Substrate assay methods

Enzymes are extremely useful analytical tools, primarily because of their specificity. Currently a very large number of enzymes are available commercially in various forms of purity and, although their cost is often high, only small amounts are usually required. Procedure 8.8 describes a fixed time method for the quantitation of D-alanine using D-amino acid oxidase. 

Procedure 8.8 Spectrophotometric determination of D-alanine using n-aniino acid oxidase 

Test reaction — catalysed by D-amino acid oxidase 

Glutamate dehydrogenase D-amino acid oxidase Standard solution D-alanine 

Into the cuvettes pipette 2.9 ml of this mixture and inomtoi the ah.xorbance for several minutes to ensure a steady reading or at least a very slow fall in absorbance. 

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Important to the performance of substrate assays is the problem of availability of well-defined enzymes fiir standardization purposes. The noted variability within and among the currently available commercial rei ents fiir thrombin. Factor Xa, and kallikrein as well as the observation that denatured or altered enzymes lose more of their biological clotting activity than their amidase potency illustrates this problem. Hemker has pointed out two examples of this latter concern vidiere an apparently fiilse higher level of activity was indicated by the substrate assay method (H3). With thrombin it... 

Assay method Natural substrate Rapid and simple Sensitive and accurate Use with variety of headgroups ... 

Bioluminescence provides the basis for sensitive enzymic assay methods both for substrate assays and coupled enzyme assays. Firefly luciferase (EC 1.13.12.5) catalyses the production of light (540-600 nm) by the oxidation of luciferin (d-LH2) (Figure 8.18). 

There are many instruments designed for either enzyme assays or substrate assays using enzymes. Information on the analytical capabilities of these instruments will be supplied by the manufacturers. This will often include protocols for specified assays using kits of commercially available pre-prepared reagents. These may be in liquid or dry form and may, for substrate assays, include immobilized enzymes. The facility to be able to develop additional automated methods on a particular instrument will depend upon its design and some instruments are dedicated solely to specified analyses. 

It is possible to bind enzymes to an insoluble matrix by a variety of methods and still retain their catalytic activity. The reusable nature of immobilized enzymes can significantly reduce costs and provides a convenient source of enzymes for performing substrate assays. Such preparations often show a greater stability and reduced inhibition effects than do soluble enzymes, although occasionally optimum pH values may be altered slightly. 

Immobilized enzymes may be used in affinity chromatographic methods but their use as catalysts may be in either the production or removal of compounds in chemical processes or as analytical tools. Many substrate assays can be performed using enzymes immobilized on a variety of surfaces, e.g. glass beads, plastic or nylon tubing. Alternatively they may be incorporated into gel or microparticulate layers on dry strips or slides. 

Substrate determinations (Table 8.10) using enzyme electrodes must be performed under controlled conditions of temperature and pH and if standard solutions are used to calibrate the electrode they must be analysed at the same time. The response time of some enzyme electrodes may be several minutes and although for many the time is shorter this factor must be considered in the design of an assay method. 

Practical Considerations. Typical absorption assay methods utilize ultraviolet (UV) or visible (vis) wavelengths. With most spectrophotometers, the measured absorbance should be less than 1.2 to obtain a strictly linear relationship (/.c., to obey the Beer-Lambert Law). Nonlinear A versus c plots can result from micelle formation, sample turbidity, the presence of stray light (see below), bubble formation, stacking of aromatic chromophores, and even the presence of fine cotton strands from tissue used to clean the faces of cuvettes. One is well advised to confirm the linearity of absorbance with respect to product (or substrate) concentration under the exact assay conditions to be employed in... 

The velocity of an enzyme-catalyzed reaction can be measured either by a continuous assay or by a stopped-time protocol. Whenever possible, the continuous measurement of a velocity (e.g., the increase or decrease in absorbance vx. time) should be utilized. In stopped-time assays, the investigator must demonstrate that the reaction is completely terminated at the specified point in time and that products are readily and quantitatively separated from substrates. In addition, one must show that the system is under initial rate conditions. Thus, at least three or four different time points should be chosen. Stopped-time assays also require an assay blank (for t = 0). In this blank, typically the quenching conditions are applied prior to the initiation step. Whenever practicable, replicate kinetic analyses should be done, even with continuous assay protocols. See Enzyme Assay Methods Basal Rate... 

MANOMETRIC ASSAY METHODS MAPPING SUBSTRATE INTERACTIONS USING KINETIC DATA MARCUS EQUATION MARCUS RATE THEORY MARKOV CHAIN Markovnikoff rule,... 

The unit is micromol/minute/milligram of protein. The commentary may contain information on specific assay conditions or if another than the natural substrate was used in the assay. Entries in Additional Information are included if the units of the activity are missing in the literature or are not calculable to the obligatory unit. Information on literature with a detailed description of the assay method may also be found. 

The most sensitive assay methods available involve the use of radioactively labeled substrates and reaction volumes of 20 to 100 piL. 

The various applications of these and several other principles have been reviewed elsewhere (67, 68). The assay methods, primarily designed for benzylpenicillin, the standard substrate, are generally suitable for other substrates, although in some cases the procedure has to be modified. Specific problems involved in the replacement of benzylpenicillin with other substrates, especially cephalosporins (69), have been recently discussed (2, 31, 32, 70). 

The most sensitive assay method for PE activity is to use, as substjgte, pectin esterified biologically (31) or chemically (32) with C methanol. In studies by Gessner (29) and Bartolome and Hoff (30) the substrate and enzyme were precipitated from the reacj on mixture with acidified ethanol or TCA before activity of the C methanol in the supernate was determined. The radioactive assay method was about 100 times as sensitive as the titrimetric method for PE activity (32). 

A fibrin clot containing adsorbed plasmin inhibitors is difficult to define in a chemical or physical sense. Generally, when enzyme reactions occur at surfaces, the porosities and adsorption properties erf which are variable, the reproducibility of enzyme assay methods is questionable. The proteinoses, to which belong the most important pharmaceutical enzymes, may present some difficulties when natural substrates (protein ) are prescribed. Here, the application of a parallel run with a reference standard is recommended. 

For enzyme inhibition assays, urine is the preferred specimen [4]. Interestingly, Bik can be measured by the inhibition of trypsin in urine but not in plasma. Urinary Bik analysis may also be performed by antibody staining, latex agglutination, and radioimmunoassay (RIA) [4]. Despite the analytical approach used, all Bik forms are measured together. The enzyme inhibition method involves adding known amounts of trypsin to the specimen and monitoring trypsin inhibition. Trypsin activity is assessed by detection of by-products from a cleavable substrate. Dipstick methods are available for the rapid detection of trypsin inhibitors in urine [15, 17 19]. 

Israel and Lesbats reported a chemiluminescence method for the determination of acetylcholine, and continuous detection of its release from torpedo electric organ synapses and synaptosomes [55], Birman described a new chemiluminescence assay method for the determination of acetylcholineesterase activity with the natural substrate [56], The method involved monitoring the increase in light emission produced by accumulation of choline, or measuring the amount of choline generated. 

Method Substrate Assay components/ Conditions Measurement Applications References... 

The properties of 7a-hydroxylase from pigeon liver microsomes305 and from rat liver306,307 have been further described, and new assay methods are available.308,309 Free cholesterol, rather than a cholesteryl ester, was the preferred substrate for the enzyme from rat liver microsomes,310 and the substrate pool for the hydroxylase was about one third of the total amount of cholesterol present in the microsomal preparation.309 Cholesterol 7a-hydroxylase activity is more sensitive to thyroid function than are the activities of the enzymes responsible for cholesterol synthesis,311 and (22f )-22-aminocholesterol, although having no effect on serum or liver cholesterol levels in rats, drastically reduced 7a-hydroxylase activity.312... 

Cholinesterase Activity. Cholinesterase activity was assayed by automatic, continuous, alkali titration of acid released from the substrate, a method previously utilized by Wilson (12), Main and Dauterman (6), Shellenberger et al. (8), and others. 

In the second category of assays, the coupled assay method, activity is measured indirectly. In this method two reactions are involved. The first is the reaction of interest, such as A - B, second, the reaction that converts B to C, might be referred to an indicator reaction, not only because it uses the product of the first reaction (i.e., B) as a substrate, but also because the... 

Figure 1.2 The assay of an enzymatic activity by the continuous assay method. In the illustration, the reaction mixture is transferred to a cuvette, which is shown in place in the light path of the spectrometer. The addition of the enzyme directly to the cuvette initiates the reaction. Product formation results in a change in absorbance, which is monitored continuously by the detector. This change signals a deflection on a recorder. Note that product formation requires neither termination of the reaction nor separation of the substrate from the product.

The discontinuous method measures activity by separating the product from the substrate. Assays characteristic of this group usually require two steps, since separation often does not include detection. Thus, first, the substrate and the product are separated, and usually the amount of product formed is measured. Assays that use radiochemical substrates are included in this group, since radiochemical detectors are unable to differentiate between the radiolabel of the substrate and that of the product. Examples of enzymes whose assay methods fall into this category are legion, and these approaches characterized by a separation step. 

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