However, for a distinct method
and its detection range, the required enzyme amount can be estimated. This will be demonstrated with the example of the UV/visible spectroscopy. The authentic absorption range is between 0 and 1, while for higher absorptions the Lambert–Beer law is no longer valid. To determine the initial velocity of an enzyme reaction, e.g. of a dehydrogenase, an absorption range of 0.1 is sufficient, and higher absorptions will easily exceed the linear phase of the progress curve. So an enzyme amount producing an absorption difference of 0.1/min will be convenient. The absorption coefficient of NADH at 340 is 6300 M−1 cm−1, 1 µmol NADH per ml has an absorption of 6.3; 0.016 µmol NADH/ml show an absorption of 0.1. To convert 0.016 µmol NADH/min in 1 ml assay mixture 0.016 IU Ibrutinib supplier respectively 0.27 nkat enzyme are required. Due to the divergent features of enzymes a general standardization of enzyme assays is not possible, rather special rules can be given as follows: 1. pH: Preferentially
the pH of the pH optimum of the respective enzyme is chosen, as far as possible at or near the physiological pH (~7.5). The author has no conflict of interest. “
“Developing sensitive enzyme assays suitable for high-throughput screening (HTS) requires identification of relevant enzyme and substrates forms, methods in purification, careful measurements of kinetic parameters, characterization of co-factors, buffers, and choice of a detection technology for the final HTS assay. find more The desired mode of action (e.g. allosteric, competitive, slow-binding ADAM7 inhibitors) for active compounds should also be considered in the assay development process. In the first part of this review we define the goals of an HTS
enzyme assay and provide an overview of the key steps in this process. In the second part we give an overview of specific technologies that have been employed to measure activity for various enzyme classes in a high-throughput setting. As well, we discuss the critical parameters that should be conveyed when reporting HTS enzyme assay data. In general, cell-free HTS assays for enzymes have been developed using three main approaches (Figure 1). These are (1) detection of substrate depletion, (2) detection of product formation and (3) detecting direct binding of a ligand to the enzyme. Methods for measuring the E·S complex, although available for many years using fast kinetic readers (Lobb and Auld, 1979), have not transitioned into HTS. For some well-explored enzyme families such as protein kinases all three methods are available and the choice of which assay to use will depend on biases towards a particular detection technology, reagent expense, the amount of enzyme required and ease of implementation within the laboratory. These considerations are discussed below along with the goals of HTS enzyme assays.