Kinetic substrate assay

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

The mode of action of inhibitors can be divided into two groups reversible or irreversible. A detailed explanation can be found in textbooks of enzyme kinetics and assays [345]. The critical aspects and kinetics of both mechanisms are summarized in Table 10.8. In brief, in all cases the presence of a substrate is a prerequisite and it has to be added at a concentration that does not hmit the reaction rate of the non-inhibited... 

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 ... 

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). 

Panteghini M, Bonora R, Pagani F. Measurement of pancreatic lipase activity in serum by a kinetic colorimetric assay using a new chromogenic substrate. Aim Clin Biochem 2001 38 365-70. 

Any analytical method should have favourable analysis efficiency, wide linear range, low cost and strong robustness. Kinetic analysis of reaction curve for Vm and So assay can have much better upper limit of linear response, but inevitably tolerates low analysis efficiency when wide linear range is required. Based on kinetic analysis of reaction curve, however, our group developed two integration strategies for enzyme initial rate and substrate assay, respectively, with both favourable analysis efficiency and ideal linear ranges. 

An excellent general coverage of lipolytic enzymes, methods of assay, and kinetics is given by Brockerhoff and Jensen (1974). Reviews of lipolytic enzymes in plants have been written by Hitchcock and Nichols (1971), Galliard (1975, 1978), and Hitchcock (1981). A useful discussion of kinetics and assays of enzymes acting on water-insoluble lipid substrates is contained in recent papers by Gatt and Bartfai (1977). 

End Point vs Kinetic Methods. Samples may be assayed for enzymes, ie, biocatalysts, and for other substances, all of which are referred to as substrates. The assay reactions for substrates and enzymes differ in that substrates themselves are converted into some detectable product, whereas enzymes are detected indirectly through their conversion of a starting reagent A into a product B. The corresponding reaction curves, or plots of detector response vs time, differ for these two reaction systems, as shown in Eigure 2. Eigure 2a illustrates a typical substrate reaction curve Eigure 2b shows a typical enzyme reaction curve (see Enzyme applications). 

Fatty Acid Transporters. Figure 2 Quencher-based real-time fatty acid uptake assay with a fluorescently labeled FFA analogue (C1-Bodipy-C12). Predominantly protein-mediated fatty acid uptake by 3T3-L1 adipocytes (diamonds) was compared with diffusion-driven uptake by fibroblasts (squares) using the QBT Fatty Acid Uptake reagent (Molecular Devices Corp., CA, USA), which contains C1-Bodipy-C12 as substrate in conjunction with a cell impermeable quencher. Uptake kinetics was recorded using a Gemini fluorescence plate reader. Error bars indicate the standard deviations from 12 independent wells. RFU relative fluorescence units. 

Literally hundreds of aldehydes have so far been tested successfully by enzymatic assay and preparative experiments as a replacement for (18) in rabbit muscle FruA catalyzed aldol additions [16,25], and most of the corresponding aldol products have been isolated and characterized. The rabbit FruA can discriminate racemic dl-(18), its natural substrate, with high preference for the D-antipode, but kinetic enantioselec-tivity for nonionic aldehydes is rather low [84,89]. 

Recently Shihabi and Bishop (93) described a refinement in the preparation of a stable substrate and demonstrated the feasibility monitoring the reaction kinetically. This procedure has been evaluated by Lifton et. al. (9 ), who found that this method correlated well (r 0.914) with the copper soap-lipase method of Dirstine. They concluded that the method was rapid (less than 5 min. per sample), accurate, precise and linear over a clinically useful range. Its simplicity allows its application as an emergency procedure. Attempts to use this assay for urine lipase activity were unsuccessful. 

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