Enzymatic reactions, tests

The activity of enzymes in the film was estimated in the following way In order to test the activity of urease, we utilized a calorimetric assay based on urea hydrolysis the enzymatic reaction was followed at 590 nm, the suitable wavelength for bromcresol purple (Chandler 1982). Urea concentration was 1.67 ts 10 M. 

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

Levels of a number of metabolites as well as a number of enzymes in body fluids are indicative of disease conditions. Many of the enzymatic reactions mentioned above have been used in solution clinical assays as well as in test strips.446,497-508 512-515 Assays for hydrogen peroxide and the enzyme peroxidase using NADH and a tetrazolium salt have been de-scribed.509,5io Assays of exogenous substances (e.g., drugs or their metabolites) also utilize this chemistry. The determination of alcohol using alcohol dehydrogenase is an example.511 As mentioned above, the assay of enzyme levels can also be achieved using tetrazolium salts.516-520... 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

In the present article, we will at first briefly overview the ONIOM methodology, with an illustration of a benchmark test of a three-layer ONIOM(QM QM MM) method, which we consider a method of future. Then we will review our recent studies of biocatalysis in which we used the ONIOM(QM MM) method to examine the effects of protein environments on the mechanisms of enzymatic reactions, with an emphasis on metalloenzymes. 

D Johnson, G Amidon. The effect of enzymatic reaction on dissolution rate Theoretical analysis and experimental test. J Pharm Sci 75 195, 1986. 

In kinetic studies of enzymatic reactions, rate data are usually tested to determine if the reaction follows the Michaelis-Menten model of enzyme-substrate interaction. Weetall and Havewala [Biotechnol. and Bioeng. Symposium 3 (241), 1972] have studied the production of dextrose from cornstarch using conventional... 

One of the established methods for finding hits is high-throughput screening (HTS). This is a powerful method enabling several thousands of compounds per day to be tested. However, there are drawbacks. In many cases the assay for the detection of a hit comprises coupled enzymatic reactions, and a test compound may falsely lead to positive results because of its interference with components of the enzyme cascade. Problems may arise further from the chemical substances that make up the compound depository which are tested in HTS. Especially for companies with historically grown collections of compounds, the quality of the chemicals is an uncontrolled parameter in the sense that it is not always known whether the compounds are still unmodified they may have decomposed, precipitated or formed aggregates (polymers). 

In order to test this hypothesis, enzymatic reactions were monitored in NMR tubes. ATP (100 p.M-3 mM) and the aminoglycosides (100 tLM-1.5 mM) were dissolved in phosphate buffer (10 mM phosphate, 2.5-5.0mM MgCl2,... 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

Complex alkaloid glycosides bearing, e.g., lactosyl (Lac), lactosaminidyl (Lac-NAc), or sialyl (Neu5Ac) moieties were required for immunomodulation tests (see below). Because of the paucity of starting material (alkaloid monoglycosides) and because of the need of regioselective glycosylation, only enzymatic reactions were practicable. 

Spectrophotometric assays can be used for the estimation of the enantiosel-ectivity of enzymatic reactions. Reetz and coworkers tested 48 mutants of a lipase produced by epPCR on a standard 96-well microtiter plate by incubating them in parallel with the pure R- and S-configured enantiomers of the substrate (R/S-4-nitrophenol esters) [10]. The proceeding of the enzyme catalyzed cleavage of the ester substrate was followed by UV absorption at 410 nm. Both reaction rates are then compared to estimate the enantiomeric excess (ee-value). They tested 1000 mutants in a first run, selecting 12 of them for development of a second generation. In this way they were able to increase the enantiomeric excess from 2% for the first mutants to 88% after four rounds of evolutive optimization. 

For a semiquantitative assay, cut each dot and put it into separate test tubes. Perform the enzymatic reaction with TMB substrate, stop with sulfuric acid and read O.D. at 450 nm. (This procedure is similar to an EIA on microtiter plates.)... 

It is stated that during an in vitro enzymatic reaction the concentration of the enzyme shall not change during the test, and that the substrate concentration exceeds the enzyme concentration in orders of magnitude in a first approximation the substrate concentration is practically constant, too. Both of these assumptions transform a reaction of 2" order into the much simpler reaction of 0 order. If the concentrations of enzyme and substrate are similar, we get a reaction of order. The reaction rate v for the association reaction... 

Both in nature and in test tubes many complicated reactions take place with apparent ease. Highly specific enzymatic reactions involving complementarity of substrate and receptor both with respect to their spatial structure and electrostatic fields lie outside the scope of this book. Here we would like only to mention a few... 

Expression of the catalytic capacity of the immobilized laccase was also observed in more than a dozen different solvents, provided that they were either saturated with water or, in the case of solvents miscible with water, small amounts of water had been added (Table III). No enzymatic reaction was observed when the solvents tested were free of water. No correlation was found between the activity of the immobilized laccase and the hydrophobicity of the solvent in which the reaction took place. The rate of laccase reaction in ethylacetate was only twice that in toluene, despite the fact that water-saturated ethylacetate contains 50 times more water than... 

As the next enzymes of the heme pathway are active, uroporphyrinogen is converted to heptacarboxyporphyrinogen during the enzymatic reaction. This can be overcome if the incubation temperature is raised to 45°C, which inactivates uroporphyrinogen decarboxylase. Yet, 37°C is the standard temperature for enzyme activity determinations. Despite our neglect of the additionally formed porphyrins by uroporphyrinogen decarboxylase in the activity calculations, we found a highly reliable test performance. 

Color tests have several advantages, including the possibility of visual prescreening on micro titer plates. Moreover, if a reliable UV/visible signal arises in consequence of an enzymatic reaction, commercially available (and fairly inexpensive) UV/visible plate readers can be used to screen thousands of mutant enzymes. 

With chemical model reactions, valuable insights can be gained about enzymatic reactions and their mechanisms. In most cases, modeling of the active center is the main target, so that simple model compounds for the decisive groups in and around the active site are sought, with which mechanism, substrate breadth, or other reaction parameters can be tested. 

The second property in particular distinguishes indicator dyes from colorforming reagents. The latter are widely used in reactions in which colors are formed by chemical or biochemical (mostly enzymatic) reactions between the analyte and added reagents. These reactions are widely used in analytical and diagnostic test kits but are normally not reversible. 

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