Enzyme Assay

97 A gear up of enzyme assays was conducted with microsomal fractions from induced Ammi majus cells and employing stereospecifically deuterated (±)-marmesin (nineteen) or (±)-2′-acetyl-2′,3′-dihydropsoralen (22) (Schemes 3 and 4).

From: Comprehensive Natural Products Chemistry , 1999

Enzyme Assays

Robert Roskoski , in xPharm: The Comprehensive Pharmacology Reference, 2007

Spectroscopic Methods

Absorbance Spectroscopy. For some enzyme assays, information technology is possible to measure the reactant or product straight based on its absorbance backdrop Fersht (1999). In the reaction catalyzed by glucose-vi-phosphate dehydrogenase (EC 1.i.1.49), one product (NADH) absorbs light at 340 nm, making it possible to monitor the reaction by following the increase in absorbance at this wavelength.

Glucose 6-phosphate + NAD+ → 6-phosphogluconate + NADH + H+

In other cases, a reactant or product is measured indirectly. With acetylcholinesterase (EC iii.1.1.7), for example, acetylthiocholine is used as a substrate that liberates thiocholine on exposure to the enzyme. The thiocholine reacts with Ellman's reagent to produce a colored product, making it possible to monitor the reaction by following the increase in absorbance.

Acetylthiocholine + H2O → acetate + H+ + thiocholine

Thiocholine + Ellman's reagent → colored derivative

Coupled assays are sometimes used to monitor enzyme activity. In this case, the enzymatic reaction of interest is paired with a second reaction that is coupled for convenient measurement. An example of this is in the assay for hexokinase (EC 2.7.ane.1). In this case, an excess of glucose-6-phosphate dehydrogenase and NAD+ is included in the analysis mixture and absorbance at 340 nm is monitored.

Reaction I: Glucose + ATP → glucose 6-phosphate + ADP

Reaction Two: Glucose 6-phosphate + NAD+ → six-phosphogluconate + NADH + H+

In this example, reaction I should be rate limiting and the concentration of glucose half-dozen-phosphate should reach a steady state post-obit a lag menstruum. Cleland Cleland (1979) formulated a procedure for ensuring that the coupled analysis is providing an accurate measurement of the reaction velocity of the enzyme. If possible, it is preferable to monitor a reaction continuously. With the assays described higher up, the spectrophotometer can be programmed to requite a continuous readout as a office of time. In separation techniques, which may include the use of radioactivity, electrophoresis, or chromatography, discontinuous or finish-point assays are employed. Having established a linear time catamenia for an assay, a parameter is measured at a unmarried fourth dimension bespeak within the linear time menstruum (most preferably, a time point well-nigh the heart of the linear phase). The velocity is then determined from the departure in signal at that time indicate and at the initiation of the reaction. Care must be exercised with end-point assays, and time courses should exist checked to confirm linearity. Changes in the incubation atmospheric condition, such as temperature, pH, or substrate concentration, tin can modify the linearity of an assay. For routine enzyme assays, the reaction vessel contains all simply one of the components, and the reaction is initiated by adding the missing component (the enzyme or one of the substrates). The other components should be equilibrated in terms of pH, temperature, and ionic force. The reaction is unremarkably initiated by the improver of a small volume of a concentrated stock solution of the missing component. A small volume ensures that this add-on does not adjy the equilibrium conditions already established. When it is not possible to add a small-scale component, the separated components should exist at the same temperature, ionic force, and p H. Although there must exist a consummate mixing of the 2 components, vigorous shaking should be avoided every bit it may denature the enzyme poly peptide. Mixing may exist accomplished by inversion of a tube with an attached stopper, or a cuvette using a Parafilm seal. Dilute proteins are often less stable than more than concentrated ones, and assays are often performed in the presence of an inert protein, such as bovine serum albumin (0.25 mg/ml), to stabilize the purified enzyme. Experiments should exist performed to ensure that the protein is inert. Because albumin, for example, may bind to hydrophobic substrates, information technology may not always be advisable for this purpose. For discontinuous assays, the timer can be fix and samples aspirated at specific time intervals after mixing. For most spectrophotometers, detection is initiated manually using an instrument panel or a computer keyboard. To add together a component to a cuvette, it requires about 20 seconds to identify the cuvette in a spectrophotometer, and to brainstorm detection past pressing a estimator key. This time is usually not of neat event because for most reactions, the analysis is run from 10 to 30 minutes. Command measurements include a non-enzyme-containing blank and a non-substrate-containing bare. Such controls ensure that adventitious reactions are not occurring. The control (not-enzyme bare) rate is subtracted from the date generated by the experimental to provide the bodily velocity of the enzymatic reaction. For many assays, it is necessary to quench or stop the reaction at a specific time to prevent further product of product. For case, samples may be taken at 5-infinitesimal intervals for a predetermined period of time and the product measured by HPLC.

Each chromatographic analysis may take thirty minutes to complete. Methods for terminating the reaction usually involve denaturation of the enzyme by calculation acid, or immersion in a boiling water bathroom. The action of some metalloenzymes tin be quenched with EDTA or other metal ion chelator. Most enzyme assays are based on spectroscopic techniques, with the two most usually used being absorption and fluorescence Fersht (1999). The wavelength used for post-obit the reaction rate should be 1 that yields the greatest difference in absorption betwixt the substrate and the product. Absorption measurements are performed with a standard spectrophotometer with the samples contained in specialized cells, or cuvettes. Dispensable plastic cuvettes that hold ane or 3 ml samples are commercially available. Their employ is restricted to the visible wavelength range (350-800 nm). Quartz cuvettes must be used (glass and plastic absorb light in the UV range) for measurements at wavelengths less than 350 nm. The path lengths for cuvettes is provided by the manufacturer. Popular path lengths are 1.00 cm, 0.40 cm, and 0.20 cm. An increasing number of assays are performed with 96 well microtiter plates with plastic or quartz bottoms. The concentration of a substance that absorbs lite at specific wavelengths can exist determined from Beer's police:

A = εcl,

where A is the absorbance of the sample at a specified wavelength, c is the concentration of the sample, l is the path length, and ε is the extinction coefficient, or molar absorptivity. ε has the units of M−ane cm−one or mM−1 cm−ane. If the value of ε is known for a particular substance, it is possible to calculate the concentration of that substance in a solution by measuring its absorption in that solution. Using Beer's law, information technology is possible to calculate the rate of modify of the concentration during the course of a reaction. A potential error using absorption measurements results from a deviation from Beer's police force because it holds just for a finite range of absorption values. Thus, because information technology may not exist applicative with absorbencies greater than i, assays should be designed to proceed experimental values less than this. It may be possible to circumvent some of these bug by using cuvettes with shorter path lengths. A sample with an absorbance of 1.00 in a i.00-cm path length cell volition have an absorbance of 0.xx in a 0.20-cm path length prison cell. In general, working with an absorbance of around 0.five represents a compromise between minimizing optical noise inherent in a spectrophotometer and having a reasonable bespeak to measure. The turbidity in a solution tin scatter light and produce apparent absorbance. Filtration or centrifugation may be used to remove these particles. Although alterations in path length circumvent some deviations from linearity (i.e., when the absorbance is so high that the spectrophotometer cannot make an accurate measurement), frequently the deviations are due to intermolecular interactions among the absorbing species. Dimerization (or germination of college order eximers) occurs at college concentrations of the absorbing species. In these cases, Beer's police force will exist obeyed if the concentration of product is decreased. This tin be accomplished by either slowing the reaction (by decreasing the enzyme concentration) or past taking measurements over a shorter catamenia of fourth dimension (before deviations from Beer's police are encountered).

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Enzyme assay techniques and protocols

Iqra Sarfraz , ... Şevki Adem , in Analytical Techniques in Biosciences, 2022

Abstract

Enzyme assays are standardized experimental protocols, which are established in guild to measure the activity or concentration of enzymes in biochemical or cell-based systems. Mostly enzymatic assays are based upon the detection of fluorescent, luminescent, or spectrophotometric endpoint point. During contempo years, florescence-based, absorbance-based, and luminescence-based high-throughput screening assays take been developed and widely used in various fields. Enzyme assays represent an important tool for screening of sample libraries in context of drug discovery. In addition, enzyme assays are valuable for diagnosis of several diseases in clinical settings due to their high sensitivity, specificity, and rapid response. In this chapter, we aim to provide an agreement of the working principle of enzymatic assays and their applications with some specific examples of enzyme assays that are used for drug discovery and diagnostic purpose.

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Drug Discovery Technologies

G.A. Holdgate , in Comprehensive Medicinal Chemistry III, 2017

two.07.ii.16 Enzyme Kinetics

Enzyme assays, which often can be followed using biophysical readouts, for example, quantification of the amount of a fluorescent production, can also be used to measure out binding kinetics. However, knowledge of the inhibitor mechanism of action (MoA) is required in order to access the relevant parameters. An case is the derivation of the kinetic parameters for inhibitors of the enoyl-acyl carrier protein reductase, FabI, from forward reaction progress curves (enzyme assay time courses) through a detailed agreement of the MoA of the inhibitors coupled with computational methods. 3

Jump dilution methods offer a more than directly mode to estimate inhibitor dissociation rate constants. In a similar way to the large dilution experiments described earlier, the enzyme is incubated with a saturating concentration of exam compound, and this mixture is rapidly diluted into a large excess of substrate. Product accumulation is then again measured as a part of fourth dimension, and the observed charge per unit of enzyme activity recovery fits to a single exponential in order to derive the inhibitor dissociation rate constant. Two important factors in conducting this blazon of experiment are that the inhibitor concentration falls well below its apparent inhibition constant, 1000i ′, upon dilution and that the final enzyme concentration, post-obit the dilution, is sufficient to generate enough production for reliable detection.

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VITAMINS | Overview

V. Spitzer , U. Höller , in Encyclopedia of Belittling Science (Second Edition), 2005

Enzyme Assays

Enzyme assays are mainly used for determination of torso status of vitamins. As vitamins usually function either every bit coenzymes or building blocks of coenzymes, the action of the vitamin-dependent enzymes is a measure of vitamin status. Usually, the assay is carried out past determining the enzyme activity with and without activation by added coenzyme. The activity tin be monitored past measuring changes in concentration of substrates or products during the reaction. An activation coefficient can be deduced, which reflects the status of the enzyme investigated, and thus the vitamin status. Virtually assays are conducted with whole blood or the separated erythrocyte fraction. They can be automated with clinical analyzers. Disadvantages include difficulties in analysis standardization, instability of the enzymes during storage, and misleading results, e.1000., due to conditions other than vitamin deficiency leading to low apoenzyme concentrations.

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Immobilized Cells

Susan Linko , ... Yi-Hong Zhu , in Progress in Biotechnology, 1996

2.iv Analytical methods

Enzyme assays . Endo-i,four-β-glucanase activity [1,iv-(1,3;1,4)-Β-D-glucan-4-glucano- hydrolase; EC three.ii.one.4] xylanase (1,4-Β-D-xylan xylanohydrolase; EC 3.2.one.8) activity and filter paper (FP) activeness were adamant spectrophotometrically at 540   nm every bit described in [13], Lignin peroxidase activeness was adamant spectrophotometrically at 310   nm at 23   °C room temperature as described by Linko and Haapala [20]. The activities were reported either as nkat ml-   1 or for lignin peroxidase equally units (U) per liter (1 U   =   16.67 nkat).

Soluble poly peptide. Soluble protein was precipitated with x% trichloroacetic acrid and determined spectrophotometrically at 750   nm according to Lowry [21], using Bovine serum albumin as standard.

Full reducing sugars. Total reducing sugars were determined colorimetrically at 540   nm past the dinitrosalicylic acid (DNS) method [22], and glucose by the method of Nelson [23].

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Clinical Enzymology

JOHN W. KRAMER , in Clinical Biochemistry of Domestic Animals (3rd Edition), 1980

F Kinetics

Enzyme assays are performed under conditions for optimal enzyme activity. Concentrations of substrate, cofactors, activators, pH, and temperature are maintained constant and so that the only variable is the enzyme. These are the weather of nil-lodge kinetics, weather at which the reaction charge per unit is independent of initial substrate concentration. Thus, an enzyme as assayed in the laboratory may not have the same natural functional action in the cell, where different atmospheric condition may exist.

The approximation of cypher-social club kinetics becomes express when the rate of enzyme activeness is very high. Reagents for an enzyme generally have but sufficient substrate concentration to maintain null-lodge kinetics for the time period of the assay or for magnitudes of enzyme activeness 2 to three times greater than the activity normally occurring in the patient's serum. When 1 is performing an enzyme assay, it is imperative that the limitations of goose egg-order kinetics be understood. If the results of the assay exceed the limits of zero-order kinetics, the enzyme activity is in mistake and should be reported simply as "greater than" that value established as the upper limit of zero-social club kinetics. Alternatively, the assay can be repeated on a diluted sample, or the size of the sample tin can be reduced and appropriate dilution corrections made.

Determination of the maximal amount of enzyme activeness that a procedure can measure requires knowledge of the equilibrium of the reaction and the concentration of substrate. An equilibrium constant (Yard eq) of 100 indicates that there will be 100 times more than production than substrate when the reaction has gone to completion. In this instance, 99% of the substrate is converted to product. However, if K eq = 1, as in the GPT reaction, but 50% of the substrate is converted to product at equilibrium and the reaction appears to stop. In some cases, if the reaction is reversible, the product may be reconverted to substrate as quickly equally product is formed.

The Chiliad eq may exist contradistinct past "trapping" the product or converting it to another form. In the reverse LDH reaction, lactate is converted to pyruvate by LDH, and, in guild to drive the reaction to pyruvate, hydrazine is used to "trap" pyruvate. This form of "trapping" permits a greater amount of action to be determined with the aforementioned amount of substrate than would exist possible if no "trapping" reagent were used.

Enzyme assays in clinical biochemistry are by and large carried out in one of two ways. The start is an "stop point" procedure analogous to a colorimetric assay. The sample is added to the reaction mixture, and, after a suitable incubation menses, the reaction is stopped by the addition of a reagent which destroys or inhibits the enzyme activity. The amount of substrate used or the amount of product produced during the incubation period is measured. This cease bespeak method is subject area to a variety of errors every bit when the action exceeds the limits of the substrate concentration, or if product inhibition occurs.

The 2nd, "kinetic" assay procedure requires sequential reading either manually or with a abiding recording device. A kinetic assay procedure is more sensitive, more authentic, and more than easily controlled than end bespeak assays, because the reaction rate can exist visualized. The constant recording device is extremely useful for this purpose because the linearity of the reaction rate can exist easily seen. The master advantages of the kinetic method in clinical enzymology are every bit follows: (1) When high enzyme activities are encountered, the reaction rate can exist determined earlier substrate is exhausted whereas, in an end betoken assay, information technology has to exist repeated when the substrate is exhausted; and (two) if an activator is present, it can exist detected on the graph (Fig. one).

Fig. 1. Illustration of potential hazards of using a end-point enzyme assay. Line ACE is a cypher-gild reaction that permits accurate determination of enzyme activity for the entire reaction time. Curve ABE initially is a zero-club reaction of high rate followed by a reduction in rate, mayhap due to exhaustion of substrate. Assay at point E would be in mistake. Curve ADE has an initial lag stage which also would be erroneous.

 (Reproduced with permission from J. B. Henry, 1963.) Copyright © 1963

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Marine Enzymes and Specialized Metabolism - Role B

Christopher R. Reisch , in Methods in Enzymology, 2018

6 Summary and Conclusions

The DmdA enzyme assay described here is relatively simple to perform and requires no special equipment with the exception of an HPLC for separation and quantification of the reaction products. The methods provided earlier tin can exist used as a framework for designing assays to suit a specific purpose. Several aspects of the method, including cell lysis, reaction volumes and timing, and details of the HPLC separation, can be adapted to work with materials on-hand. Though the enzyme substrate THF is irreversibly oxidized in air, the assay tin be performed nether aerobic conditions with the protocol supplied here. Lastly, the enzyme was stable with improver of EDTA to the buffers, which should enable easy workflows for prison cell lysis and enzyme analysis.

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Cellulases

James E. McDonald , ... Alan J. McCarthy , in Methods in Enzymology, 2012

4.half-dozen Cellulase activity assays

Several enzyme assays are bachelor for the detection and quantification of cellulolytic activeness in isolated strains. Three principal approaches are used:

(1)

Measurement of hydrolysis products such as reducing sugars and total sugars: Reducing saccharide assays include the dinitrosalicyclic acid (DNS) method (Miller, 1959) and the Nelson–Somogyi method (Somogyi, 1952). The anthrone–H2So4 (Viles and Silverman, 1949) and phenol–H2SOiv (Dubois et al., 1956) methods tin be used to measure full soluble sugars released by extracellular enzymes in civilisation supernatants.

(two)

Measurement of reductions in substrate quantity via total carbohydrate assays: The anthrone–HiiAnd sofour (Viles and Silverman, 1949) and phenol–H2SOfour (Dubois et al., 1956) methods are the almost widely used. However, these techniques are limited to the study of pure celluloses, equally derivatives of other carbohydrates may interfere with the quantification of cellodextrins (glucose equivalents).

(3)

Measuring changes in the physical properties of substrates: Historically, physical characteristics including turbidity, viscosity, bloated cistron, disruption of cellulose structure, and strength of cellulose fibers have also been used to assess cellulase activeness.

For a comprehensive clarification of these methods, the reader is referred to the review of Zhang et al. (2006).

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Biotechnological applications of enzymes and future prospective

Seema Anil Belorkar , Sudisha Jogaiah , in Protocols and Applications in Enzymology, 2022

12.2.4 Coupled enzyme assay

The coupled enzyme assay is all-time exemplified by a reaction converting phosphoenolpyruvate to pyruvate. This reaction is catalyzed by enzyme 1 pyruvate kinase. Clinically, the rate of the reaction catalyzed past enzyme 1 is to be assayed for which a measurable and detectable metabolite is required. The subsequent reaction is the conversion of pyruvate to lactate that is associated with oxidation of NADH that is easily measurable past UV spectrophotometry ( Kiianitsa et al., 2003). Fig. 12.four explains the detection of activity of phosphoenol pyruvate.

Figure 12.4. Coupled enzyme assay.

In that location are diverse challenges in successfully accomplishing the coupled assays, as two reactions are simultaneously addressed ane after the other in the same reaction medium. The general issues encountered are differences in the optimal atmospheric condition of both enzymes like pH change. Studies are now focused to handle and improvise these coupling outputs (Moisa et al., 2020).

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Theory of Extraction Techniques

1000.Westward. Ducey , ... South.M. Lunte , in Comprehensive Sampling and Sample Preparation, 2012

2.25.4.2.vi Enzyme Assay

The use of enzyme assays (peculiarly coupled assays) allows for the rapid and, in some cases continuous, assay of Medico perfusate with very depression detection limits, and without the need for a separation step (imparted by the high specificity of the enzyme). To date, most Doc-coupled enzyme assays employ nicotinamide adenine dinucleotide (NAD)-dependent enzymes and produce fluorescent or electrochemical active products. These assays accept been reviewed past Obrenovitch and Zilkha. 113 In full general, the employ of MD-coupled enzyme assay allows for temporal resolution on the club of 2 min and is amenable to analytes that can serve as substrates for an NAD- or NADP-dependent enzyme.

To provide i example, Obrenovitch and coworkers examined changes in L-glutamate in ischemic rat brains. 114 Using glutamate dehydrogenase and NAD+, NADH was produced and quantified by fluorescence detection. Glutamate concentrations were monitored continuously over a xxx min perfusion of 100 mM potassium ion. The authors were able to demonstrate the relationship between glutamate concentration and epileptic episodes with much greater temporal resolution in comparison to an off-line HPLC method.

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