Enzymes assays

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 ... 

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

A, basically the KB for an antagonist but specifically measured in a biochemical binding study (or enzyme assay). 

Another reason is that xylo-oligosaccharides of defined structure are very important substrates that serve as model compounds for the optimization of hydrolytic processes and in enzymic assays. The enormous development... 

Reymond, J.-L. (ed.) (2006) Enzyme Assays High-Throughput Screening, Genetic Selection and Fingerprinting, Wiley-VCH, Weinheim, Germany. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

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.

Possibility of prenatal diagnosis by appropriate enzyme assays... 

Enzyme Assay. Na , K -ATPase, and sarcoplasmic reticulum Ca - ATPase were prepared from rat hearts (22) and dog hearts (23), respectively. Bovine heart cyclic AMP phosphodiesterase was purchased from Sigma. The enzyme reaction was carried out after 5-min pretreatment with the drug, and the amount of inorganic phosphate liberated during the reaction period was determined. 

Fig. 2) and b) discrete sample processing using small test tubes, in order to ensure the physical separation of each enzyme assay (i.e., the LKB-8600) (Table I). 

Temperature Control. While it was well known that enzyme catalysis is a direct function of temperature, little attention was paid to its control in kinetic enzyme assays until the pioneer work of Schneider and Willis (11). These workers showed that the temperature compartment of the Beckman DU spectrophotometer varied widely as a function of room temperature and of the number of times the cuvet compartment was opened. Thus, while most authors have assumed that they were conducting their assay at room temperature (i.e., a nominal 25 ) direct measurements showed that the cuvette temperature was closer to 32 C. Schneider and Willis suggested that thermospacers, hollow plates adjacent to each side of the cuvette compartment through which water at a constant temperature is circulated, be used in order to standardize clinical enzyme assay temperatures. 

When linked enzyme assays are used, the exogenous added enzymes may also be contaminated with small traces of the primary enzyme whose activity is measured, thereby leading to falsely high activities In this instance it is also desirable to make certain that the added enzymes are free of any undesirable activity, i.e, pig heart malic dehydrogenase should be free of GOT activity when used for GOT assays (17),... 

Product Acceptors. Many enzyme assays use acceptors, as for instance 2-ethylaminoethanol and other aminated alcohols iihich act as acceptors for the phosphoryl product of the reaction catalyzed by alkaline phosphatase (25) (Fig. 4). Hydroxylamine can act as an acceptor for the hydroxyacetone produced by eno-lase and semicarbazide can act as an acceptor for the pyruvate produced by LD. It is necessary to optimize the concentration of such an acceptor before using it routinely as often what may be a theoretically desirable acceptor is in practice superfluous. 

Dinovo, E. C. Miyada, D. S. and Nakamura, R. H. Evaluation of direct and indirect compled enzyme assay systems for measurements of creatine kinase activity. 

Russell, C. D. Cotlove, E. Serum glutamic-oxaloacetic transaminase Evaluation of a coupled-reaction enzyme assay by means of kinetic theory. Clin. Chem. (1971), 17, 1114-1122. 

Enzyme Assay The activity was assayed by the determination of substrate viscosity diminishing using Ostwald viscometer (10). The enz5une reaction was done at 37°C in 0.05 N acetate buffer pH 5.25. One unit of enzyme was defined as the amount of enzyme that could reduce the viscosity of 2% pectin by 50% in 10 min. 

Biochemical characterisation of PemB development of a double enzyme assay to measure PME activity... 

The mycelia, of six days old culture, were harvested by filtration through Whatman paper n 1 and kept at -80 C for RNA assays. The culture filtrates were used for enzyme assays. 

Extracellular PG activity was not detected in cultures on glucose, a similar situation to that of Fusarium moniliforme (18). In contrast, all our data confirm the presence of the protein and the mRNA of PG in non-inducing conditions our assays did reveal no differences between FORL PG growing on both conditions, regarding migration or other detectable characteristics that could justify the presence of the enzyme and its lack activity. It is posible that a very low concentration of the enzyme results in undetectable activity in enzyme assays. 

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