Fixed change enzyme assay

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. 

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... 

Fixed-change assays are relatively uncommon, and are used for enzyme quantitation. These assays monitor the time required for the generation of a given concentration of product. The enzyme concentration is inversely related to the time required for this extent of reaction to occur, so that a linear plot of 1/f against [E] is used as a calibration curve. 

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. 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

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...

EMIT Using Hapten-Enzyme Conjugate. In this assay, a fixed amount of hapten-enzyme conjugate is incubated with a fixed amount of antibody to the hapten together with a variable amount of free hapten. The antibody will cause a change in enzymic activity upon reaction with one or more haptens on the enzyme. In this assay, equilibrium does not necessary have to be attained before enzyme measurement, no separation of free from antibody-bound hapten is required, and the rate of enzyme reaction is measured, rather than an end point. 

Another way to use the technique is to fix the colloid onto the distance layer s surface. If the height of distance layer itself is changed specifically via. an enzymatic assay shrinking or swelling the distance layer, for example, a color shift is observed. By this, the presence of an enzyme substrate or a reactive chemical is transduced by the system into an optical signal. The effect of moderate to high analyte concentrations can be observed with the naked eye. Applications and details of this technique are discussed below. 

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