Assay Method Information

Assay Name:  Determination of Enzyme Inhibition in SARS-CoV-2 3-Chymotrypsin-Like Protease (3CLP) Assay
Description:  A synthesized fluorescent substrate containing the cleavage site (indicated by the arrow, ↓) of SARS-CoV-2 3CLP (2-Abz-SVTLQ↓SG-Tyr(NO2) R NH2) was used for the fluorescence resonance energy transfer (FRET)-based cleavage assay. The protease reaction of SARS-CoV-2 3CLP towards fluorescent substrate was performed in activity buffer (20 mM Bis Tris, pH 7.8, 1 mM DTT) at 37 C. for 10 min. The final concentration of protease used in the assay was fixed at 80 nM and the concentrations of the substrate were varied from 0.1 to 500 μM. Reaction was started with the enzyme and the fluorescence signal of the Abz-SVTLQ peptide cleavage product was monitored at an emission wavelength of 420 nm with excitation at 320 nm, using an Flx800 fluorescence spectrophotometer (BioTek). Before kinetic calculations, it was verified that the proportionality between the fluorescence emitted and the amount of the substrate used in the assay was linear. The minimal concentration of the enzyme and time of reaction that gave a linear dependence of amount of generated product with time was chosen. Initial velocities in corresponding relative fluorescence units per unit of time (ARFU/s) were converted to the amount of the cleaved substrate per unit of time (M/s) by fitting to the calibration curve of free Aminobenzoyl-SVTLQ. All data are corrected for inner filter effects by an adopted literature protocol. In short, the fluorescence signal (RFU) at each substrate concentration was determined and defined as f(FRET). Then, 5 μL free Aminobenzoyl-SVTLQ at final 5 μM was added to each concentration and fluorescence was taken f(FRET+Aminobenzoyl-SVTLQ).
Affinity data for this assay

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