Assays of enzymes

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

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. 

The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is generally more difficult. In some instances, the product or remaining substrate can be transformed into a more readily detected compound. In other instances, the reaction product may have to be separated from unreacted substrate prior to measurement—a process facili-... 

ASSAYS OF ENZYME-CATALYZED REACTIONS TYPICALLY MEASURE THE INITIAL VELOCITY... 

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. 

White blood cells, red blood cells and cultured fibroblasts are commonly used to measure enzyme activities, especially for the diagnosis of inherited enzyme abnormalities. Leukocytes may be collected by sedimentation in viscous media such as Fycol. The collection of red cells presents no problem following centrifugation of anticoagulated blood. The assay of enzymes and fibroblasts requires appropriate tissue culture facilities and extensive experience in dealing with cultured human cells. 

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... 

Volume II. Preparation and Assay of Enzymes Edited by Sidney P. Colowick and Nathan O. Kaplan... 

Volume VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques... 

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. 

Colorimetric Assays Involving Redox Reactions, and (iv) Colorimetric Assays of Enzyme Levels. 

A few typical examples of colorimetric assay of enzyme levels will be discussed briefly hereunder ... 

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. 

Isolation and Assays of Enzymes or Substances Resulting in Formation or Removal of Oxygen Radicals Biological sources of Of, 105, 59 overview of superoxygenase, 105, 61 methods for the study of superoxide chemistry in nonaqueous solutions, 105, 71 generation of superoxide radicals in aqueous and ethanolic solutions by vacuum-UV photolysis, 105, 81. 

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).

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