Fixed-time assay

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

For fixed time assays this most frequently involves the use of standards and a calibration graph. Some methods, e.g. the use of the molar absorbance coefficient in spectrophotometry, do not requite standards and giiNometric methods permit the calculation of molar concentration from the volume of gas (1 gram mole of gas occupies 22.4 litres at standard temperature and pressure, STP). 

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. 

Fluorimetric methods are useful for monitoring reactions involving the nucleotide coenzymes. The natural fluorescence of the reduced forms in the region of 460 nm can be used in kinetic assays. However, this fluorescence is destroyed at pH values below 2.0, whereas any oxidized forms of the coenzymes present are stable. If the pH of the solution is then raised above 10.5 and heated, the oxidized forms are themselves converted to fluorescent derivatives. This latter procedure lends itself to fixed time assays such as is illustrated in Procedure 8.6. 

PROCEDURE 8.6 Fluorimetric assay of u-amino acid oxidase (fixed time)... 

Li PK, Lee JT, Mac Gillivray MH, Schaefer PA, Siegel JH (1980) Direct, fixed-time kinetic assays for beta-hydroxybutyrate and acetoacetate with a centrifugal analyser or a computer-backed spectrophotometer. Clin Chem 26 1713-1717... 

Isal Standard addition graph. An assay for substance X is based on its ability to catalyze a reaction that produces radioactive Y. The quantity of Y produced in a fixed time is proportional to the concentration of X in the solution. An unknown containing X in a complex, unknown matrix with an initial volume of 50.0 mL was treated with increments of standard 0.531 M X and the following results were obtained. Prepare a graph of Equation 5-9 and find [X]... 

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 ... 

The greatest advantage of the spectrophotometric method is that it is direct and rapid, requires no sample workup, and allows for continuous assays of lipase activity compared to the multiple fixed-time-point analyses incumbent within Basic Protocols 1 and 2. The spectrophotometric method can also be done using very small volumes (as small as 1 ml) and is suitable for following the course of purification (such as in chromatographic fractions) or adaptable to 96-well plates (and subject to automation, if available). Thus, it is the method of choice for screening several samples or preparations for lipase (esterase) activity. 

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.

In the Anfinsen procedure (43) the acid-soluble nucleotides produced after a fixed time of RNA digestion are measured. The undegraded RNA is precipitated with perchloric acid or a mixture of uranyl acetate and trichloroacetic acid. The absorbance of the supernatant solution after filtration or centrifugation is used as a measure of activity. Here also the assay is a measure largely of step 1 activity. 

Fig. 4. Various ways of quantitation of TAC in inhibition assays measurement of induction time, absorbance (fluorescence) after fixed time, and area the kinetic curve of time course of changes in absorbance or fluorescence. Dashed line, reference solid line, sample measured. Differences between the areas under curves for sample and reference (protection area) indicated only.

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. 

Assay A laboratory test to identify and/or measure the amount of a particular substance in a sample. Types of assays include endpoint assays, in which a single measurement is made at a fixed time kinetic assays, in which increasing amounts of a product are... 

This procedure is used to determine the lipase activity in preparations derived from microbial sources. The assay is based on the measurement of the amount of free fatty acids formed from an olive oil emulsion in the presence of sodium taurocholate over a fixed time interval. This assay is particularly used for measuring lipase activity in foods. 

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). 

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. 

Some of the kinetic properties of ATCase described above will be examined in this experiment. The assay to be used is a fixed-time, colorimetric procedure. Carbamyl aspartate accumulated in the first step of the procedure is assayed in a second step (Fig. 9-3). 

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. 

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