Enzyme assay 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 ... 

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.

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... 

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). 

Coupled enzyme assays have been developed for the determination of substances as diverse as glucose, uric acid, and cholesterol, the principal application being quantitation in biological fluids such as blood, plasma, and urine. Typical examples are illustrated by Eqs. (9)-(12). 

A protocol for continuous enzyme assay that involves one or more auxiliary enzymes to convert a product of the primary reaction in a second or auxiliary reaction that produces a change in absorbance or fluorescence. As noted below, coupled enzyme assays, while convenient, are fraught with experimental limitations that must be overcome in order to obtain valid initial velocity data. 

McClure was among the first to treat the kinetics of coupled enzyme assays, and others have discussed essential aspects of experimental design. Rudolph et al provided an extensive list of coupled-enzyme assays for selected enzyme reaction products, and several examples are shown in Table I. 

The components of the coupling system should neither inhibit nor activate the primary enzyme. Moreover, care must be exercized to ascertain that the auxiliary enzyme (s) is not contaminated with other minor enzyme activities capable of influencing the primary enzymatic activity. The results from any coupled enzyme assay must exactly match the results obtained with other valid initial rate assays to ensure that the presence of the auxiliary system in no way affects the activity of the primary enzyme. This is typically accomplished by comparing data obtained from the coupled assay with stopped-time assay results to ensure that similar results are obtained. 

Easterby proposed a generalized theory of the transition time for sequential enzyme reactions where the steady-state production of product is preceded by a lag period or transition time during which the intermediates of the sequence are accumulating. He found that if a steady state is eventually reached, the magnitude of this lag may be calculated, even when the differentiation equations describing the process have no analytical solution. The calculation may be made for simple systems in which the enzymes obey Michaehs-Menten kinetics or for more complex pathways in which intermediates act as modifiers of the enzymes. The transition time associated with each intermediate in the sequence is given by the ratio of the appropriate steady-state intermediate concentration to the steady-state flux. The theory is also applicable to the transition between steady states produced by flux changes. Apphcation of the theory to coupled enzyme assays makes it possible to define the minimum requirements for successful operation of a coupled assay. The theory can be extended to deal with sequences in which the enzyme concentration exceeds substrate concentration. 

The assay protocol should measure true initial rates (See Initial Rate Condition). For most systems, this represents a time period in which less than ten percent of the substrate concentration has undergone conversion. However, if a reaction is not significantly favored thermodynamically or if product inhibition is particularly potent, then a much smaller percentage of substrate conversion may be needed such that true initial rate conditions are obtained. Addition of an auxiliary enzyme system may prove necessary to avoid product accumulation. See Coupled Enzyme Assays... 

COUNTING STATISTICS COUNTING STATISTICS COUPLED ENZYME ASSAYS Coupling enzymes,... 

EXPERIMENTAL DESIGN OF INITIAL RATE ENZYME ASSAYS COUPLED ENZYME ASSAYS... 

Scheme 4. Coupled enzyme assays shifting the fluorinase reaction in favour of 5 -chloro-5 -deoxyadenosine (5 -CIDA) 14 synthesis [15].

The substrate for MurD (UDP-MurNAc-L-Ala) and purified Mur enzymes were required to develop a coupled enzyme assay. None of the Mur enzymes or their substrates were available commercially, although purification procedures for the... 

Figure 2 SDS-PAGE of purified Mur enzymes used in the coupled enzyme assay. A Novex 10-20% Tricine gel was used and stained with Coomassie blue. Lane 1 contains the molecular-weight standards (kDa) lanes 2-4 contain 3.5 p.g of MurD, MurE, and MurF, respectively.

Since MurD was the primary target, it was made the rate-limiting step in the coupled enzyme assay and conditions were optimized for that catalytic reaction. The kinetic parameters were determined for MurD. The Km for UMA and d-G1u guided the choice of substrate concentrations for the MurD reaction so the enzyme was optimally efficient [31], The kinetic parameters for the amino acid or dipeptide and tripeptide substrates for MurE and MurF were then determined... 

Figure 3 Determination of S. pneumoniae MurD kinetic parameters. Initial velocities as a function of substrate concentration were measured using the ADP coupled enzyme assay with PK and LDH. Data were fit with a nonlinear regression analysis using GraphPad Prism. Top panel is for UMA and bottom panel is for d-G1u.

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