PH stat

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 ... 

Garcia-Lopez JA, Monteoliva M. 1988. Physiological changes in human erythrocyte cholinesterase as measured with the "pH-stat". Clin Chem 34 2133-2135. 

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

The cysteinyl residue in the core was oxidized with performic acid according to the method of Hirs ( ) and the oxidized core was digested with chymotrypsin for 8 hours at room temperature in a pH stat at pH 8.9. Enzyme equal to 0.5% of the substrate by weight was added at 0 and at 2 hr. The chymotryptic peptides were also separated by Dowex 50-X4 chromatography. 

Marsh grapefhiit (MGF) pulp was homogenized in 5 volumes of extraction buffer at 4 C and maintained at pH 8.0 (28). The homogenate was stirred for one hour, centrifuged and the supernatant used as the PE extract. Activity was measured by titration with a Brinkman (Westbury, NY) pH stat titrator at pH 7.5 and 30°C in 25 mL of 1 % high methoxyl pectin (Citrus Colloids Limited, Hereford, UK) with O.IM NaCl. PE units are expressed as the microequivalents of ester hydrolysed per minute. Uronic acid analyses were conducted based on the m-phenyl phenol (4) as modified for microplate reading (30). 

Especially in dicotyledonous plant species such as tomato, chickpea, and white lupin (82,111), with a high cation/anion uptake ratio, PEPC-mediated biosynthesis of carboxylates may also be linked to excessive net uptake of cations due to inhibition of uptake and assimilation of nitrate under P-deficient conditions (Fig. 5) (17,111,115). Excess uptake of cations is balanced by enhanced net re-lea,se of protons (82,111,116), provided by increased bio.synthesis of organic acids via PEPC as a constituent of the intracellular pH-stat mechanism (117). In these plants, P deficiency-mediated proton extrusion leads to rhizosphere acidification, which can contribute to the. solubilization of acid soluble Ca phosphates in calcareous soils (Fig. 5) (34,118,119). In some species (e.g., chickpea, white lupin, oil-seed rape, buckwheat), the enhanced net release of protons is associated with increased exudation of carboxylates, whereas in tomato, carboxylate exudation was negligible despite intense proton extrusion (82,120). 

The stoichiometries of both oxygen consumption and of proton release subsequent to Fe(III) hydrolysis have been determined by using a combined oximeter and pH stat (Yang et ah, 1998). The overall reaction at the ferroxidase centre is postulated to be ... 

Honda, H. Sugiyama, H. Saito, I., and Kobayashi, T., High cell density culture of Rhodococcus rhodochrous by pH-stat feeding and dibenzothiophene degradation. Journal of Fermentation and Bioengineering, 1998. 85(3) pp. 334—338. 

To each ml of QD solution, add 50 pi of the EDC/sulfo-NHS stock solution. Maintain the pH at 7.0 by the addition of base, if necessary. Small volume reactions may be controlled using a pH stat. 

Fixed-activity and sliding-activity paths (Sections 14.2-14.3) are analogous to their counterparts in fugacity, except that they apply to aqueous species instead of gases. Fixed-activity paths are useful for simulating, for example, a laboratory experiment controlled by a pH-stat, a device that holds pH constant. Sliding-... 

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. 

The titrant used in pH-stat titration is usually a dilute (perhaps 0.01 M) acid or base. Two different sets of data can be obtained from a pH-stat titration. The amount of a substrate consumed or product formed can be determined by the total amount of titratant used. Because the titrant is added over a period of time, the rate of reaction can also be determined. 

Even a small soil sample can be considered a bioreactor, and so the method of pH-stat is applicable to soil. Although this method is applicable to the study... 

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

Ficara E, Rozzi A, Cortelezzi P. Theory of pH-stat titration. Biotechnol. Bioeng. 2003 82 28-37. 

Various devices can be used to determine the kinetics and rates of chemical weathering. In addition to the batch pH-stats, flow through columns, fluidized bed reactors and recirculating columns have been used (Schnoor, 1990). Fig. 5.15a illustrates the fluidized bed reactor pioneered by Chou and Wollast (1984) and further developed by Mast and Drever (1987). The principle is to achieve a steady state solute concentration in the reactor (unlike the batch pH-stat, where solute concentrations gradually build up). Recycle is necessary to achieve the flow rate to suspend the bed and to allow solute concentrations to build to a steady state. With the fluidized bed apparatus, Chou and Wollast (1984) could control the AI(III) concentration (which can inhibit the dissolution rate) to a low level at steady state by withdrawing sample at a high rate. 

The Rate of reductive Dissolution of Hematite by H2S as observed between pH 4 and 7 is given in Fig. 9.6 (dos Santos Afonso and Stumm, in preparation). The HS" is oxidized to SO. The experiments were carried out at different pH values (pH-stat) and using constant PH2s- 1.8 - 2.0 H+ ions are consumed per Fe(II) released into solution, as long as the solubility product of FeS is not exceeded, the product of the reaction is Fe2+. The reaction proceeds through the formation of inner-sphere =Fe-S. The dissolution rate, R, is given by... 

E Schnabel. A better synthesis of tert-butyloxycarbonylamino acids through a pH-stat reaction. Liebigs Ann Chem 702, 189, 1967. 

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