Fixed time enzyme assay

Fixed time enzyme assays measure the amount of substrate used or product produced in a Fixed time. 

NOTE Two dilutions of the enzyme were used at each pH because this is a fixed time-point assay. Rather than continuously measuring the change in absorbance at 420 nm over time, you stopped each reaction at 4 min by increasing the pH. If the enzyme dilution that showed linear kinetics over 4 min at pH 7.7 did not show linear kinetics at a different pH, the solution with a lower concentration of the enzyme (/3-gal 2) will produce linear kinetics over 4 min. If /3-gal 1 and /3-gal 2 both displayed linear kinetics over 4 min at a particular pH, then the initial velocity of the solution containing /3-gal 1 will be twice that of the solution containing /3-gal 2. 

Fined charge enzyme assays measure the time taken for a fixed amognt.of aubtftrato to be used or product to be... 

Table 8.6 Examples of fixed time spectrophotometric methods of enzyme assay... 

The use of fluorescent substrates or products permits sensitive kinetic measurement of enzyme reactions to be undertaken and although there are relatively few natural fluorescent substrates, analogues can sometimes be used (Table 8.7). Some products can be converted to fluorescent compounds and can be used in fixed time assays. 

If an enzyme assay involves continuous monitoring of substrate or product concentration, the assay is said to be kinetic. If a single measurement of substrate or product concentration is made after a specified reaction time, a fixed-time assay results. The kinetic assay is more desirable because the time course of the reaction is directly observed and any discrepancy from linearity can be immediately detected. 

Figure E5.7 displays the kinetic progress curve of a typical enzyme-catalyzed reaction and illustrates the advantage of a kinetic assay. The rate of product formation decreases with time. This may be due to any combination of factors such as decrease in substrate concentration, denaturation of the enzyme, and product inhibition of the reaction. The solid line in Figure E5.7 represents the continuously measured time course of a reaction (kinetic assay). The true rate of the reaction is determined from the slope of the dashed line drawn tangent to the experimental result. From the data given, the rate is 5 jumoles of product formed per minute. Data from a fixed-time assay are also shown on Figure E5.7. If it is assumed that no product is present at the start of the reaction, then only a single measurement after a fixed period is necessary. This is shown by a circle on the experimental rate curve. The measured rate is now 16 jumoles of product formed every 5 minutes or about 3 /rmoles/minute, considerably lower than the rate derived from the continuous, kinetic assay. Which rate measurement is correct Obviously, the kinetic assay gives the true rate because it corrects for the decline in rate with time. The fixed-time assay can be improved by changing the time of the measurement, in this example, to 2 minutes of reaction time, when the experimental rate is still linear. It is possible to obtain...

B 9. Study Figure E5.7, which displays the kinetic progress of an enzyme-catalyzed reaction. What time limit must be imposed on rate measurements taken using the fixed-time assay Why ... 

Figure C4.1.3 Range-finding and the effect of enzyme concentration on the time course of a reaction. The dotted lines show the true initial rate. Only assay C will be reliable if a fixed-time assay is used.

During (he measurement of die enzyme reaction, the reaction velocity ideally should remain constant. Case of proteases or hydrolases ate known where the reaction rate gradually decreases as a result of an inhibitory effect of the reaction products, Therefore it is recommended that an enzyme assay be based, when feasible, upon a measurement of the initial reaction rate. This initial reaction rate can in most cases be obtained by extrapolation, a minimum reaction time being required for obtaining a sufficiently precise titration of the molecules removed or produced during this fixed time span. 

A so-called kinetic assay, in which the reaction rate is followed continuously, is advantageous because it is possible to observe directly the linearity or nonlinearity of the response with respect to time. Many enzyme assays, however, are based on a single measurement at a defined time, a so-called fixed-time assay. It is usually not possible to predict the appropriate amount of enzyme in either kinetic or fixed-time assays to obtain an optimum velocity like that of Assay 2 in Figure 11-14. This may be empirically determined by a dilution experiment in two stages. At first, constant volumes of serial 10-fold dilutions of enzyme are assayed to find the range of dilution in which the calculated activity is maximal and constant (see Figure 11-15). 

The prime test of the validity of v0 assays is a graphical test to establish that the rates observed are a linear function of enzyme concentration, as illustrated in Figure 11-16. This test should be applied to all assays in which reaction rates are used to measure enzyme concentrations. This procedure is especially important when fixed-time assays are used (see Figure 11-14). 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

Enzymatic assay methods are classified as fixed-time assays, fixed-change assays, or kinetic (initial rate) assays. Kinetic assays continuously monitor concentration as a function of time pseudo-first-order conditions generally apply up to 10% completion of the reaction to allow the initial reaction rate to be determined. If the initial substrate concentration is > 10Km, then the initial rate is directly proportional to enzyme concentration. At low initial substrate concentrations (< 0.1 Km), the initial rate will be directly proportional to initial substrate concentration (cf. Chapter 2). For enzyme quantitation, a plot of initial rate against [E] provides a linear... 

Product inhibition is a cause of nonlinearity of reaction progress curves during fixed-time methods of enzyme assay. For example, oxaloacetate produced by the action of aspartate aminotransferase inhibits the enzyme, particularly the mitochondrial isoenzyme. The inhibitory product may be removed as it is formed by a coupled enzymatic reaction malate dehydrogenase converts the oxaloacetate to malate and at the same time oxidizes NADH to NADL... 

Methods in which some property related to substrate concentration (such as absorbance, fluorescence, chemiluminescence, etc.) is measured at two fixed times during the course of the reaction are known as two-point kinetic methods. They are theoreticahy the most accurate for the enzymatic determination of substrates. However, these methods are technically more demanding than equifibrium methods and all the factors that affect reaction rate, such as pH, temperature, and amount of enzyme, must be kept constant from one assay to the next, as must the timing of the two measurements. These conditions can readily be achieved in automatic analyzers. A reference solution of the analyte (substrate) must be used for calibration. To ensure first-order reaction conditions, the substrate concentration must be low compared to the K, (i.e., in the order of less than 0.2 X K, . Enzymes with high K , values are therefore preferred for kinetic analysis to give a wider usable range of substrate concentration. 

All assay methods are based on the forward ALD-catalyzed reaction. Both photometric fixed-time and continuous-monitoring procedures have been developed. In the analytical approach on which all the commonly used procedures and kits are based, the ALD reaction is coupled with two other enzyme reactions. Triosephosphate isomerase (EC 5.3.1.1) is added to ensure rapid conversion of all GLAP to DAP. Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is added to reduce the DAP to glycerol-3-phosphate, with NADH acting as hydrogen donor. The decrease in NADH concentration is then measured. 

Two situations must be distinguished, (i) assays of pure enzyme and (ii) assays of cell extracts. When purified enzyme preparations are available, no labeled substrate is required. Natural or synthetic sphingomyelin is prepared, pure or mixed with other lipids, in the form of extruded large unilamellar vesicles (LUV) ca. 100 nm in diameter. When pure sphingomyelin vesicles are used extrusion must take place at a temperature close to or above the gel-fluid transition temperature of the lipid, i.e. often 45-50 °C. LUV and enzyme are mixed in the appropriate assay buffer and aliquots are removed at fixed time intervals. The aliquots are mixed with chloroform-methanol and, after phase separation, phosphorous (from phosphorylcholine) is assayed in the aqueous phase. The procedure has been described in detail by Ruiz-Arguello et al. [91]. 

One of the most valuable applications of electronic data-processing is in enzyme assays. Kinetic measurements of enzyme activity in which the rate of reaction is monitored (usually using UV measurement) are more specific than endpoint colorimetric methods, in which the development of color in a coupled reaction is measured after a fixed time. Modern systems continuously or intermittently monitor the growth in concentration of the reaction product or the decrease in concentration of one of the reactants (the substrate). From the rate of change in concentration or the average change in concentration over several fixed time-intervals, the circuitry calculates the activity in reportable units. Other ramifications of these systems will be discussed below. 

Some enzyme reactions can be studied colorimetrically when either the substrate or product can be converted chemically to a coloured product suitable for measurement in a u.v. or visible light spectrophotometer. In the case of alanine aminotransferase, the pyruvate formed in the reaction can be converted to pyruvate-2,4-dinitrophenylhydrazone by the addition of 2,4-dinitrophenylhydrazine (DNP). Addition of sodium hydroxide yields a product with an absorption maximum at 505 nm. Other examples of colorimetric procedures will be found in the last section. Colorimetric procedures are used for enzyme assays in the sampling mode, whereby samples of the reaction mixture are analysed at certain fixed times after starting the reaction. Graphs depicting the reaction rate must then be constructed by plotting amount of substrate transformed against time. 

In competitive immunoassays, a standard of enzyme-labeled Ag is added to the sample for competitive equilibration with the Ab. Depending on the analyte and the configuration of the reaction vessel used, equilibration can take several minutes to hours, but adequate results may be obtainable before this. The unbound Ag and Ag are then rinsed from the tubes and the enzyme substrate S added. At a fixed time, the sample is analyzed for the elec-troactive product (P), whose concentration shall be proportional to the Ag in the well if the enzymatic reaction is carried out under substrate saturation conditions. Because of the competitive binding, a typical standard plot of the current versus the concentration of Ag has an inverse, linear relationship. The assays of this type in Table 2 are denoted by E (enzyme) and C (competitive) under the assay type. 

Electrochemical detection also offers an alternative approach to amine enzyme activity measurement as the assays for the precursors, the amines, and their metabolites are adequately sensitive. Modifications to the normal assay are required especially if the substrate, which will be added in excess, is electroactive and separated on the same LC column. For example, dopamine-p-hydroxylase activity is measured by incubating dopamine with the required cofactors for a fixed time and then measuring the amount of norepinephrine formed (Davis and Kissinger, 1979). However, sub-... 

A review of enzyme analysers has described the automation of fixed-time and continuous-monitoring assays and the uses of partly or completely automated analysers and multi-channel systems. Automated analyses of polysaccharide hydrolases, in soluble or insoluble form, have been based on determination of the liberated reducing sugars with 3,5-dinitrosalicylic acid. A sensitive and specific method for locating glycoside hydrolases (e.g. oc-D-mannosidases) in polyacryl-... 

The hallmark of slow binding inhibition is that the degree of inhibition at a fixed concentration of compound will vary over time, as equilibrium is slowly established between the free and enzyme-bound forms of the compound. Often the establishment of enzyme-inhibitor equilibrium is manifested over the time course of the enzyme activity assay, and this leads to a curvature of the reaction progress curve over a time scale where the uninhibited reaction progress curve is linear. We saw... 

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