Enzyme-based spectrophotometric assays

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

The rate of hydrolysis of DNA, RNA, and polynucleotides can be measured by a sensitive spectrophotometric assay which is based on the hyperchromicity that occurs upon hydrolysis of these substrates (S). The enzyme has a 7-fold greater affinity for denatured DNA than for RNA (8). No inhibitory products accumulate during the course of the reaction. The pH optimum for RNase and DNase activities is between 9 and 10, depending on the Ca2+ concentration. At higher pH values less Ca2+ is required. The inhibitory effect of high Ca2+ observed consistently by many investigators is more pronounced at higher pH values (S). 

In the case of enzymes involved in biochemical pathways, the isolation is often based on activity assays. The nature of the activity assay depends on the enzymatic reaction and can involve, for example, the detection of a product on a thin-layer chromatography (TLC) plate (see Chapter 4, Section 1.2.1), the appearance or disappearance of a specific absorbance in a spectrophotometric assay, or a coupled assay involving the oxidation or reduction of a co-factor such as nicotinamide dinucleotide (NAD(H)), which can be measured by changes in fluorescence. 

The of the MF + C02/formyl-MF couple is —497 mV and that of HVH2 is —414 mV [184]. Thus, in vitro H2 is not a good electron donor for Reaction (48). However, an HPLC-based assay in the direction of C02-reduction, using Ti(III)-citrate ( 0 = —480 mV) as an artificial electron donor, has been described [185]. In the opposite direction, the enzyme is easily assayed spectrophotometrically using the artificial electron acceptor methylviologen ( = —446 mV) ... 

The enzyme activity was measured by a continuous spectrophotometric assay (see Methods), active site concentration was determined by FAD absorption at 452 nm (8 = 12.83 mM-icm-i) as described by Frederick et al. (1990) and the protein concentration was measured by the Bradford assay (BioRad reagent) using bovine serum albumin as standard, or by its absorption at 280 nm using a published factor of 1.67 O.D. per mg (Swoboda Massey, 1965). The specific activity was 430 U/mg, and the overdl yield of enzyme, based on active sites measurement (452 nm absorption), was about 40%. 

Kinetic spectrophotometric assays for Na are based on activation of the enzyme p-galactosidase by Na" to hydrolyze o-nitrophenyl-[3-D-galactopyranoside (ONPG). The rate of production of o-nitrophenol (the chromophore) is measured at 420 nm. 

The spectrophotometric assay is based on a POase indicator reaction to measure the amount of H2O2 liberated. For this reason an excess of POase and H-donor (Section 10.2.1.4.2) has to be used. One unit (U) is then expressed as the amount of enzyme liberating 1 pmole of H2O2 per min at 25°C. 

A second commonly used approach employs synthetic substrates for spectrophotometric or fluorometric assays [3,4]. These substrates permit a continual assay well suited for kinetic studies and provide reasonable sensitivity. The major drawback is that the substrates are not natural substrates and as such, their use should be considered as a model that may or may not reflect the enzyme s kinetic properties in biological systems. The thioacylester analogs of phospholipids provide a sensitive spectrophotometric assay for some PLA, or PLAj assays based on the reaction of released thiol with Ellmann s reagent. 

COMPOUNDING OF ERRORS. Data collected in an experiment seldom involves a single operation, a single adjustment, or a single experimental determination. For example, in studies of an enzyme-catalyzed reaction, one must separately prepare stock solutions of enzyme and substrate, one must then mix these and other components to arrive at desired assay concentrations, followed by spectrophotometric determinations of reaction rates. A Lowry determination of protein or enzyme concentration has its own error, as does the spectrophotometric determination of ATP that is based on a known molar absorptivity. All operations are subject to error, and the error for the entire set of operations performed in the course of an experiment is said to involve the compounding of errors. In some circumstances, the experimenter may want to conduct an error analysis to assess the contributions of statistical uncertainties arising in component operations to the error of the entire set of operations. Knowledge of standard deviations from component operations can also be utilized to estimate the overall experimental error. 

Spectrophotometric methods are, of course, not restricted to assays based on the NAD(P)+/NAD(P)H pair of coenzymes, and there are many natural and synthetic substrates whose reactions can be assayed in this way. The p-nitrophenyl group forms the basis of many convenient spectroscopic assays, for example for the enzyme glucuronidase ... 

For example, when a heart attack occurs, a lack of blood supplied to the heart muscle causes some of the heart muscle cells to die. These cells release their contents, including their enzymes, into the bloodstream. Simple tests can be done to measure the amounts of certain enzymes in the blood. Such tests, called enzyme assays, are very precise and specific because they are based on the specificity of the enzyme-substrate complex. If you wish to test for the enzyme lactate dehydrogenase (LDH), you need only to add the appropriate substrate, in this case pyruvate and NADH. The reaction that occurs is the oxidation of NADH to NAD+ and the reduction of pyruvate to lactate. To measure the rate of the chemical reaction, one can measure the disappearance of the substrate or the accumulation of one of the products. In the case of LDH, spectrophotometric methods (based on the light-absorbing properties of a substrate or product) are available to measure the rate of production of NAD+. The choice of substrate determines what enz)rme activity is to be measured. 

The proteins, avidin and streptavidin, are widely utilized in biotin analysis due to their outstanding aiSnity and specificity toward the binding of biotin (Zempleni et al. 2009). Generally an avidin-binding assay for the determination of biotin operates through the competition of sample biotin and labelled biotin (such as isotope-labelled biotin or biotinylated enzyme) with the limited number of avidin. Finally, the signals are acquired spectrophotometrically or electrochemically from the reaction of labelled enzyme with corresponding substrate or based on radioactivity counts. 

The activity of the cholinesterases can be determined directly using traditional spectrophotometric methods and also electrochemical techniques. Electrochemical methods for cholinesterase activity assay that are based on pH-shift potentiometry have been described (5-11). Conventional pH electrodes (7-11) and pH sensitive field effect transistors (5, 6) were employed as transducers coupled with cholinesterase enzymes. The main disadvantage of the pH-shift based method is a strong requirement for low buffer capacity of the sample. In addition, the sensitivity of pH based analytical techniques, in general, is less than that based on amperometric assay. The theoretical threshold of pH based assay methods is as low as 58 mV per decade of analyte concentration. Ion-selective membranes (12) and mediator-assisted potentiometry (13) have also been proposed for assays of cholinesterase inhibitors. 

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