Kinetic assay methods, enzyme

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. 

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

End Point vs Kinetic Methods. Samples may be assayed for enzymes, ie, biocatalysts, and for other substances, all of which are referred to as substrates. The assay reactions for substrates and enzymes differ in that substrates themselves are converted into some detectable product, whereas enzymes are detected indirectly through their conversion of a starting reagent A into a product B. The corresponding reaction curves, or plots of detector response vs time, differ for these two reaction systems, as shown in Eigure 2. Eigure 2a illustrates a typical substrate reaction curve Eigure 2b shows a typical enzyme reaction curve (see Enzyme applications). 

Experimental studies on the effect of substrate concentration on the activity of an enzyme show consistent results. At low concentrations of substrate the rate of reaction increases as the concentration increases. At higher concentrations the rate begins to level out and eventually becomes almost constant, regardless of any further increase in substrate concentration. The choice of substrate concentration is an important consideration in the design of enzyme assays and an understanding of the kinetics of enzyme-catalysed reactions is needed in order to develop valid methods. 

Table 8.3 Examples of kinetic spectrophotometric methods of enzyme assay... 

Direct kinetic methods comparable to those used for enzyme assays are generally feasible only with automated instrumentation because of the difficulty in measuring the rapidly falling reaction rate as the low concentration of substrate is further depleted. 

The velocity of an enzyme-catalyzed reaction can be measured either by a continuous assay or by a stopped-time protocol. Whenever possible, the continuous measurement of a velocity (e.g., the increase or decrease in absorbance vx. time) should be utilized. In stopped-time assays, the investigator must demonstrate that the reaction is completely terminated at the specified point in time and that products are readily and quantitatively separated from substrates. In addition, one must show that the system is under initial rate conditions. Thus, at least three or four different time points should be chosen. Stopped-time assays also require an assay blank (for t = 0). In this blank, typically the quenching conditions are applied prior to the initiation step. Whenever practicable, replicate kinetic analyses should be done, even with continuous assay protocols. See Enzyme Assay Methods Basal Rate... 

P 75] A static enzyme assay experiment was carried out using a stopped-flow method [161]. This is commonly used for monitoring reaction kinetics. P-Galacto-sidase was used as model enzyme to convert the substrate fluorescein mono-p-D-galactopyranoside (FMG) via hydrolysis into fluorescein. As buffer solution 10 mM potassium phosphate at pH 7.2 with 1 mM ascorbic acid was used to minimize photobleaching. The enzymatic reaction is accompanied by a change in fluorescence intensity which can be monitored with a microscope. 

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... 

A commercial serum creatine kinase assay8 employs the kinetic method for enzyme quantitation. This three-enzyme, coupled assay involves the following sequence of reactions ... 

When setting up methods of enzyme assay, it is necessary to (1) explore the relationship between reaction velocity and substrate concentration over a wide range, (2) determine K and (3) detect any inhibition at high substrate concentrations. Zero-order kinetics are maintained if the substrate is present in large excess (i.e., concentrations at least 10 and preferably 100 times that of the value of K ,). When [S] = 10 X K V is approximately 91% of the theoretical y,nax. The K , values for the majority of enzymes are of the order of 10 to 10" mol/L therefore substrate concentrations are usually chosen to be in tlie range of 0.001 to O.lOmol/L. On occasion, the optimal concentrations of substrate cannot be used (e.g., when the substrate has limited solubility or when the concentration of a given substrate inhibits the activity of another enzyme needed in a coupled reaction system). 

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. 

After the glycosyltransferase of interest has been pmchased or isolated, it is desirable to verify the activity of the enzyme with a standard substrate and the compound of interest under the conditions of the preparative synthesis. Methods for assaying glycosyltransferase activity are summarized in Table 1, and a suitable method should be selected based on the characteristics of the substrate and the required sensitivity of the assay. Kinetic studies to obtain Km and Kiax values for the substrate and optimization of reaction conditions, including buffer and pH, should also be carried out. 

There are several reasons to work with dilute solutions of enzyme. First, there is the obvious practical issue of conserving what is often a precious supply of enzyme that has been obtained with some labor and cost. Second, dilution can aid in eliminating unwanted interactions, thereby linearizing the rate vs enzyme curve as described above. Finally, it may be difficult to make measurements of initial rates in steady state unless the enzyme preparation is sufficiently dilute. If too much substrate is converted in the time required to make the measurement, then one must slow the reaction, and this is typically done by reducing the amount of enzyme in the assay. Transient kinetic methods typically require the use of concentrated enzyme solutions. Hence, these methods are seldom used until after basic understanding of the reaction mechanism has been obtained through steady-state kinetic methods, and critical tests can be designed to elucidate further the mechanism by transient kinetic methods. 

Kulys et al. (1986b) studied urea determination by difference measurement between two antimony electrodes covered with exchangable membranes (Fig. 68). Urease was attached in the pores of a macroporous membrane (thickness, 10 pm, pore diameter, 0.1 pm) by glutaraldehyde. This layer was covered with a monoacetylcellulose membrane. The membrane for the auxiliary electrode was prepared analogously, but using BSA instead of urease. The assay of urea was carried out with a differential amplifier which simultaneously differentiated the time course of the potential difference between enzyme and auxiliary electrode (kinetic method). Thus, a response time of only 20 s was possible. 

In enzyme assays, the polymer substrate is added to a buffered system, containing one or several types of purified enzymes. These assays are very useful in examining the kinetics of depolymerisation, or oligomer or monomer release fi om a polymer chain under different assay conditions. The method is very rapid (minutes to hours) and can give quantitative information. However, mineralisation rates cannot be determined with enzyme assays. 

The second technique employed for substrate determination is the measurement of the rate of an enzymatically catalyzed reaction, as is used to determine enzyme activity. The kinetic assay may be successfully used for determination of substances that do not react in a manner that can be detected directly, or in cases where the quantity of the substrate is too low to measure with the simple endpoint method. The determination of substrate using kinetic methods is possible only when the substrate concentration is low enough compared with the Michaelis constant. Thus, the following is required to perform the assay ... 

The high sensitivity of fluorescence spectroscopy and the selectivity of enzymatic assays are responsible for the increasing use of fluorimetric methods in enzymology. Enzyme determinations usually involve the use of kinetic methodology for measuring the rate of formation of the fluorescent product, while both equilibrium and kinetic methods are used to determine the substrates. Fluorimetric measurements on enzyme-catalyzed reactions have been used for a long time to determine a variety of enzymes and substrates (Figure 2). 

This enzyme assay method was recently used to kinetically characterize a synthetic enzyme, isomelezitose synthase, which was prepared by site-directed mutagenesis of sucrose isomerases. Continuous measurement of fluorescence ehanges in a 384-well plate, containing 4,4 -o-BBV/HPTS and isomelezitose synthase, allowed these authors to measure the concentration of fructose and isomaltulose in real time and to kinetically follow the conversion of sucrose into fructose and isomaltulose. ... 

Allison, R. D. and D. L. Purich, 1979. Practical considerations in the design of initial velocity enzyme rate assays, in Methods in Enzymology, Vol. 63, Enzyme Kinetics and Mechanism, Part A, Initial Rate and Inhibitor Methods (D. A. Purich, Ed.), pp. 3-22, Academic Press, San Diego, CA. 

Amylase can be measured by kinetic methods using coupled enzyme assay systems. The maltose formed by the action of amylase is converted to glucose, by including maltase in the reaction mixture. Glucose oxidase which is also added converts glucose to gluconic acid as follows ... 

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