Discontinuous assays

The major advantage associated with continuous assays is that the initial rate of product formation can be determined with complete confidence, and any unusual behavior of the enzyme would be immediately apparent. The major disadvantage is a question of throughput an instrument such as a platereader would remain dedicated to the reading of a single plate for the duration of the enzyme-substrate incubation period, compared with an equivalent discontinuous assay where an entire plate may be measured in a... 

The reason for these substantial errors is quite simply that in a discontinuous assay, the researcher assumes that product formation remains (approximately) linear for the duration of the incubation period. Although this may hold true for high substrate concentrations (in this example), it is clear from continuous data that deviation from linearity is substantial at lower substrate concentrations. Accordingly, what must be done prior to use of a discontinuous assay protocol is a timecourse assessment, in which the concentration of product formed from both low and high concentrations of substrate is determined at various time... 

In addition, unlike many other discontinuous assays that focus on only one of the components of the reaction, the HPLC assay offers the potential to monitor several. For example, consider adenosine kinase, the enzyme that uses two substrates and forms two products according to the reaction Ado + ATP —> AMP + ADP. Since HPLC can readily separate all four compounds (see Fig. 1.4), and all four compounds can be detected at 2S4 nm, it is apparent that with the HPLC method, the level of each component can be monitored during the course of the reaction, providing a complete analysis of each time point. ... 

The need to use a discontinuous assay method does not automatically mean that the HPLC method is the procedure of choice. For HPLC to be suitable, it must be possible to separate the components, and some method for detection and quantitation must be available. Next, neither the ingredients in the reaction mixture nor those used to terminate the reaction should produce problems for the separation and detection. Finally, the enzyme itself should be considered. 

Figure 9.140 HPLC elution profile of a sample taken from a discontinuous assay of crude mouse spleen extract, using iV,-fluorenylmethoxycarbonyl-EEY(P)AA [Fmoc-EEY(P)AA] as substrate. Chromatography was performed on a C g Novapak column (10 cm X 8 mm) eluted isocratically with 36% acetonitrile-water (0.1% TFA) (v/v) at a flow rate of 2 mL/min. Peaks are due to (A) methanol and methanol-soluble compounds derived from the sample of crude homogenate, (B) Fmoc-EEY(P)AA (1238 pmol), and (C) Fmoc-EEYAA (195 pmol). Appropriate controls showed that no interfering compounds eluted in the position of the peptides. Inset Fluorescence monitoring of HPLC of Fmoc-EEYAA (75 fmol) eluted isocratically with 36% acetonitrile-water (0.1% TFA). Excitation and emission wavelengths, 268 and 307 nm, respectively, with gain X 100 and 10 mV chart scale. (From Nash et al., 1993.)...

Pre-steady-state kinetics using discontinuous assays (rapid chemical quench) 355... 

Figure 15-8 Effect of the ratio of Fe(II) atoms per molecule on the rate of iron oxidation by some ferritins and their variants. Remaining Fe(II) was measured in a discontinuous assay by removing an aliquot and adding it to a solution of ferrozine. Protein solutions were in 0.1m Mes buffer pH 6.5. Fixed iron concentration of 48pM (NH4)2 Fe (804)2 at 1 pM protein. Y-axes indicate the fraction of remaining Fe(II). (A) EcFTNa -f 48 Fe atoms per molecule (B) EcFTNa - - 480 Fe atoms per molecule (C) EcFTNa - - 980 Fe atoms per molecule (D) HuHF + 40 Fe atoms per molecule (E) HuHF -(- 500 Fe atoms per molecule (F) HuHF -1- 2000 Fe atoms per molecule.

Enzyme activity can be determined by a continuous or discontinuous assay. If the analytical device is provided with a reeorder that register the course of reaction, the initial rate could be easily determined from the initial slope of the product (or substrate, or coupled analyte, or coenzyme) concentration versus time curve. It is not always possible or simple to set up a continuous assay in that case, the course of reaction should be monitored discontinuously by sampling and assaying at predetermined time intervals and samples should be subjected to inactivation to stop the reaction. This is a drawback, since the enzyme should be rapidly, completely and irreversibly inactivated by subjecting it to harsh conditions that can interfere with the... 

The enzyme activity is used as an index to estimate the enzyme s potential to produce the desired product. Different physicochemical techniques are available to measure the activity, either by substrate consumption or product formation (Smeltzer et al, 1992), For a good estimate of the activity, the range of substrate concentrations should be carefully selected and accurately measured. Continuous assays, which continuously monitor changes in the reaction solution s physical properties, such as light absorbance (colorimetric), fluorescence (fluorometric), or heat release/ absorbance (calorimetric), are among the possible techniques. Discontinuous assays, where samples from a reaction solution are collected at intervals, and the amount of substrate or product concentrations are measured, are also frequently used. 

Boeker, E. A. (1987). Analytical methods for fitting integrated rate equations. A discontinuous assay. Biochem. J. 245, 67-74. 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

Operational schematic of discontinuous biosensing system (assay), 16 3-3 Key characteristics of sensor systems, 17... 

FIGURE 3-2 Operational schematic of discontinuous biosensing system (assay). 

The initial velocity of reaction is defined by the slope of a linear plot of product (or substrate) concentration as a function of time (Chapter 2), and we have just discussed the importance of measuring enzymatic activity during this initial velocity phase of the reaction. The best measure of initial velocity is thus obtained by continuous measurement of product formation or substrate disappearance with time over a convenient portion of the intial velocity phase. However, continuous monitoring of assay signal is not always practical. Copeland (2000) has described three types of assay readouts for measuring reaction velocity continuous assays, discontinuous... 

The method of Kato and Nakai (27) for determining protein surface hydrophobicity was adapted for evaluating procyanidin binding to BSA and Gl. The procedure is based on the fact that the fluorescence quantum yield of cis-parinaric acid increases 40-fold when cis-parinaric acid enters a hydrophobic environment from a hydrophilic environment. The digestion of BSA by trypsin in the presence of procyanidin dimer, procyanidin trimer and black bean procyanidin polymer was evaluated by discontinuous sodium dodecyl sulfate (SDS) slab gel electrophoresis and a picryl sulfonic acid (TNBS) assay (28). 

The demonstration of genotoxic activity in a pharmaceutical molecule may not necessarily translate into discontinuation of development and ultimate licensing of the product to be marketed. A survey of the Physicians Desk Reference reveals many examples of pharmaceutical products that have been shown to elicit genotoxic activity in one or more gene-tox assays.41 This observation indicates that other factors (such as risk, benefit, seriousness of ailment, target patient population, dosage, and frequency of administration, among others) are taken into consideration. 

The major advantage associated with the discontinuous approach is that only a single measurement is made, facilitating data analysis. In addition, for spectrometer- and platereader-based assays, many more samples can be measured in unit time, compared with the equivalent continuous assay system. 

Discontinuous approaches are used through necessity in radiometric assays (Oldham, 1993) and in liquid- and gas-chromatography-based protocols (Syed, 1993). Many commercially available kits designed to measure a particular enzyme activity combine absorbance and fluorescence platereader-based technologies with assay protocols that could be done continuously. However, since many users of these kits have little experience in enzymology, assay instructions usually outline a simplified procedure whereby a... 

Classical low values for the mammalian enzyme that have appeared in the literature are the result of enzyme inactivation by hydrogen peroxide when measurements were carried out with peroxide levels in excess of 10 mM over time scales of 10 minutes or longer. The rapid sampling/titration method of Bonnichsen overcame the inactivation problem and permitted a satisfactory correlation of the overall catalytic measurements and Chance s observations on the intermediate complex (compound 1). Eventually, the introduction of the UV detector/spectrophotometer and the consequent assay based upon the UV absorbance of peroxide (35) further simplified the process by eliminating the discontinuous titrimetric assay. 

TTie major disadvantage of the GeMSAEC is that although several analyses are run in parallel, the system is discontinuous and the rotor has to he stopped for reloading it is also difficult to carry out more than one type of assay in a rotor at any one time. This problem was overcome in the DACOS approach (Discrete Analyser with Continuous Optical Scanning) described by Snook et al. [17]. In this approach, reaction tubes are situated at the periphery of the rotor, which turns in discrete steps, a few degrees at a time, to enable... 

Traditionally, HAT activity is measured with a discontinuous radioactive filterbinding assay, which uses pH]acetyl-CoA as a histone acetyltransferase substrate [46]. The transfer of [ H]acetyl-groups to the histone substrate by histone acetyltransferases is detected by liquid scintillation counting of pHjacetylated histones, which are retained on a phosphocellulose disk. Due to its discontinuous character, this assay is technically problematic and not ideal for kinetic analysis. Hence, other assays that work with radiolabeled acetyl-CoA have been described that are suitable for a higher throughput. These work with streptavidin-covered beads [47] or a variant of the SPA with microtiter plates that contain a scintillant (FlashPlates) [48]. But as all these protocols are based on radioactively labeled substrates, they apparently show the same disadvantages that were described for the radioactive HDAC assay protocols. Therefore, nonradioactive assays have been developed to study histone acetyltransferase activity. 

Plotting the number of compounds as a function of percent inhibition at 10 M results in a distribution curve with a slope break point that corresponds to a discontinuity in the first derivative ( DFD ). A change in the distribution density suggests that there are different populations within the percent inhibition values. The DFD point is numerically calculated from the distribution curve and gives an assessment of the suitable threshold for each assay. Most often it is located in the vicinity of 30% inhibition at 10 M (unpublished work). 

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