PH-stat method

Potentiometry is another useful method for determining enzyme activity in cases where the reaction Hberates or consumes protons. This is the so-called pH-stat method. pH is kept constant by countertitration, and the amount of acid or base requited is measured. An example of the use of this method is the determination of Hpase activity. The enzyme hydroly2es triglycerides and the fatty acids formed are neutralized with NaOH. The rate of consumption of NaOH is a measure of the catalytic activity. 

The kinetics of alkaline hydrolysis of an ester can be followed by the pH-stat method, in which the pH is held constant by adding a solution of strong alkali to the reacting ester solution. The volume of alkali added in order to keep the pH constant is recorded as a function-time. Find the volume function needed to determine the rate constant that is, given the volume-time data for this type of experiment, what plot or calculation will yield the rate constant ... 

Nabb DP, Whitfield F. 1967. Determination of cholinesterase by an automated pH-stat method. Arch Environ Health 15 147-154. 

A titrametric assay of PLCSc, alternatively called the pH-stat method, was the workhorse in early studies [28]. This method simply involves titrating the acidic product of the PLC reaction as it is formed with a solution of standard base. An advantage of this continuous assay is that it can be used to detect the turnover of both synthetic and natural substrates, and its sensitivity has been estimated to be in the 20-100 nmol range. However, the pH-stat assay has low throughput capability, and it cannot be easily performed in a parallel fashion with multiple substrate concentrations. It is also necessary to exclude atmospheric carbon dioxide from the aqueous media containing the enzyme and substrate. 

Describe the two types of information that can be obtained about a reaction by using the pH-stat method of titration. 

Reaction Eq. (6) has been studied in the pH range 8-10 by the pH-stat method (211). The reaction was found to be first order in the mononuclear species, and the rate-determining step was proposed to involve a dissociation reaction of the tr[Pg.86]

The base hydrolysis of (en)2Co(OH)2Co(en)24+ studied by the pH-stat method (pH 9.3-10.4) revealed two consecutive first-order reactions which could, however, be interpreted in terms of quite different reaction schemes (359). 

An automated pH-stat method gives a direct measurement of the percentage of hydrolyzed peptide bonds, the degree of hydrolysis (DH). The DH is calculated using the equation ... 

The pH-stat method can be modified and applied to a particular enzyme or substrate to assay any enzyme/substrate combination. The substrate can come from any source of protein such as poultry, milk, soybean, or fish processing byproducts. The amount of protein in the reaction should not exceed 8% (as calculated in Basic Protocol 1). The enzyme can be any alkaline endopeptidase such as Alcalase, trypsin, or chymotrypsin, and should be used in the proportions indicated in Basic Protocol 1. The selection of the appropriate enzyme depends on its efficiency and cost. 

An example of direct difference counting, in a situation where the overall titration curve is not known, is provided by Fig. 7. It shows the difference between the titration curves of active carboxypeptidase A, which contains Zn++, and the inactive zinc-free protein, as determined by Coleman and Vallee (1961). The measurements were made by the pH-stat method, and confined to a relatively narrow pH range. The figure shows a difference of two groups per bound Zn++ ion, with pK values of 7.7 and 9.1. The two groups involved lose their protons when Zn++ is bound. In the absence of a complete titration curve a completely unequivocal interpretation... 

The rate of dissolution of synthetic and deep sea biogenic (pteropods) aragonite in seawater have also been determined in the laboratory by the pH-stat method (57). The results of the experiments to determine the change in the rate of dissolution as a function of undersaturation are presented in Figure 16. The pteropods were found to dissolve at. only about 3% the rate, per unit surface area, of the synthetic aragonite. The results also indicate a change in the empirical reaction order from 2.92 to 7.37 at = 0,44. The rate equations for pteropod dissolution are ... 

Experimental. We studied the dissolution of semi-optical grade crystals of Iceland spar (44.5 cm g and 96.5 cm g ) in dilute solutions as a function of pH, PCO2 and temperature. The pH-stat method was used to identify forward reactions far from equilibrium (in the near absence of backward reaction). The "free drift" method was used to study the reaction near equilibrium where both forward and backward rates must be considered. Details of the experimental procedures are given elsewhere ( ). 

The net proton charge of proteins in pure water at +20°C is calculated with ZH = 0 at the isoelectric point. In mixed solvent, at +20°C, the net proton charge is determined by the pH stat method relative to the ZH value in water at a given pH. The glass electrode is calibrated in pure water at a given pH, then recalibrated in mixed solvent at the... 

One example is the pH-stat method, which was developed by Obermann and Cremer (1992) and commissioned by the Office of Water and Waste of the State of North-rhine-Westphalia, Germany. 

These methods are suitable for both AChE and BuChE and are applicable to any substrate because hydrolysis of an ester always results in acid formation. The increase in acid concentration can be measured poieniiometrically by continuous titration with sodium or potassium hydroxide at a constant pH (pH-stat method) (Jeasen-Holm et a ., 1959), or one can measure the decrease in pH after a selected time of assay ApH). The ApH method is an end point method introduced by Michel (1949), and it is used for routine or field measurements, pH can be measured colorimetrically with a pH indicator, which is a simple protocol. 

The rate constants for the dissolution of cellulose acetate phthalate in solutions of different basic salts were determined using an automatic pH-stat method. A linear relationship between the logarithm of the rate constants and the pRg of the basic salts was observed. Evidence was given that the rate of proton transfer, which leads to the dissociation and, consequently, to the dissolution of the polymer, is the rate determining step and is governed by the Bronsted catalysis law."... 

Pig pancreatic lipase added to a mixture of ethyl butyrate, ethyl isobutyrate, and ethyl pivalate in phosphate buffer (pH 8), and quenched with 10 M NaOH when reaction complete (pH-stat method) - butyric acid. Y 65% (> 95% purity). The enzyme only hydrolyzed straight-chain esters. F.e., preferential cleavage of functionalized esters, and comparison with horse liver acetonic powder, s. B. De Jeso et al., Synth. Commun. 18, 1699-705 (1988) preferential hydrolysis of dicarboxylic acid esters s. ibid. 1691-7. 

The hydrolysis kinetics of coordinated esters have been measured by pH-stat methods for a series of a-aminoacid ester complexes [equations (3) and (4) L = NH2CHRCOOR A = NH2CHRCOO ]/ The results... 

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