Substrate 1 was dissolved at a concentration of 1.0 mM in DMF containing TFA (0.1% v/v). Fluorescence of the EDANS moiety was measured on a M1000 Pro plate reader from Tecan (Maennedorf, Switzerland) by excitation at 340 nm and observation of emission at 490 nm. A fluorophore calibration was performed to enable quantitation of assay data. The product exhibits a fluorescence of 70 RFU/nM at a gain setting of 216, and all assays were performed at this gain setting unless indicated otherwise. Assays were performed in a Corning black, flat bottom, non-binding surface, 96-well plate. Assays were conducted at room temperature in 200 μL of 50 mM sodium acetate buffer, pH 5.0, containing NaCl (0.10 M), DMF (2% v/v), substrate 1 (1–40 μM), and HIV-1 protease (25 pM–6.5 nM). Assays with 30 and 40 μM of substrate 1 required 3% and 4% v/v DMF, respectively. Inhibition assays were conducted with picomolar–nanomolar inhibitor (depending on the enzyme concentration and Ki value) and 10 μM substrate 1 . Inhibition assays were monitored for until ≤7% of the substrate was converted to product. Initial velocities were measured in quadruplicate.
Solution concentrations of HIV-1 protease (10.7 kDa) was determined by measuring the absorbance at 280 nm and estimating the extinction coefficient as 12,500 M−1cm−1 with software from ExPASy28 (link). The fraction of active enzyme was determined by active-site titration using darunavir as the titrant and found to be 76% with respect to the value based on the A280 nm. Fluorescence was monitored over the linear range of the detector, which corresponds to 700 nM of product formation at a gain setting of 216.
Solution concentrations of HIV-1 protease (10.7 kDa) was determined by measuring the absorbance at 280 nm and estimating the extinction coefficient as 12,500 M−1cm−1 with software from ExPASy28 (link). The fraction of active enzyme was determined by active-site titration using darunavir as the titrant and found to be 76% with respect to the value based on the A280 nm. Fluorescence was monitored over the linear range of the detector, which corresponds to 700 nM of product formation at a gain setting of 216.
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