Enzymic assay methods

Bioluminescence provides the basis for sensitive enzymic assay methods both for substrate assays and coupled enzyme assays. Firefly luciferase (EC 1.13.12.5) catalyses the production of light (540-600 nm) by the oxidation of luciferin (d-LH2) (Figure 8.18). 

The velocity of an enzyme-catalyzed reaction can be measured either by a continuous assay or by a stopped-time protocol. Whenever possible, the continuous measurement of a velocity (e.g., the increase or decrease in absorbance vx. time) should be utilized. In stopped-time assays, the investigator must demonstrate that the reaction is completely terminated at the specified point in time and that products are readily and quantitatively separated from substrates. In addition, one must show that the system is under initial rate conditions. Thus, at least three or four different time points should be chosen. Stopped-time assays also require an assay blank (for t = 0). In this blank, typically the quenching conditions are applied prior to the initiation step. Whenever practicable, replicate kinetic analyses should be done, even with continuous assay protocols. See Enzyme Assay Methods Basal Rate... 

Auxiliary Enzyme Assay Methods for Initial Rate Analysis ... 

Enzyme assay methods for screening have been augmented by new screening technologies that allow homogeneous assay formats. Scintillation proximity... 

A fibrin clot containing adsorbed plasmin inhibitors is difficult to define in a chemical or physical sense. Generally, when enzyme reactions occur at surfaces, the porosities and adsorption properties erf which are variable, the reproducibility of enzyme assay methods is questionable. The proteinoses, to which belong the most important pharmaceutical enzymes, may present some difficulties when natural substrates (protein ) are prescribed. Here, the application of a parallel run with a reference standard is recommended. 

The HPLC enzyme assay method provides an alternative procedure with which to approach this problem. Clearly, to be able to assay these activities by the HPLC method, it is necessary to separate ATP, ADP, and AMP. This separation can be easily accomplished by ion-exchange HPLC eluted isocratically with a mobile phase containing a phosphate buffer and sufficient concentration of salt to elute the ATP. Under these conditions, the order of elution of the compounds would be AMP first, ADP next, and ATP last. 

Koda N, Tsutsui Y, Niwa H, Ito S, Woodward DF, Watanabe K (2004) Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme new enzyme assay method using LC/ESI/MS. Arch Biochem Biophys 424 128-136... 

Solution-Based Enzyme Assay Methods Other Than Absorbance... 

SOLUTION-BASED ENZYME ASSAY METHODS OTHER THAN ABSORBANCE... 

Reference Stem cell type Culture format Differentiation factors % ALB -i-ve HLCs (assay) Enzyme (assay method) %hPH comparator Other comparators... 

This enzyme assay method was recently used to kinetically characterize a synthetic enzyme, isomelezitose synthase, which was prepared by site-directed mutagenesis of sucrose isomerases. Continuous measurement of fluorescence ehanges in a 384-well plate, containing 4,4 -o-BBV/HPTS and isomelezitose synthase, allowed these authors to measure the concentration of fructose and isomaltulose in real time and to kinetically follow the conversion of sucrose into fructose and isomaltulose. ... 

A review on the subject of immobilized enzymes in biochemical analysis covers preparation and properties of immobilized enzymes assay methods using immobilized enzymes (spectrophotomatic assays, automated methods in biochemical analysis, enzyme electrodes, and colorimetric analyses using immobilized enzymes) and applications of immobilized enzymes in biochemical analysis. ... 

Ricinoleate has many industrial uses, but the only commercial source is castor bean, which is hazardous. It is thus desirable to produce ricinoleate in a transgenic plant. The cDNA for oleoyl-12-hydroxylase, the key enzyme in the biosynthesis of ricinoleate, has been cloned recently (1). In order to reach the 90% ricinoleate level provided by castor bean, it will require understanding of ricinoleate production, the enzymes that move it into triglyceride (TG) and the enzymes that keep oleate available as hydroxylase substrate. We have recently developed an enzyme assay method to characterize oleoyl-12-hydroxylase using the putative substrate, 1-acyl-2-oleoyl-.yn-glycero-3-phosphocholine (2). We report here the in vitro metabolism of 2-oleoyl-PC in the microsomes isolated from immature castor bean. 

Cultivation of the bacterium, purification of amylase, enzyme assay method, basic analytical methods such as determination of protein and sugars, polyacrylamide gel electrophoreses with or without sodium dodecyl sulfate (SDS) and thin layer chromatographic method were described in previous papers (1,3,7). 

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

HPLC, high-petfomtance liquid chromatogtaphy ELISA, enzyme-linked immunosorbent assay method RIA, radioimmunoassay method. 

J. Strahan, Development and application of an enzyme-linked immunosorbent assay method for the determination of multiple sulfonylurea herbicides on the same microwell plate, in Environmental Immunochemical Methods, ed. J.M. Van Emon, C.L. Gerlach, and J.C. Johnson, American Chemical Society, Washington, DC, pp. 65-73 (1996). 

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

Urdea, M.S., Warner, B.D., Running, J.A., Stempien, M., Clyne, J., and Horn, T. (1988) A comparison of non-radioactive hybridization assay methods using fluorescent, chemiluminescent and enzyme-labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 16, 4937-4956. 

Creaser reported that staphlococcus aureus forms an inducible f5-galactosidase166. The production of this enzyme is inhibited by the addition of gramicidin. Strictly speaking, gramicidin does not inhibit the enzyme directly but this method could be used as the basis of a sensitive assay method. 

Wilson, S.D., Munson, A.E. and Meade, B.J., Assessment of the functional integrity of the humoral immune response the plaque-forming cell assay and the enzyme-linked immunosorbent assay, Methods, 19, 3, 1999. 

The assay methods reported in the literature vary with respect to the use of methylene blue or another dye, and to the coenzyme used, depending on enzyme source, etc. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

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