Enzymatic coupled assay

Coupled assays have been used to monitor a variety of different enzymatic reactions and can be split into two types chemically coupled and enzymatically coupled assays. In the former type, the product of the enzymatic reaction under study is detected by reaction with a reactive chemical to allow easy detection of the analyte of interest.23 In enzymatically coupled reactions, the products or substrates of the reaction of interest are acted upon by a second enzyme, creating a tangible readout. One advantage of using such a standardised assay format is that the development of such a method allows the activity of a family of enzymes to be monitored by a single detection method.24,25 The... 

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. 

As the enzyme itself is usually the focus of interest, information on the behavior of that enzyme can be obtained by incubating the enzyme with a suitable substrate under appropriate conditions. A suitable substrate in this context is one which can be quantified by an available detection system (often absorbance or fluorescence spectroscopy, radiometry or electrochemistry), or one which yields a product that is similarly detectable. In addition, if separation of substrate from product is necessary before quantification (for example, in radioisotopic assays), this should be readily achievable. It is preferable, although not always possible, to measure the appearance of product, rather than the disappearance of substrate, because a zero baseline is theoretically possible in the former case, improving sensitivity and resolution. Even if a product (or substrate) is not directly amenable to an available detection method, it maybe possible to derivatize the product with a chemical species to form a detectable adduct, or to subject a product to a second enzymatic step (known as a coupled assay, discussed further later) to yield a detectable product. But, regardless of whether substrate, product, or an adduct of either is measured, the parameter we are interested in determining is the initial rate of change of concentration, which is determined from the initial slope of a concentration versus time plot. 

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. 

Glucose was determined by the glucose oxidase-peroxidase method. Cellobiose (liberated enzymatically from methylcellotrioside) was determined in a coupled assay using cellobiose dehydrogenase from Sporotrichum thermophile (4). 

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

In the case of enzymes involved in biochemical pathways, the isolation is often based on activity assays. The nature of the activity assay depends on the enzymatic reaction and can involve, for example, the detection of a product on a thin-layer chromatography (TLC) plate (see Chapter 4, Section 1.2.1), the appearance or disappearance of a specific absorbance in a spectrophotometric assay, or a coupled assay involving the oxidation or reduction of a co-factor such as nicotinamide dinucleotide (NAD(H)), which can be measured by changes in fluorescence. 

In this laboratory exercise, you will study the effects of phosphorylation and allosteric regulation on the activity of glycogen phosphorylase. In the first experiment, you will phosphorylate glycogen phosphorylase b in vitro using y-[32P]ATP and glycogen phosphorylase kinase. In the second experiment, you will study the effect of phosphorylation on glycogen phosphorylase activity, as well as the effect of AMP on glycogen phosphorylase b activity with the use of a coupled enzymatic (kinetic) assay (Fig. 15-3). 

Fig. 8.3. Coupled assay for amidohydrolase activity. In the enzymatic reaction (A) an amide bond is cleaved releasing carboxylic acid and amine. The free amino group of the amine is then reacted with fluorescamine (B) in a rather fast reaction yielding fluorescent compounds. The excess of fluorescamine hydrolyzes spontaneously within seconds leaving only non-fluorescent compounds.

Potentiometric methods are less useful in metabolite assays because of the instability of the signals and the need of calibration. A special form of a potentiometric enzyme electrode, the enzymatically coupled field effect transistor is reviewed by Caras and Janata [31]. 

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... 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

When coupled with an optical microscope, CL imaging is a potent analytical tool for the development of ultrasensitive enzymatic, immunohistochemistry (IHC) and in situ hybridization (ISH) assays, allowing spatial localization and semiquantitative evaluation of the distribution of the labeled probe in tissue sections or single cells to be performed. 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

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