Assays of enzyme activity

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. 

Urine may be collected for assays of enzyme activities following cleansing of the genitalia with mild antiseptic soap followed by rinsing with water. The urine is collected in a chemically clean container with no preservative. As the activity of urinary enzymes is a function of the volume of the specimen it is important to time the collection accurately. A collection period of 8 hours is quite adequate, and the use of longer periods is not desirable because enzyme activities can rapidly decrease in the relatively hostile medium of the urine. The urine should be refrigerated and transferred promptly to the laboratory, where it should also be processed promptly. 

Delays of more than two hours after the collection is completed may lead to markedly erroneous results. Urines with bacterial overgrowth, hemolyzed blood, or those obtained following any type of instrumental examination of the urinary tract may also lead to erroneous results, and should not be used for assays of enzyme activities. The stability of some clinically important enzymes in the various body fluids is given in Table IV. 

Galactose tolerance tests have also been used in attempts to detect heterozygous carriers for galactosemia (D5, H6) this is dealt with in Section 5.3.1. Some heterozygotes are made ill by the galactose load (D3, H3, H16). Galactose tolerance tests have now been replaced, for this purpose, by the more specific and sensitive method of assay of enzyme activity. 

O Figure 4-2a shows theoretical output from a continuous platereader assay of enzyme activity at five substrate concentrations between 1 and 15 pM. It is clearly apparent that the rate of product formation... 

In assays of enzyme activities a cofactor, but not a prosthetic group, can be easily lost from the enzyme by dilution during extraction or purification, or removed by agents that will bind the cofactor. For these reasons, an excess of cofactor is routinely added to the assay medium (e.g. in kinase assays) for the measurement of enzyme activity. 

The methods of gel synthesis, immobilization of monomer conjugated enzyme, assay of enzyme activity, and determination of gel water content have been published elsewhere (4,5). A schematic of the synthesis is shown in Fig. 1. The gel compositions are identified as NA-100" (100% NIPAAm), "NA-95" (95% NIPAAm, 5% AAm), NA-90 (90% NIPAAm, 10% AAm) and "NA-85" (85% NIPAAm, 15% AAm) all are based on mole percents of monomers. Total monomer concentration was always 1.75 M. The experiment to determine the temperature dependence of enzyme activity was carried out after the enzyme reversibility experiment. 

B. Most assays of enzyme activity depend on the assumption that very little of the substrate, S, has been converted into product, P, at the time of measurement. 

Aspartate a-decarboxylase 753, 755 Aspartate p-decarboxylase 746 Aspartate racemase 741 Aspartic acid (Asp, D) 52, 53s biosynthesis 517 pXa value of 293, 487 Aspartic proteases 621-625 Aspartyl aminopeptidase 621 p-Aspartyl phosphate 539, 540s Assays of enzyme activity 456 Assembly core of virus shell 365 Assembly pathway... 

Above pH 6 the release of p-nitrophenyl phosphate from synthetic substrates (Table I) results in a shift in the ultraviolet spectrum of the chromophore which may be followed spectrophotometrically (Fig. 3). This forms the basis for a sensitive and convenient assay of this enzyme (61). Perhaps the substrate best suited for routine assay of enzymic activity is nitrophenyl-pdTp, since it has highly favorable kinetic constants. 

Details of experimental methods are not included. Original papers or appropriate books (2, 3) should be consulted for methods of enzyme preparation and for assays of enzymic activity. For earlier surveys the chapters by G. Schmidt and M. Laskowski, by H. G. Khorana, and by C. F. Anfinsen and T. H. White, Jr., in the second edition of The Enzymes, Vol. 5, and specialized monographs (1, 4) should be consulted. 

Figure 3.2 Condensation of genomic libraries and example assay of enzyme activity (Robertson, 1996).

The land of substrate used must give a relevant specificity to the reaction catalyzed, and this may intlunice the reaction tale itsett as well as the optimal pH value and ionic arength, Whenever possible the substrate should be a pure, well-defined chemical entity. This also means that for a quantitative assay of enzyme activity, the highest substrate purity should be required as well as precise control of the external factors. 

The combination of horizontal slab-gel electrophoresis and in situ assay of enzyme activity is a versatile and powerful method for detecting protein variants (isozymes and allozymes) in plants. Such variants are potentially useful as genetic markers for mapping chromosomes and in studies of breeding systems,1 population structure,2,3 gene flow,4 polyploidy,5,6 and systematics.7-9... 

Sugars labeled with tritium have been used for the assay of enzyme activity where no other method exists. For instance, UDP-2-acet-... 

Strategy for Design of an HPLC System for Assay of Enzyme Activity... 

STRATEGY FOR DESIGN OF AN HPLC SYSTEM FOR ASSAY OF ENZYME ACTIVITY... 

Figure 9.96 Separation and determination of 2 (3 )-AMP. Assay conditions sample, 1 /xL column, 100 mm X 0.5 mm, silica gel-NH2 (ODS-C 8 Merck LiChrosorb NH2) mobile phase, 8 /xL/min range, 0.16 (absorbance) = 100 chart speed, 1 mm/min. (A) Assay of standard solutions. (B) Assay of enzyme activities. Enzyme Rat cerebral homogenate (9.5 yxg protein). Substrate 20 mM 2, 3 -cAMP. Preincubation 5 minutes at 37°C. Incubation 37°C for 5,10,15, and 20 minutes. (From Tsukada et al., 1980.)...

For disorders characterized by an underlying enzyme deficiency (e.g., Gaucher disease, Fabry disease, Tay-Sachs, Hurler syndrome), assays of enzyme activity in blood and/or tissues is generally available (Meikle et al., 2004). Mutation analysis is also available, particularly for populations in whom the common disease alleles are known (e.g., mutations among Ashkenazi Jews for Gaucher, Tay-Sachs, Niemann-Pick type A, and mucolipidosis type IV Ostrer, 2001). In other cases, analysis of the gene defect responsible for rare subtypes is available through specialized laboratories. 

Place 1 qt methanol (tightly capped) in a - 20°C freezer overnight. 10-4. Place at least 2 liters glass-distilled water at 4°C overnight. Figure 10-17 is a schematic flow chart of the operations involved in partial purification of acid phosphatase. Supernatant solutions enclosed in rectangles on the flow chart are to be sampled for later assay of enzyme activity and protein concentration. 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

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