Substrate assay

Identification by nuclear magnetic 19A Chromogenic substrate assay... 

The CTAD additive mixture has found application in the monitoring of heparin therapy by either the chromogenic substrate assay or the APTT and in the measurement of platelet markers such as P-selectin (CD62) by flow cytometry (108, 109). 

The only rate-limiting factor in a coupled assay should be the concentration of the initial and linking products and all other reagents should be in excess. The role of the auxiliary and indicator enzymes is essentially that of a substrate assay system and under optimum assay conditions the rate of the indicator reaction should be equal to the rate of formation of the initial product. The indicator reaction must be capable of matching the different test reaction rates and its velocity can be defined by the Michaelis-Menten equation in the usual way ... 

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). 

Total AH = —208 kJ mol-1 Because the energy changes are so small, microcalorimetry lends itself to substrate assays in the presence of excess enzyme rather than to enzyme assays and is particularly useful when immobilized enzymes are used. 

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. 

Table 8.10 Substrate assays using enzyme electrodes...

The hbraries of enzyme substrates were obtained by spht-pool synthesis to yield one-bead-one-compound hbraries. The substrate assay was performed with a range of proteolytic enzymes such as subtilisin Carlsberg [26], cruzipain [27], protein disulfide isomerase [28-29], matrix metalloprotease MM P-9 [30], papain [31],... 

Trypsin and factor Xa (fXa) are two members of the chymotrypsin family that have 38% sequence identity on the amino acid level and have distinguishable substrate specificities. Recently, the N-terminal 13-barrel of fXa and the C-terminal /3-barrel of trypsin were fused at a rationally designed site in the linker region between the two domains in order to create a hybrid fXa-trypsin protease (Hopfner et al., 1998). The fXa-trypsin hybrid was highly active and more active than either parent on three of the ten substrates assayed, as determined by k /Km. For most substrates, the activity of fXa-trypsin was an admixture of the two parents, probably because trypsin had higher activity than fXa for all the substrates tested. 

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

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. 

For which type of substrate assay, the kinetic or the endpoint, is it necessary to maintain the substrate-analyte concentration at levels well below the Km of the enzyme used ... 

Soluble enzymes are employed in a wide variety of substrate and enzyme activity assays, and specialized instrumentation has been developed to automate reagent addition and quantitation. However, several disadvantages exist in the analytical use of soluble enzymes for substrate assays. Soluble enzymes are not reused or recycled, unless the cost of the enzyme justifies the lengthy repurification procedure. Furthermore, the activities of soluble enzymes decreases significantly with time, so that fresh assay solutions are frequently required. For these reasons, many assays that employ soluble enzymes have been adapted for use with immobilized enzymes. These immobilized enzymes are usually incorporated onto or into a stationary phase in a flow system substrate is introduced via a mobile, or buffer phase, and conversion into products occurs as the mobile phase flows through a column containing immobilized enzyme. A postcolumn detector allows product quantitation. 

SYNTHETIC PEPTIDE SUBSTRATE ASSAYS FOR HEMOSTASIS TESTING... 

Results from a substrate assay may be reported in several different ways. For example, six different ways to report the level of the thrombin inhibitor, antithrombin III, have been used (S25). One system fiimiliar to biochemists would be nanokatals (nkat) per unit of volume. A nkat is defined as the amount of enzyme which converts one nanomole of substrate per second. The second system, which would be more fiimiliar to the coagulationist, reports results in terms of percent of normal or plasma equivalent units (PEU), one PEU being the amount of a substance present in 1 ml of a standard laboratory plasma pool drawn from normal donors. This question on reporting units becomes more important when the substrate assays are substituted in the global assays, PT and PTT. The latter clot-endpoint tests measure the total effect of many fiictors and it is known that the PT and PTT are insensitive to changes in some of the fectors over a wide range, i.e., 60-120%. This point will be discussed later in Sections 6 and 7. 

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... 

Before discussing the various assays which have developed using synthetic peptide substrates, one should review briefly the basic principles for these procedures. In order to introduce substrate assays into a laboratory, a thorough knowledge of enzyme kinetics is desirable. Several excellent reviews on this subject are available (C4, S22, W4). 

---