Enzyme assays equilibrium method

The majority of the many methods used to study the composition of equilibrium solutions of carbohydrates examine the mixture without separating the individual components. With the discovery that the anomeric forms of sugars could be readily separated by gas chromatography of their tri-methylsilyl ethers, a new approach to the problem was found. A protocol was developed for the direct gas chromatographic analysis of the amount of each anomer present in an aqueous solution. The protocol can be used on the micro scale and can be used in enzyme assays such as that for mutarotase. The method has been made more effective by combining gas chromatography with mass spectrometry. It is shown how mass spectral intensity ratios can be used to discriminate anomers one from another. The application of these methods to the study of complex mutarotations is discussed. 

Similarly, with the integration strategy for enzyme substrate assay, we also use twice the lower limit of the equilibrium method as the lower limit by the integration strategy if the standard error of estimate is much larger or else, three times the standard error of estimate by the integration strategy is taken as the lower limit of linear response. 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

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.

This soluble enzyme is specially abundant in skeletal muscle, only one quarter as plentiful in the myocardium and brain, and practically absent from other tissues (C13), so that hemolysis does not affect its activity in serum in the study of muscle disease this distribution offers great advantages. Its function is specifically the equilibration of creatine phosphate and ADP with creatine and ATP, with equilibrium heavily in favor of the latter compounds. Since its activity in serum is some million times lower than in skeletal muscle, serum assay offers certain difficulties three reliable methods, however, are available. 

Equilibrium Denaturation. A variety of different techniques can be employed to monitor protein conformational changes in the presence of denaturants. Activity measurements reflect the extent of alterations of the active site environment. However, enzyme activity measurements may be affected the presence of denaturant in the assay mixture. The denaturation curves obtained by this method are difficult to inte ret and can only be taken as a first approximation of the unfolding transition. U.V. difference spectra indicate conformational changes by monitoring the degree of solvent exposure of aromatic amino-acid side chains. Finally, fluorescence intensity measurements can reveal the nature of the environment (polar, non-polar) of the four tryptophans of p-lactamase. 

Modem methods for study of metal-activated enzymes include NMR and ESR spectroscopy, water relaxation rates by pulsed NMR (PRR), atomic absorption, Mbssbauer, X-ray and neutron diffraction, high-resolution electron microscopy, UV/visible/IR spectroscopy, laser lanthanide pertubation methods, fluorescence, and equilibrium and kinetic binding techniques. Studies with Mg(II)-activated enzymes have been hampered by the lack of paramagnetic or optical properties that can be used to probe its environment, and the relative lack of sensitivity of other available methods initial velocity kinetics, changes in ORD/CD, fluorescence, or UV properties of the protein, atomic absorption assays for equilibrium binding, or competition with bound Mn(II) °. Recent developments in Mg and 0-NMR methodology have shown some promise to provide new insights . ... 

Some of the most definitive studies of Mg(II)-activated enzymes have been performed by mangetic resonance (NMR, ESR) methods with the Mn(ll)-substituted species. An integrated picture of the role of the metal ion in catalysis in almost all cases also includes data from kinetics (steady state and pre-steady state), equilibrium binding, and optical spectroscopic methods. As stated above, there are but a few examples of true Mn-containing enzymes, especially in mammalian sytems. Table 1 provides a non-exhaustive list of examples of both Mn-specific and Mn/Mg-activated enzymes. Within the latter category are enzymes that show a preference for but not absolute specificity for one ion or the other. The distinction between these categories is not simple, often being dependent upon the source or form of the enzyme and various parameters as the type of assay used, temperature, pH, and others. 

The high sensitivity of fluorescence spectroscopy and the selectivity of enzymatic assays are responsible for the increasing use of fluorimetric methods in enzymology. Enzyme determinations usually involve the use of kinetic methodology for measuring the rate of formation of the fluorescent product, while both equilibrium and kinetic methods are used to determine the substrates. Fluorimetric measurements on enzyme-catalyzed reactions have been used for a long time to determine a variety of enzymes and substrates (Figure 2). 

An issue that has recently emerged was a microscale method to assay bioprocess options to improve bioconversion reactions yields by overcoming thermodynamic and kinetic limitations [110]. In this study they observed that the choice of the amino donor and the equilibrium shift via second enzyme reactions are the best options to increase the product yield. 

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