PH-stat assay

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. 

Perhaps of first concern in determining the overall design of a particular assay is the actual method used for product identification (or for substrate depletion) per unit time. Many different methods have been utilized (e.g., radiometric, spectrophotometric, fluorometric, pH-stat, polarimetric, etc.) No matter which method is used, the product has to be clearly identified (or substrate, if substrate depletion is being measured). With stopped-time assays, it may be necessary to separate product(s) from substrate(s) prior to determination of the amounts of the metabolite(s) present (as well as demonstration that product(s) and substrate(s) are truly separated). If so, the investigator should be able to demonstrate that the assay procedure clearly measures true initial rates (see below). Closely related to these issues are concerns about purity (See Substrate Purity Enzyme Purity Water Purity, etc.) and stability (See Substrate Stability Enzyme Stability, etc.. If the components of the assay mixture are not stable over the time course of the experiment (or, if certain side reactions occur), then corrections have to be made in analyzing the rate behavior. 

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. 

A somewhat more elaborate assay has been proposed by Blakeley et al. (13) employing a recording pH stat. In this assay no buffer is used, but dithiothreitol (DTT) is present during the titration (that maintains the pH at 7.0). The unit, IU, is defined as the amount that causes the decomposition of 1 jumole of urea per minute (38°, pH 7.0, 0.05 M urea, 2 t M DTT). One Sumner unit = 17.6 IU/mg = 11 Uf/mg. Specific... 

The enzyme is routinely assayed by a modification of the Fiske and SubbaRow technique for measuring Pi production from PP (5). Cooper-man has developed a continuous automated pH-stat pyrophosphatase assay which is very convenient for repetitive analyses (0). Kinetic studies over a wide range of conditions require a more sensitive assay such as the production of 32Pi from 32PP, as used by Moe and Butler (9). 

Much interest for ion transport has its origin in the field of crown ether chemistry. Therefore, most model studies of ion channels have been more or less based on crown ether chemistries. Pioneering work has been undertaken by Fyles, who not only synthesized varieties of gigantic molecules starting from crown ethers,88-91 but established a method of the rate assay for ion transport across lipid bilayer membranes, a pH stat technique.92-95 Vesicles having different inside and outside... 

Potentiometry. Potentiometric methods rely on the logarithmic relationship between measured potential and analyte concentration. The most common involves an instrument called a pH-Stat , in which a glass (pH) electrode follows reactions that either consume or produce protons. Since pH changes cause changes in enzyme activity, the pH is maintained at a constant value by the addition of acid or base. The rate of titrant addition is then proportional to the rate of the enzymatic reaction. Precise measurements using the pH-Stat require low buffer concentrations in the enzymatic assay mixture. 

With method D, the inhibitor was previously dissolved into the substrate before carrying out the lipase assay (Fig. 9.5 D). This inhibition method was used with the pH-stat, rnonomolecular films and oil drop techniques. Lipase hydrolytic activity was then continuously recorded as described below. 

From [51]. (B) Effects of increasing orlistat concentration on HPL residual activity as measured, using the pH-stat technique, on tributyrin ( ) or PSO (A) according to method C. Kinetic assays were performed in a ther-mostated (37°C) vessel containing 0.5 mL TAG substrate mechanically emulsified in... 

Solutions of a pancreatin FIP standard preparation in physiological saline were diluted 1 1 with a solution of 8% BSA, 0.9% NaCl and measured with this fast, completely automated method. The Ortho method yielded an excellent correlation with the FIP pH-stat reference method Ortho U/L = 0.968 FIP U/L + 4.232 (r = 0.999). The recoveries in the low (294 FIP U/L), medium (440 FIP U/L), and high (734 FIP U/L) activity ranges were 101.1, 99.9, and 96.5%, respectively the intra-assay CVs were 3.0, 1.9, and 1.6% (n = 5). The detection limit, determined by a two-step dilution method and defined as the highest dilution that can be distinguished from the zero solution with 95% confidence, was 3.2 Ortho U/L. 

The assay based on the observation of NADH production from lactate and NAD+ has also been used 101). The NADH can be detected from its ultraviolet spectrum, but may also be coupled with phenazine metho-sulfate and a tetrazolium salt to give a blue color suitable for automated analysis and histological stains 102). The equilibrium is vmfavorable at neutral pH and the reaction is best carried out at pH 9-10. The reaction can also be followed by observing the proton uptake in a pH-stat 103). 

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. 

In a separate assay, the rate of hydrolysis of each substrate was analyzed using an automated Sargent-Welch pH Stat system (Sargent-Welch Scientific Company, Skokie, ID. By stepwise addition of a previously standardized O.OIN NaOH solution, the acid generated upon hydrolysis was measured. Titrations were carried out at pH 7,6 and 30 C. 

---