Coupled assay method

In the second category of assays, the coupled assay method, activity is measured indirectly. In this method two reactions are involved. The first is the reaction of interest, such as A - B, second, the reaction that converts B to C, might be referred to an indicator reaction, not only because it uses the product of the first reaction (i.e., B) as a substrate, but also because the... 

Figure 7.20 The multivalent surface of dendrimers can be used to couple biotin groups and labels for detection in immunoassays. One such conjugate was made by coupling NHS-biotin and a maleimido-iron chelate to an amine-dendrimer for use in an unique carbonyl metallo assay method.

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. 

The viral load test quantifies viremia by measuring the amount of viral RNA. There are several methods used for determining the amount of HIV RNA reverse transcriptase-coupled polymerase chain reaction, branched DNA, and nucleic acid sequence-based assay. Each assay has its own lower limit of sensitivity, and results can vary from one assay method to the other therefore, it is recommended that the same assay method be used consistently within patients. 

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 enzyme is activated by magnesium ions, has an optimum pH of about 7.5 and offers a very sensitive assay method for ATP but has much wider applications when used in coupled assays with ATP-converting 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. 

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.

The poor accessibility of the a-nitrogen and its reduced nucleophilicity due to Np-protection requires strong N -acylating conditions. Coupling assays of Boc-Pro-OH or Fmoc-Pro-OH with H-(ZNH)Gly-OEt under various conditions show that the best yield is obtained with a symmetric anhydride or an acyl chloride.[93 The symmetric anhydride method affords Z-Protp[CO-N(NH2)]Ala-NHiPr (78) in 55% yield (Scheme 22)P1 ... 

The advantage of fluorescence-based assays is their high sensitivity. It is therefore perhaps surprising that few such systems have been developed for evaluating the enantioselectivity of enzyme-catalyzed reactions. Fluorescence as a detection method is used in an enzyme-coupled assay [26] (see Section 9.3.4.3) and in the capillary array electrophoresis [25] (see Section 9.3.6.5). If several substrates need to be screened simultaneously, fluorescence-based substrate arrays as enzyme fingerprinting tools can be used, although enantioselectivity still needs to be addressed [26e],... 

In 1926 Minot and Murphy (4) announced that whole liver was effective in the treatment of pernicious anemia. The initial assay methods, which were clinical (5), coupled with what we now know are the exceptionally small amounts of Bi2 (even in a relatively rich source such as liver) required that two more decades pass before Folkers (6) and Smith (7) in 1948 simultaneously isolated crystalline vitamin Bi2 (1, R = CN). A further decade passed before it was realized that the so-called vitamin (cyanocobalamin) was an artifact of the isolation procedure and that the enzymatically active species is the vitamin Bi2 coenzyme (5 -deoxyadenosylcobalamin, 1, R = 5 -deoxyadenosyl). This initial observation arose during Barkers study on the conversion... 

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... 

Information gained from assays and future potential So far, few field measurements have been made of caspase-like activities (Berman-Frank et al., 2004 Vardi et al., 1999), but the assay methods appear to be sensitive enough to allow use in natural communities. As work using cultures proceeds, and our understanding of cell death processes improves, assays of capase-like activity may offer an important means to distinguish different forms of cell mortality. Aside from bulk in vitro assays, the availability of ceU-permeable substrates, coupled with flow cytometry will provide improved resolution and specificity (e.g. Bidle and Bender, 2008). 

Early work demonstrated the use of enzymes coupled to antibodies or antigens as reagents in immunoassay. Enzyme activity can be measured in a variety of ways, each with certain advantages, which makes a variety of enzymes good labeling substances. Most assay methods are based on spectroscopic properties derived from an enzymatically transformed substrate. These methods are colorimetry, flurorometry, luminometry, and electrometry. 

A commercial serum creatine kinase assay8 employs the kinetic method for enzyme quantitation. This three-enzyme, coupled assay involves the following sequence of reactions ... 

Numerous photometric, fiuorometric, and coupled enzyme methods have been developed for the assay of CK activity, using either the forward (Cr —> CrP) or the reverse (Cr < CrP) reaction. Analytically the reverse reaction is preferred because it proceeds about six times faster than the forward reaction, although the cost of the starting chemicals, CrP and ADP, is greater than the cost of creatine and ATP. 

All assay methods are based on the forward ALD-catalyzed reaction. Both photometric fixed-time and continuous-monitoring procedures have been developed. In the analytical approach on which all the commonly used procedures and kits are based, the ALD reaction is coupled with two other enzyme reactions. Triosephosphate isomerase (EC 5.3.1.1) is added to ensure rapid conversion of all GLAP to DAP. Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is added to reduce the DAP to glycerol-3-phosphate, with NADH acting as hydrogen donor. The decrease in NADH concentration is then measured. 

Methods were described for the estimation of GP activity in serum on the enzymic determination of glucose-1-phosphate in a coupled assay system and for the electrophoretic separation of GP isoenzymes. More recently, an immu-noenzymometric assay for the measurement of the isoenzyme GP-BB was developed. The upper reference limit of this research assay was 7 pg/L. 

Catecholamines and Metabolites HPLC, coupled with electrochemical or fiuorometric detection, now provides the most widely used assay method for measurements of urinary or plasma catecholamines in the routine clinical laboratory. Once equipment is pur-... 

Jones DP (2002) Redox potential of GSH/GSSG couple assay and biological significance. Methods Enzymol 348 93-112... 

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