Kinetic assays

In this paper we review the fluorescence and spectroscopic assays commonly used in our lab to study neutrophil activation. The advantages of these assays are threefold. (1) These assays are very sensitive. (2) These are kinetic assays which can be measured with a time resolution of 1 s or less. This is important since many of these responses occur within seconds of stimulation and are... 

Diacylglycerol has long been known to be a weak competitive inhibitor of PLC/fc, whereas phosphorylcholine shows very little inhibition [40, 49, 116]. Recent kinetic assays of PLCB(. activity in the presence of DAG indicate that it is a competitive inhibitor with a Kl of the order of 10 mM, whereas phosphorylcholine was found to be an extremely weak (K = 30-50 mM), mixed inhibitor of PLC/J( [34]. Because diacylglycerol is a competitive inhibitor of the enzyme, the nature of the catalytic cycle dictates that it must be the last product to leave the enzyme active site. 

The evaluation of results of assay optimization experiments such as those described above (see Section 6.4.2.1) also provides valuable information about enzymatic kinetic behavior. For example, the results shown in Figures 6.45 and 6.46 already provide information on enzymatic activity at each time point. In general, when evaluating enzyme kinetics, assays are designed to yield a measured conversion close to initial velocity.32... 

The kinetic assay of LDH is based on the conversion of lactic acid to pyruvic acid, in the presence of nicotinamide adenine dinucleotide (NAD), and is closely monitored at intervals of 30 seconds or 1 minute by measuring the increase in absorbance at 340 nm. In this particular instance lactic acid available in an excess to ensure that the increase in pyruvic acid is linear with time, i.e., directly proportional to time. The reaction involved may be expressed as follows ... 

Figure 7.15 Enzyme-multiplied immunoassay (EMIT). The three reactants, test (or standard) antigen, enzyme-labelled antigen and a limited amount of antibody are allowed to react and reach an equilibrium position. The unbound labelled antigen which remains is the only source of enzyme activity, the bound enzyme being inactivated. This free enzyme can be quantitated using a direct kinetic assay method and is proportional to the amount of unlabelled antigen originally present.

Fluorimetric methods are useful for monitoring reactions involving the nucleotide coenzymes. The natural fluorescence of the reduced forms in the region of 460 nm can be used in kinetic assays. However, this fluorescence is destroyed at pH values below 2.0, whereas any oxidized forms of the coenzymes present are stable. If the pH of the solution is then raised above 10.5 and heated, the oxidized forms are themselves converted to fluorescent derivatives. This latter procedure lends itself to fixed time assays such as is illustrated in Procedure 8.6. 

Direct kinetic assays are the only valid methods for the measurement of activators and inhibitors and calibration plots of the percentage activation or inhibition by known amounts of the substance can be made. Examples of inhibition assays include the quantitation of organophosphorus pesticides using the inhibition of cholinesterase (EC 3.1.1.7) while manganese can be measured in amounts as low as 1 X 10-12 mol using its activating effect on isocitrate dehydrogenase (EC 1.1.1.41). 

Figure 8.21 A kinetic assay of NAD+. The rate of increase in absorbance at 550 nm as cytochrome c is reduced is a measure of the steady-state concentration of NAD+.

It has already been stated that a suitable quantitative assay technique must be available to measure the reaction of interest and it is assumed that the experimenter has determined optimal reaction conditions for the enzyme of interest. All kinetic assay techniques assume that v is a variable and that [S] is known as such, preparation of substrate must be meticulous in terms of ensuring that concentrations are correct, and this in turn will rely upon factors such as good weighing and pipetting techniques with calibrated instruments capable of precise, accurate, and sufficiently sensitive measurement. 

If an approximate Km value for the enzyme-substrate combination of interest is known, a full-scale kinetic assay may be done immediately. However, often an approximate value is not known and it is necessary first to do a range finding or suck and see preliminary assay. For such an assay, a concentrated substrate solution is prepared and tenfold serial dilutions of the substrate are made so that a range of substrate concentrations is available within which the experimenter is confident the Km value lies. Initial velocities are determined at each substrate concentration, and data may he plotted either hyperholically (as V versus [S]) or with [S] values expressed as logio values. In the latter case, a sigmoidal curve is fitted to data with a three parameter logistic equation (O Eq. 4) ... 

If an approximate Km value is known, a kinetic assay may be done to obtain an accurate determination of Km and This is achieved simply by determining values for v (initial velocities) for a range of appropriate substrate concentrations. 

There is also some merit in choosing substrate concentrations that result in incremental increases in v of similar magnitude. O Table 4-2 indicates suitable substrate concentrations, expressed as fractions or multiples of the Km value, leading to consistent increases in y, and this could be used as a template when designing a kinetic assay. 

ELISA assays may be competitive or noncompetitive. As the name imphes, in a competitive ELISA, enzyme-labeled antigen competes with free antigen (the analyte of interest) for a fixed and limited quantity of immobihzed antibody binding sites. After incubation, the microtiter plate (sohd support) is rinsed to remove all unbound species and the enzyme substrate is added in saturating concentration. The conversion of substrate to produce can be measured continuously (kinetic assay) or, more commonly. 

The data presented in Figs. 3 and 4 are examples of the types of kinetic binding data that are readily acquired with commercially available flow cytometers. Quantitative, real-time analysis of fluorescent A-formyl peptide association with neutrophil receptors has been described by Fay et al. (4), and this publication should be consulted for detailed protocols required for quantitative kinetic assays (see Notes 5 and 6). 

Kinetic assays give access to the binding reaction s forward and reverse rate constants, i.e. the association rate constant fe+i and the dissociation rate constant fe i that characterize the association and the dissociation of the target-marker complex and the Kj [see Eq. (4)]. 

Other miscellaneous assays for penicillin or other 3-lactams in milk is the Penzyme Test which uses cell wall enzjrmes inhibited by 3-lactam drugs in a kinetic assay. This test system is purported to be able to detect 0.005 units penicillin/mL and requires approximately 30 min to complete. It, like many other assays, detects 3-lactam antibiotics only. 

Selected entries from Methods in Enzymology [vol, page(s)] Dilution of enzyme samples, 63, 10 lipolysis substrate effect, 64, 361, 362 dilution jump kinetic assay, 74, 14-19, 28 dilution method [for dissociation equilibria, 61, 65-96 continuous dilution cuvette, 61, 78-96 data analysis, 61, 74, 75 equations, 61, 70-74 errors, 61, 76-78 experimental procedures, 61, 69, 70 merits, 61, 75, 76 theory, 61, 68, 69... 

Because ATP and GTP hydrolysis play such central roles in biochemical processes, kinetic assays of orthophos-... 

Using a rapid quench-fiow kinetic assay for post-complex fragment formation, Nair and Cooperman showed that the ET encounter complex of serpin and enzyme forms both E I and the post-complex fragment with the same rate constant, indicating that both species arise from ET conversion to E I. These results support the conclusions (a) that the peptide bond remains intact within the ET complex, and (b) that E I is likely to be either the acyl-enzyme or the tetrahedral intermediate formed after water attack on acyl-enzyme. 

E2. Ellis, G., and Goldberg, D. M., Optimal conditions for the kinetic assay of serum glutamate dehydrogenase activity at 37°C. Clin. Chem. 18, 523-527 (1972). 

PROCEDURE SGPT Preliminary Trayser, Seligson Kinetic Assay Calbiochem Kit... 

Li PK, Lee JT, Mac Gillivray MH, Schaefer PA, Siegel JH (1980) Direct, fixed-time kinetic assays for beta-hydroxybutyrate and acetoacetate with a centrifugal analyser or a computer-backed spectrophotometer. Clin Chem 26 1713-1717... 

Prokaryotic DNA polymerases are so accurate that special kinetic assays have had to be introduced to detect errors in vitro. These depend on replicating under controlled conditions the circular DNA of a small bacteriophage that contains a... 

If an enzyme assay involves continuous monitoring of substrate or product concentration, the assay is said to be kinetic. If a single measurement of substrate or product concentration is made after a specified reaction time, a fixed-time assay results. The kinetic assay is more desirable because the time course of the reaction is directly observed and any discrepancy from linearity can be immediately detected. 

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...

Part B Tyrosinase level for kinetic assay—1 hour. 

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