Assays coupled enzyme, development

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). 

Before an immobilized enzyme can be used for an industrial process, it is essential to characterize it in terms of its catalytic and kinetic properties. A quantitative assay must be developed to measure the activity, kinetic parameters, and stability of the enzyme. In a coupling reaction, H202 rapidly reacts with phenol and 4-aminoantipyrine (electron donor) in the presence of peroxidase to produce a quinoneimine chromogen (Equation E12.2, Figure El 1.2), which is intensely colored with a maximum absorbance at 510 nm. (This is the same as the product formed in the analysis of cholesterol in Experiment 11.)... 

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... 

An enzyme-linked immunosorbent assay (ELISA) was developed by Van der Schans and colleagues for detection of the mustard adduct within DNA, using monoclonal antibodies raised against N7-HETE-guanosine-5 -phosphate coupled to keyhole limpet hemocyanin (n). The ELISA was successfully applied in toxicokinetic studies in which levels of adducted DNA were followed in conjunction with measurement of intact sulfur mustard (12). 

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. 

Hariharan, J., Rane, R., Ayyanathan, K., Philomena, V. P., Kumar, V. P., Prahlad, D., and Datta, S. (1999). Mechanism-based inhibitors Development of a high throughput coupled enzyme assay to screen for novel antimalarials. ]. Biomol. Screen. 4,187-192. 

The product of the reaction of ATC oxygenase is DHTC, the last intermediate in the tetracycline biosynthetic pathway before tetracycline dehydrogenase converts it to tetracycline. A coupled enzyme system has been developed to assay conversion of DHTC (produced continuously using ATC oxygenase in vitro) to tetracycline (79.86). Tlte system has also been used to investigate the role of S -deazaflavin—the unusual co ctor involved in electron transfer for this last step in tetracycline biosynthesis (87). 

Recently, a universal enzyme-coupled fluorescence assay for glycosyl transferases was developed. This method is extremely cost-effective and is based on the quantification of nucleotides produced in the glycosyl transfer reaction. The guanosine diphosphate (GDP), uridine diphosphate (UDP), and cytidine monophosphate (CMP) are phos-phorylated with nucleotide kinase in the presence of excess of ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase,glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for the conversion of resazurin to resorufin, which is then quantified by fluorescence measurement. 

Numerous biochemical tests are suitable as detection methods. The assay may be developed in-house. In this case, sufficient know-how shotdd be available to perform and optimize the respective tests. In addition, enzyme inhibition tests are well-suited for HPLC coupling. Even bioassays (e.g., toxicity tests) shotdd be suitable for this purpose. However, in this case, the toxicity of the solvent should be considered. Evaporation might be the best solution here as well. Users with less or even no experience of biochemical assays are not necessarily excluded from their application. Eor some analytical parameters, commercial test kits are available, which are not only useful for the analysis of the respective analytes in... 

V. Bioluminescence-enhanced assay. This assay, originally developed for im-munoblot detection (20), utilizes coupled enzyme reactions initiated by APase that releases D-luciferin from D-luciferin O-phosphate (Novabiochem AG). The D-luciferin subsequently reacts with ATP and oxygen in a reaction catalyzed by luciferase (from firefly Photinus pyralis), resulting in light emission that can be detected on a sensitive photographic film (see Section D, Chapter 8). The reaction is performed in a pH 8.0 buffer. In immunoblot detections, this assay achieves a sensitivity of 5—50 pg protein (or 2—20 x 10 IgG molecules). 

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

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]. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

A wide range of coupled assays involving ATP, NAD(P)H and FMN can be developed using these two enzymes and provide increased levels of sensitivity over other coupled assays (Procedure 8.7). 

Furthermore, Meldal et al. developed the idea to combine the substrate cleavage on the solid phase with an inhibitor library assay in which a substrate is synthesized and coupled to a library of putative inhibitors (34) (Fig. 10.4). The incubation of such an inhibitor library with enzymes led to cleavage and hence fluorescence in beads containing weak inhibitors. Beads containing strong inhibitors re-... 

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