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High Throughput Determination of Enzyme Kinetic Parameters with Microplate Photometry

Marika Raitio, Reija-Riitta Harinen and Jorma Lampinen, Thermo Fisher Scientific, Sample Preparation & Analysis, Vantaa, Finland

Determination of enzyme kinetic parameters Km and Vmax as well as inhibition constant Ki is a very common assay in modern drug discovery. This assay require by its nature concentration series of either enzyme reaction substrate or possible inhibitor, or both. Therefore, the assay is ideal to be performed in high density microplate formats where high number of samples can be easily measured. Anyhow, when one performs kinetic readings with high number of samples one easily has to sacrifice the quality of the resulting kinetic curve due to low sampling frequency of the same sample. If the time difference between two consecutive readings becomes too long, then the reaction rate calculations easily loose their accuracy. These enzyme kinetic assays should therefore be performed with an instrument that can read the plate as fast as possible. The obvious solution for this are CCD based imaging devices that can read the whole plate simultaneously, but these devices are quite expensive, therefore not very good choices for those who need this kind of capacity every now and then.
This paper shows how enzyme kinetic assays can be performed with high throughput using 384 -well microplates and affordable microplate photometers with the sampling frequency that is sufficient for the excellent result quality. The instrument used were Thermo Scientific Multiskan GO and Multiskan FC that can read the whole 384-well plate in ten seconds. The performance was tested with a common beta-galactose / ONPG enzyme reaction and it was inhibited with PETG (2-Phenylethyl β-D-thiogalactoside) or lactose. The assay were performed in standard 384 well plates and 96-well plates for the comparison.
These results show that high sampling frequency produces accurate results. Calculated kinetic parameters correlate well with the literature references and using much more expensive readers does not improve the result quality. It is also possible to reach high throughput with these low cost photometers. For example, when one does inhibition efficiency measurement it is possible to have 16 inhibitors within one 384 well plate (6 inhibitor concentrations + blank + zero inhibition control, all with three replicates). When the assays can well be performed with low volume 384 well plates using a low assay volume, it is also possible to keep the reagent consumption and costs reasonable even if one does few replicates is every assay.


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