Assay enzymatic

In the enzymatic assays of cholesterol, glucose, and urea, oxygen is used and H2O2 is formed. The reaction for uric acid [69-93-2] is... 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

A solution of 2.25 g (25 mmol) of D-glyccraldehyde in 300 mL of water is combined with a solution of 20 mmol of dihydroxyacetonc phosphate (DIIAP) in 200 mL of water freshly adjusted to pH 6.8. The mixture is incubated with 100 U of L-rhamnulose 1-phosphate aldolase at r.t. for 24 h with monitoring of conversion by TLC (2-propanol/sat. ammonia/water 6 4 2) and by enzymatic assay for DHAP55. 

FIGURE 6-22 Schematic of a microchip system for enzymatic assays. Hie channels terminate at reservoirs containing the indicated solutions. (Reproduced with permission from reference 81.)... 

Literally hundreds of aldehydes have so far been tested successfully by enzymatic assay and preparative experiments as a replacement for (18) in rabbit muscle FruA catalyzed aldol additions [16,25], and most of the corresponding aldol products have been isolated and characterized. The rabbit FruA can discriminate racemic dl-(18), its natural substrate, with high preference for the D-antipode, but kinetic enantioselec-tivity for nonionic aldehydes is rather low [84,89]. 

Antibiotics may be assayed by a variety of methods (see Chapter 8, pages 166-188, in Pharmaceutical Microbiology, 5th edition 1992). Only microbiological and radio-enzymatic assays will be considered briefly here see Figs 25.5 and 25.6 and sections 4.1.1 and4.1.2. 

White L.O. Reeves D.S. (1983) Enzymatic assay of aminoglycoside antibiotics. In Antibiotics Assessment of Antimicrobial Activity and Resistance (eds A.D. Russell L.B. Quesnel), pp. 199-210. Society for Applied Bacteriology Technical Series No. 18. London Academic Press. 

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

Subsequently, proteolytic fragments of the rabbit renal 25-kDa amiloride-binding protein were micro-sequenced and found to have high sequence homology with rat and human NAD(P)H quinone oxidoreductase. Indeed, enzymatic assays revealed that renal brush border membrane vesicles contain significant NADPH quinone oxidoreductase activity. Presumably NAD(P)H quinone oxidoreductase coincidentally binds amiloride analogs with the same rank order as the Na /H exchanger [39]. 

The metabolic and pharmacokinetic profile of sucralose (this is a novel intense sweetener with a potency about 600 times that of sucrose) in human volunteers was studied by Roberts and coworkers [82]. Part of this study was realized using PLC in the following chromatographic system in which the stationary phase was silica gel and the mobile phase was ethyl acetate-methanol-water-concentrated ammonia (60 20 10 2, v/v). Separated substances were scraped off separately, suspended in methanol, and analyzed by filtration, scintillation counting, or enzymatic assay. It was shown that the characteristics of sucralose include poor absorption, rapid elimination, limited conjugative metabolism of the fraction absorbed, and lack of bio-accumulative potential. 

Enzymatic Assay. The enzymatic (luciferase) assay for adenosine triphosphate (ATP) is one of the methods applied to areas of biocidal control in oil production operation [1454]. A reliable method for the determination of ATP is the measurement of bioluminescence produced by the luciferin luciferase system. 

SDS-PAGE, MALDI TOP MS, 2-D Gel HPLC, GC, GC-MS GC, GC-MS HPLC, FTIR Diastase analysis GC, enzymatic assay LC, LC-MS, CE Pollen analysis AES and ICP-AES... 

In general, CE is simple, rapid, and low cost because it needs neither laborious treatment of the samples nor long times of analysis. However, its high detection limit is a major limitation of CE. CE is often poorly reproducible. Enzymatic assay is more suitable for quantifying one organic acid in honey samples because it is specific, precise, and accurate. GC is more suitable for analyzing volatile or semivolatile chemicals. HPLC is versatile and reproducible. However, common HPLC detectors such as UV-VIS are not very sensitive for organic aliphatic acids. 

A group of benzimidazole-2-pyrazole PHD2 inhibitors appeared in a recent patent application, with IC50 values as low as 16 nM for compound 43 in a [14C]-based enzymatic assay [64]. Furthermore, members of this class of compounds were reported to stimulate erythropoietin secretion from PIep3B cells by up to 150% at 100 pM concentrations. 

Apart from immunoassays, enzyme assays can also be used to detect certain substrates in a clinical diagnostic setting. The benefits of performing enzymatic assays on microchips are the analytical power and minimal reagent use in microfluidic systems combined with the selectivity and amplification factors that come with biocatalysis. 

D.C. Duffy, H.L. Gillis, J. Lin, N.F. Sheppard Jr, and G.J. Kellogg, Microfabricated centrifugal microfluidic systems characterization and multiple enzymatic assays. Anal. Chem. 71, 4669-4678... 

Enzymatic assays EROD Bg, Cc Liver Oxidative stress, dioxin-like compounds... 

The diagnosis of lysosomal disorders is usually based on enzymatic assays in white blood cells or cultured skin fibroblasts [ 1 ]. Molecular studies required for identification of carriers. Initial diagnosis of peroxisomal disorders... 

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. 

This section describes recent applications of jitPEC methodologies for separation-based enzymatic assays. It covers the most common applications (1) those involving the development and optimization of assays (2) those in which jitPLC is use to evaluate real-time enzyme kinetics and (3) those in which /./PEC is used to determine substrate specificity. 

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