Enzyme-based activity assays

Many of the P-carboline and isoquinoline alkaloids described here display potent, and often selective, cytotoxicity (Table 1, 2) or exhibit antimicrobial activity (Table 3). Specific activity in enzyme-based assays and other activity is described below. 

The activity of enzymes in the film was estimated in the following way In order to test the activity of urease, we utilized a calorimetric assay based on urea hydrolysis the enzymatic reaction was followed at 590 nm, the suitable wavelength for bromcresol purple (Chandler 1982). Urea concentration was 1.67 ts 10 M. 

The ultimate goal of lead optimization is to produce compounds that will elicit the desired cellular and organismal phenotype when dosed at appropriate concentrations. During the course of lead optimization activities it is common for pharmacologists to evaluate compounds not only using in vitro enzyme activity assays but also in cell-based assays as well. A question that often arises at this stage of drug discov-... 

Protein fragment complementation assays are based on an enzyme reassembly strategy whereby a protein-protein interaction promotes the efficient refolding and complementation of enzyme fragments to restore an active enzyme. The approach was initially developed using the reconstitution of ubiquitin as a sensor for protein-protein interactions (Johnsson and Varshavsky, 1994). Ubiquitin is a 76 amino acid protein that... 

We have found that many compounds identified in our screen are nonspecific inhibitors of luciferase enzyme activity. To eliminate these, we test the hits in a luciferase enzyme-based counterscreen. Firefly and renilla luciferase are produced in vitro by programming Krebs-2 extracts with FF/HCV/Ren mRNA and allowing the translations to proceed at 30° for 1 h. Ten microliters are then pipetted into a 96-well plate and compound is added to a final concentration of 20 /iM (1% DMSO). Luciferase activity is then determined as described previously in step 3. Since compound is added only after the translation reaction is complete, inhibitors of translation should not score positively in this assay. Typically, a 1-ml in vitro translation reaction is sufficient to screen 45 candidate hits in duplicate for nonspecific luciferase inhibitory activity. Compounds that inhibit in this counterscreen are eliminated from future analysis. 

For biochemical assays, /iPLC allows direct quantification of substrates and products using a much-valued separation-based approach that allows development and optimization of challenging enzymatic assays faster and with fewer false positives. The separation-based approach employed by /iPI. C dramatically reduces assay development time from months to a few days. Since substrate and enzymatic products are separated prior to detection, /iPLC enables development of difficult assays, such as analyzing enzymes with low kinetic activities and enzymes that cannot be analyzed on existing platforms. 

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]. 

Theory The method of LDH assay is based on kinetic analysis. In a kinetic enzymatic assay a unit of enzyme activity is defined as the quantity of enzyme that brings about a certain absorbance increase in 30 seconds or 1 minute at a fixed temperature (for instance 25 0.2°C) ... 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

GTPCH (EC 3.5.4.16) converts the substrate GTP to 7,8-dihydroneopterin triphosphate (H2NTP) and formate. GTPCH activity is determined by measuring neopterin, the completely oxidized and dephosphorylated H TP-product of the enzyme reaction. Conversion of H2NTP to neopterin is carried out after the enzymatic reaction in presence of iodine at pH 1.0, followed by dephosphorylation with alkaline phosphatase at pH 8.5-9.0. Neopterin is detected fluorimetrically at 350/440 nm upon HPLC separation. The assay is based with some modifications on the methods published by Viveros et al. and Hatakeyama and Yoneyama [15,16]. 

As depicted in Figure 6.8 the stability screening was based on DERA activity assay, the retro-aldol reaction of 2-deoxy-D-ribose 5-phosphate to acetaldehyde and D-glyceraldehyde 3-phosphate. D-glyceraldehyde 3-phosphate is further converted by the auxiliary enzymes triose phosphate isomerase and glycerol phosphate dehydrogenase. As the latter reaction consumes NADH it can be measured spectro-pho to metrically by the decrease in absorbance at 340 nm. 

The presence or absence of an enzyme is typically determined by observing the rate of the reaction(s) it catalyzes. Quantitative enzyme assays are designed to measure either the total amount of a particular enzyme (or class of enzymes) in units of moles or, more commonly, the catalytic activity associated with a particular enzyme. The two types of assays differ in that those in the latter category measure only active enzyme. The assays contained in this section are concerned primarily with the measurement of catalytic activity, or active enzyme. The assays are based on kinetic experiments, as activities are calculated from measured reaction rates under defined conditions. The basic Premise for these assays is that the amount of enzyme in a reaction mi xture can be determined from the rate at which the enzyme-catalyzed reaction occurs. 

Substrate concentration is yet another variable that must be clearly defined. The hyperbolic relationship between substrate concentration ([S ) and reaction velocity, for simple enzyme-based systems, is well known (Figure C1.1.1). At very low substrate concentrations ([S] ATm), there is a linear first-order dependence of reaction velocity on substrate concentration. At very high substrate concentrations ([S] A m), the reaction velocity is essentially independent of substrate concentration. Reaction velocities at intermediate substrate concentrations ([S] A"m) are mixed-order with respect to the concentration of substrate. If an assay is based on initial velocity measurements, then the defined substrate concentration may fall within any of these ranges and still provide a quantitative estimate of total enzyme activity (see Equation Cl. 1.5). The essential point is that a single substrate concentration must be used for all calibration and test-sample assays. In most cases, assays are designed such that [S] A m, where small deviations in substrate concentration will have a minimal effect on reaction rate, and where accurate initial velocity measurements are typically easier to obtain. 

A nonlinear relationship between enzyme concentration and measured activity is indicative of a more complex reaction system. Complications of this nature may arise from such things as changes in the composition of the reaction mixture (e.g., pH due to the addition of increasing amounts of enzyme solution), assay limitations (e.g., insufficient substrate), limited coupling-enzyme (where assays are based on coupled enzyme systems), the presence of inhibitors, and enzyme-cofactor or enzyme-enzyme dissociation phenomena. Nonlinear relationships may also be an inherent... 

The reaction conditions chosen for the assays are based on published optimal conditions for PGase enzymes. These enzymes typically have maximal activities at slightly acidic pH (Tucker and Seymour, 2002) and, in general, appear to be relatively stable at temperatures from 30° to 40°C. Optimal reaction conditions are likely to be enzyme specific, so one may have to alter the conditions to match the properties of the enzyme of interest. In all cases, the analyst should take into account the properties of the substrate, particularly its solubility, as well as the properties of the enzyme. For example, because solutions of polygalacturonic acid tend to gel as the pH is lowered below 3, viscometric assays (Basic Protocol 2) at these relatively low pHs are often not feasible. 

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. 

Sensors of this type were constructed as multi-electrode arrays bearing chohnesterases of differing sensitivity to organo-phosphates, and the assay extended to milk [35]. Both spiked milk and milk from shops inhibited the activity of the electrodes. In two instances, estimates of the level of organo-phosphates in spiked milk made using sensors were very close to those made using GC-MS. This appears to have been a fortunate co-incidence as the response curves used for calibration were not linear. Nine out of ten milk samples from shops inhibited at least some members of the array. In only one case did the GC-MS assay find insecticides in the samples, but the insecticides were not organo-phosphates. The inhibition shown by the enzyme-based arrays was reversible by pyridine-2-aldoxime... 

This sssay is used to investigate kysliiiBsidsss activities from a variety of sources or to estimate the inhibitory capacities of compounds on the enzyme. The assay is based on (he determination of the liberated Af-acetyl-D-glucosamine end groups... 

Cyclic nucleotides are purinic base derivatives with powerful biological activity. It is widely accepted that cyclic nucleotides mediate many of the intracellular biochemical events triggered by neurotransmitters and hormones (1,2). Therefore, the analysis of these compounds carries special relevance in biological sciences. A wide variety of techniques has been developed for cyclic nucleotide assays including binding to phosphokinase (3,4) or to antibodies (5) activation of enzymes... 

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