Enzyme assay spectrophotometric

Butler, E. G., Gfll, R., and Garych, L. (1988). A semiautomated system for measurement of 96 simultaneous spectrophotometric enzyme assays. Anal. Biochem. 170, 402—408. 

Table 8.3 Examples of kinetic spectrophotometric methods of enzyme assay... 

Enzyme Assays. Procedures for the HPLC assay of prephenate aminotransferase (32), the spectrophotometric assay of shikimate dehydrogenase... 

For example, when a heart attack occurs, a lack of blood supplied to the heart muscle causes some of the heart muscle cells to die. These cells release their contents, including their enzymes, into the bloodstream. Simple tests can be done to measure the amounts of certain enzymes in the blood. Such tests, called enzyme assays, are very precise and specific because they are based on the specificity of the enzyme-substrate complex. If you wish to test for the enzyme lactate dehydrogenase (LDH), you need only to add the appropriate substrate, in this case pyruvate and NADH. The reaction that occurs is the oxidation of NADH to NAD+ and the reduction of pyruvate to lactate. To measure the rate of the chemical reaction, one can measure the disappearance of the substrate or the accumulation of one of the products. In the case of LDH, spectrophotometric methods (based on the light-absorbing properties of a substrate or product) are available to measure the rate of production of NAD+. The choice of substrate determines what enz)rme activity is to be measured. 

The enzyme catalyzes the incorporation of one atom of oxygen into imidazoleacetate, and the accumulation of imidazoloneacetate was observed. The reaction product was unstable and decomposed spontaneously. NADH (nicotinamide adenine dinucletotide hydrate) was consumed as a hydrogen donor. The enzyme assay was carried out either by the spectrophotometric measurement of NADH oxidation or... 

CDs also can be used as stabihzersfor color indicators to increase the stability of indicators used for the spectrophotometric determination of hydrogen peroxide in body fluids. In addition, CDs and their derivatives also have applications in enzyme assays and enzyme activity measurement. For example, glucosyl- or maltosyl-a-CD have been used to increase the accuracy and sensitivity of the assay of amylase. 

Sugar nucleotides 1-3 were found to act as donors for the a(l- 4)FucT. Their relative rates of reaction were estimated using a novel spectrophotometric coupled enzyme assay (22) which correlated the rate of production of GDP (equation I) with the rate of glycosyl transfer. The results, presented in Figure 3, show that 3-deoxy-L-fucose and D-arabinose were transferred at 1.7 and 6.0%, respectively, of the rate for L-fucose. The structures of the products were identified by an ELISA assay and scaled-up reactions ( 2 mg) allowed the product trisaccharides to be characterized by NMR (22)- 7 he natural Lewis-a trisaccharide determinant as well as two of its analogs could thus be prepared enzymatically. 

The crude fish enzyme extracts were prepared as per the method of Baranowski et al. (1984), and stored in ice for use in the pressure treatments and subsequent enzyme assays. The spectrophotometric methods of Hummel (1959) and Erlanger et al (1961) were used to assay for chymotrypsin-like and trypsin-like enzyme activities using BTEE and BAPNA as substrates, respectively. Cathepsin C activity was assayed using gly-phe-NA as substrate (Lee et al, 1971), and collagenase activity in the fish extracts were assayed as per the method of Wunsch and Heidrich (1963). Protein content of the crude enzyme extracts from fish was determined by the method of Hartree (1972). 

In an enzymatic assay, spectrophotometric or electrochemical determination of the reactant or the product is the preferred approach. When this is not applicable, the determination is performed by a coupled enzyme assay. The coupled reaction includes an auxiliary reaction in which the food constituent is the reactant to be converted to product, and an indicator reaction which involves an indicator enzyme and its reactant or product, the formation or breakdown of which can be readily followed analytically. In most cases, the indicator reaction follows the auxiliary reaction ... 

Enzyme assays of the solutions containing unbound human neutrophil elastase were conducted in pH 7.6 buffer composed of O.IM sodium phosphate, 0.5 M NaCl, and 3.3 % DMSO and subjected to spectrophotomeric measurement of the release of p-nitroaniline at 410 nm from the enzymatic hydrolysis of MeOSuc-Ala-Ala-Pro-Val-pNA (Sigma) (9). The spectrophotometric kinetic assays were performed in a Bio-Rad Microplate Reader (Hercules, CA) with a 96-well format. Two hundred microliter iquots of a elastase solution (0.2 units) were assayed per welt, and 20 microliters of a 475 micromolar substrate solution was added to initiate the enzyme reaction. 

The physicochemical properties of the reactants in an eiKyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the abihty of a substrate or product to absorb hght. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb hght at a wavelength of 340 run, whereas their oxidized forms NAD(P) do not (Figure 7—9). When NAD(P)+ is reduced, the absorbance at 340 run therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 run will be observed. In each case, the rate of change in optical density at 340 nm will be proportionate to the quantity of enzyme present. 

AE catalyses the cleavage of acetyl groups from different substrates. The enzyme activity was determined by measuring the release of acetic acid. The amount of acetic acid was measured spectrophotometrically using an acetic acid analysis kit (Boehringer, Mannheim). The activity of AE was measured in 0.6% sugar beet pectin solubilised in 25 mM Na-succinate pH 6.2 and incubated with enzyme fraction in total 500 nl assay. The samples were incubated at 40°C and aliquots were examined after 0, 1, 2 and 3 hours of incubation. The enzyme reaction was stopped by incubating the samples at lOO C for 5 min. Precipitated... 

Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HCl buffer (pH 8.0) with ImM CaCl2, were incubated at 37 C for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms Ipmol of 4,5-unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M cm [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37°C. Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HCl buffer (pH 8.0) with 0.5 mM CaCl2. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min under the conditions of reaction. Specific viscosity (n p) is (t/to)-l, where t is the flow time (sec) of the reaction mixture and t is the flow time of the buffer. The inverse pecific viscosity (n p ) is proportional to the incubation time and the amount of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. 

Fig. 20c. 1. ELISA assay, (a) Antibodies to the drug of interest are secured to a solid substratum such as a test tube or micro-well plate. The sample containing the analyte antigen is added to the reaction surface, (b) After the analyte has bound to the antibody, the vessel is rinsed to remove unbound antibody. A second antibody to the analyte is added. This antibody has a bound enzyme which has been chosen because its reaction produces a colored product which can be detected spectrophotometrically. (c) After this second antibody has bound to the first antibody-antigen complex, the surface is again rinsed to remove unbound-antibody enzyme. The enzyme substrate is added in sufficient excess such that the rate of product formed is proportional to the amount of enzyme present. The enzyme-linked assays are very sensitive, since each enzyme can rapidly catalyze thousands of substrate to product reactions.

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

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