Enzyme coupled

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 ... 

Table 3. Clinically Important Substances Detected Using Coupled Enzyme Reactions...

Enzyme electrodes are based on the coupling of a layer of an enzyme with an appropriate electrode. Such electrodes combine the specificity of the enzyme for its substrate with the analytical power of electrochemical devices. As a result of this coupling, enzyme electrodes have been shown to be extremely useful for monitoring a wide variety of substrates of analytical importance in clinical, environmental, and food samples. 

Figure 7-10. Coupled enzyme assay for hexokinase activity. The production of glucose 6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP". When an excess of glucose-6-phosphate dehydrogenase is present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose 6-phosphate by hexokinase.

A wide variety of enzymes have been used in conjunction with electrochemical techniques. The only requirement is that an electroactive product is formed during the reaction, either from the substrate or as a cofactor (i.e. NADH). In most cases, the electroactive products detected have been oxygen, hydrogen peroxide, NADH, or ferri/ferrocyanide. Some workers have used the dye intermediates used in classical colorimetric methods because these dyes are typically also electroactive. Although an electroactive product must be formed, it does not necessarily have to arise directly from the enzyme reaction of interest. Several cases of coupling enzyme reactions to produce an electroactive product have been described. The ability to use several coupled enzyme reactions extends the possible use of electrochemical techniques to essentially any enzyme system. 

The final method of coupling enzyme reactions to electrochemistry is to immobilize an enzyme directly at the electrode surface. Enzyme electrodes provide the advantages already discussed for immobilization of enzymes. In addition, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near to the electrode. The concept of combining an enzyme reaction with an amperometric probe should offer all of the advantages discussed earlier for ion-selective (potentiometric) electrodes with a much higher sensitivity. In addition, since the response of amperometric electrodes is linear, background can be selected. 

J.W. King and K.S. Nam, Coupling enzyme immunoassay with supercritical fluid extraction, in Immunoassays for Residue Analysis, ed. R.C. Beier and L.H. Stanker, American Chemical Society, Washington, DC, Chapter 34, pp. 422-438 (1996). 

A common characteristic of metabolic pathways is that the product of one enzyme in sequence is the substrate for the next enzyme and so forth. In vivo, biocatalysis takes place in compartmentalized cellular structure as highly organized particle and membrane systems. This allows control of enzyme-catalyzed reactions. Several multienzyme systems have been studied by many researchers. They consist essentially of membrane- [104] and matrix- [105,106] bound enzymes or coupled enzymes in low water media [107]. 

Recently [63], we studied the behavior of two-enzyme system catalyzing two consecutive reactions in a macroheterogeneous medium (modified Lewis cell). The system consisted of lipase-catalyzed hydrolysis of trilinolein and subsequent lipoxygenation of liberated fatty acids (Fig. 3). Our approach compared the kinetic behavior of coupled enzymes in the Lewis cell with the sequential study of separated phenomena presented before ... 

Finally, one must take into account that in using a coupled enzyme assay one must produce, or purchase, not only the target enzyme of interest but also the coupling enzymes and any co-substrates required for these additional protein reagents. Hence a coupled enzyme assay can be quite expensive to implement, especially for large library screening. In some cases the cost may be prohibitive, precluding the use of a particular coupled enzyme assay for HTS purposes. 

In carrying out an enzyme assay it may be convenient to introduce an auxiliary enzyme to the system to effect the removal of a product produced by the first enzymatic reaction. McClure [Biochemistry, 8 (2782), 1969] has described the kinetics of certain of these coupled enzyme assays. The simplest coupled enzyme assay system may be represented as... 

A coupled enzymic high-performance liquid chromatographic method has been used to determine down to 0.1-0.8 xM of formate in sea water [130]. 

Microtiter plates HRP/H202/luminol AP/dioxetanes Firefly luciferin/luciferase Bacterial luciferin/luciferase Detection of enzymes and metabolites by direct or coupled enzyme reactions Determination of antioxidant and enzyme inhibitory activities Immunoassay... 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

An indicator enzyme is the enzyme that catalyses the final measurable reaction in a coupled enzyme assay. 

Figure 8.13 Typical reaction trace of a coupled enzyme assay. The indicator reaction in many coupled assays will often show a demonstrable change after the addition of the sample but before the addition of the substrate for the test enzyme. This blank reaction may be due to the presence of endogenous substrates in the sample and its rate (B) must be measured in order to be able to calculate the activity of the test enzyme (T —B) from the total rate of reaction (7) which results from adding the substrate.

Coupled enzyme assays provide a good alternative to chemical modification and permit a kinetic technique to be employed. In a coupled assay, the rate at which the product is formed is measured by using the product of the reaction as a substrate for a second enzyme reaction which can be monitored more easily. Coupled assays offer great flexibility in enzyme methodology while still retaining all the advantages of continuous monitoring techniques and are illustrated by Procedure 8.5. 

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). 

End-point assays can also use coupled enzyme systems in which the product of the test reaction provides the substrate for the subsequent auxiliary and indicator reactions. The choice of pH is less critical than with the measurement of enzyme activity frequently a compromise pH is used and... 

Biocatalytic approaches to cofactor regeneration can be divided into coupled-enzyme methods and coupled-substrate methods.In the coupled-enzyme method, the oxidized cofactors (NAD+ and NADP+) are recycled in situ by performing an oxidation reaction using a second enzyme and an inexpensive auxiliary substrate. This second enzyme must employ the same cofactor, but neither enzyme should be able to accept the same substrate. 

Davis et adopted the coupled-enzyme method to access the (5)-hydroxyester... 

Recombinant cells expressing a cloned ADH have also been used in a coupled enzyme method to efficiently produce the (R)-2-chloromandelate intermediate in the synthetic route to clopidogrel in 90 % yield and >99 % ee at 200 gL substrate concentration (Scheme 1.53). This procedure does not use hydrogen cyanide and, therefore, represents... 

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