acid). Thus, conditions of pH which favour the formation of the ions of these compounds will also reduce their activity. The effect of pH on the ability of acetic acid and phenol to inhibit the growth of a mould is shown in Fig. 11.4. 

Effect of pH on the activity of PNL. The enzyme exhibited maximum activity at pH 9.5 (figure 6). 

Figure 6. Effect of pH on the activity. Reaction mixtures, buffered at different pH values 7-9 (tris/HCl), 9-10 (glycine), were incubated under standard conditions. Both buffers were 0.05M of final concentration in the reaction mixture.

The pH of the finished product may have a strong influence on the type of preservative used. A good example of this can be seen with the use of organic acids which may exist in a predominantly dissociated or an undissociated form as a consequence of the product pH. The undissociated form is considered to confer the antimicrobial activity and the effect of pH on benzoic, sorbic and dehydroacetic acid is described in the graph below. It can be seen that, at the normal pH of most personal care products ie. 5.5 to 7.0, there is little activity remaining. Hence organic acids would be suitable preservatives for predominantly acidic products, such as astringent washes made with lemons. 

Several studies have been carried out to investigate the effect of pH on azo dye decolorization. In these assays, the decrease of absorbance at the wavelength corresponding to the maximum absorption for each dye is used as the method to evaluate the effectiveness of decolorization. Unfortunately, in most cases it is not clear if the isosbestic point of each dye was taken into account, and so it cannot be well understood if the different decolorization rate at different pH is due to a physical factor or to a differently influenced metabolic activity. 

The most conventional investigations on the adsorption of both modifier and substrate looked for the effect of pH on the amount of adsorbed tartrate and MAA [200], The combined use of different techniques such as IR, UV, x-ray photoelectron spectroscopy (XPS), electron microscopy (EM), and electron diffraction allowed an in-depth study of adsorbed tartrate in the case of Ni catalysts [101], Using these techniques, the general consensus was that under optimized conditions a corrosive modification of the nickel surface occurs and that the tartrate molecule is chemically bonded to Ni via the two carbonyl groups. There were two suggestions as to the exact nature of the modified catalyst Sachtler [195] proposed adsorbed nickel tartrate as chiral active site, whereas Japanese [101] and Russian [201] groups preferred a direct adsorption of the tartrate on modified sites of the Ni surface. 

Figure 8.2 The effect of pH on the enzyme lactate dehydrogenase (EC 1.1.1.27). The enzyme shows maximum activity at pH 7.4 (A). When stored in buffer solutions with differing pH values for 1 h before re-assaying at pH 7.4, it shows complete recovery of activity from pH values between 5 and 9 but permanent inactivation outside these limits (B).

The effect of pH on in vitro aldrin epoxidase activity was established over a pH range 6.5-8.5 (Figure 1). A pH of 7.5 was used as optimum. The effect of temperature on in vitro aldrin epoxidase activity was determined over a range of 20°-40°C (Figure 2). An optimum incubation temperature of 30°C was used. The maximum epoxidase activity was attained at a Tris-HCl buffer concentration of 5.0 X 10"1 M (Figure 3). 

It is appropriate now to return to the effect of pH on the [Co(phen)3] oxidation of PCu(I). If protonation at the remote site influences the reaction of PCu(II), then a similar effect might be expected for the reaction of PCu(I) with positively charged complexes. In the case of PCu(I) the kinetics are dominated by the inactivation resulting from the active site protonation. Whereas the pK for the [Fe(CN)g] oxidation is in good agreement with the HNMR independently measured value, the apparent pK obtained with [Co(phen)3] " is significantly higher, an effect which is clear from an inspection of Fig. 10. A two pK fit is possible in the case of [Co(phen)3], as has been illustrated [1,100],... 

The hydrogenase film on the electrode was very stable, and this allows the study of active/inactive interconversion under strict potential control. By comparing cyclic voltammetry and potential step chronoamperometry, we were able to integrate energetics, kinetics and H e stoichiometry of the reaction. The effects of pH on these processes could also be conveniently observed. 

Fig. 2. Effects of pH on Enzyme Activity. Symbols ( ), P-mannanase M-I ( ), P-mannanase M-II (A), P-mannanase M-III.

Studies on the effect of pH on peroxidase catalysis, or the heme-linked ionization, have provided much information on peroxidase catalysis and the active site structure. Heme-linked ionization has been observed in kinetic, electrochemical, absorption spectroscopic, proton balance, and Raman spectroscopic studies. Kinetic studies show that compound I formation is base-catalyzed (72). The pKa values are in the range of 3 to 6. The reactions of compounds I and II with substrates are also pH-dependent with pKa values in a similar range (72). Ligand binding (e.g. CO, O2 or halide ions) to ferrous and ferric peroxidases is also pH-dependent. A wide range of pKa values has been reported (72). The redox potentials of Fe3+/Fe2+ couples for peroxidases measured so far are all affected by pH. The pKa values are between 6 and 8, indicative of an imidazole group of a histidine residue (6, 31-33),... 

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. 

The pHopt may not reflect the effect of pH on protein activity (or on biological processes) but may be a result of the effect of pH on the ionization(s) of the sub-strate(s). This may be particularly true when utilizing artificial substrates having value(s) significantly dif-... 

Fig. 2.2.4.3 Effect of pH on the activity of recombinant ADH. The enzymatic activity was measured using the following 250 mM buffers citrate/Na2HP04, pH 4.0-7.0 TEA/HCl, pH 7.0-8.6 and glycine/NaOH, pH 8.6-11.0.

The effect of pH on the apparent catalytic activity of cupric ions in a solution containing glycine is depicted in Fig. 2. The activity passes through a maximum at a pH of about 2. This has been interpreted (Peters and Halpern, 21) in terms of the following pH-dependent equilibria ... 

Istarova, T.A., Semenova, M.G., Sorokoumova, G.M., Selishcheva, A.A., Belyakova, L.E., Polikarpov, Yu.N. (2005). Effect of pH on caseinate interactions with soy phospholipids in relation to surface activity of their mixtures. Food Hydrocolloids, 19, 429-440. 

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