Enzyme assay optimization

The pH optimum for the lactate-to-pyruvate (L—>P) reaction is 8.8 to 9.8, and an assay mixture, optimized for LD-1 at 37 °C, contains NAD% 9mmol/L, and L-lactate, 80mmol/L. For the P —> L assay, at 37 °C, the pH optimum is 7.4 to 7.8, NADH 300fJ.mol/L, and pyruvate 0.85mmol/L. The optimal pH varies with the predominant isoenzymes in the sample and depends on the temperature and on substrate and buffer concentrations. The specificity of the enzyme extends from L-lactate to various related 2-hydroxyacids and 2-oxo-acids. The catalytic oxidation of 2-hydroxybutyrate, the next higher homologue of lactate, to 2-oxobutyrate is referred to as 2-hydroxybutyrate dehydrogenase (HBD) activity. LD does not act on n-lactate, and only NAD serves as a coenzyme. 

For the sulfhydration reaction, C>-phosphohomoserine is the most active substrate under assay conditions optimal for activity with this compound. When the activities were determined under conditions optimal for O-acetylhomoserine, activity with O-acetylhomoserine was increased to 17% of that with O-phosphohomoserine, while the relative activity with O-succinylhomoserine remained unchanged. Sulfhydrase activities with O-phosphohomoserine and O-acetylhomoserine showed different patterns of development during early growth of excised barley embryos, suggesting that activities with the two substrates may be catalyzed by separate enzymes (Datko et al., 1977). Purification of these activities will be required to determine whether this suggestion is correct. 

Intact plastids were isolated from early, early to mid and mid to late cotyledon embryos (1.5, 2.5 and 3.5 mg embryo fresh weight) of B. napus cv. Topas (according to [1]). Isolated plastids were incubated with " C-labelled metabolites and the incorporation of C into fatty acids determined [1]. The activities of enzymes were measured using standard assays [1] optimized for plastid extracts. The proportions of glycolytic enzyme activities attributable to the plastid were determined by adding increasing concentrations of cytosol (supernatant) back to a plastid pellet. The plastidial activity of each enzyme was then calculated from the relationship between its total activity and that of a cytosolic and plastidial marker enzyme essentially as previously described [3]. Short term assays for the rate at which isolated plastids take up Relabelled metabolites were performed using silicone oil filtration [4]. 

The measure of the catalytic activity of an enzyme is the rate of the reaction catalyzed by the enzyme. The conditions of an enzyme activity assay are optimized with relation to type and ionic strength of the buffer, pH, and concentrations of substrate, cosubstrate and activators used. The closely controlled assay conditions, including the temperature, are critical because, in contrast to substrate analysis, the reliability of the results in this case often can not be verified by using a weighed standard sample. 

Another reason is that xylo-oligosaccharides of defined structure are very important substrates that serve as model compounds for the optimization of hydrolytic processes and in enzymic assays. The enormous development... 

Product Acceptors. Many enzyme assays use acceptors, as for instance 2-ethylaminoethanol and other aminated alcohols iihich act as acceptors for the phosphoryl product of the reaction catalyzed by alkaline phosphatase (25) (Fig. 4). Hydroxylamine can act as an acceptor for the hydroxyacetone produced by eno-lase and semicarbazide can act as an acceptor for the pyruvate produced by LD. It is necessary to optimize the concentration of such an acceptor before using it routinely as often what may be a theoretically desirable acceptor is in practice superfluous. 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

In some cases one s best guess at physiological conditions does not support sufficient catalytic activity to make a screening assay feasible. In this situation one has no choice but to compromise in favor of more optimal laboratory conditions. Nevertheless, one should attempt, whenever possible, to come as close as feasible to assay conditions that reflect the physiological context in which the target enzyme operates. 

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-... 

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