Papain-like proteinase (PLpro) 40 μl volume of 142 nM PLpro in bufer A [50 mM HEPES, pH 7.5; 0.1 mg/ml bovine serum albumin (BSA), and 5 mM Dithiothreitol (DTT)] was dispensed in 96 well plat and then incubated with 100 μl of different concentrations of the tested compounds for 5 min. Reactions were initiated by the addition of a fluorogenic substrate, Arg-Leu-Arg-Gly-Gly-AMC (Enzo Biochem, USA) (RLRGGAMC, 10 μl of 250 μM) in buffer A, shaken vigorously for 30 s, and then incubated for 6 min. The reactions were subsequently quenched with 10 μl acetic acid (0.5 M), shaken for 30 s, and measured for fluorescence emission intensity (excitation λ: 360 nm; emission λ: 460 nm). Finally, percentage of inhibition (%) was detected.
Arg leu arg gly gly amc
Manufactured by Enzo Life Sciences
Sourced in United States
Arg-Leu-Arg-Gly-Gly-AMC is a fluorogenic peptide substrate used for the detection and measurement of enzyme activities. The substrate consists of the peptide sequence Arg-Leu-Arg-Gly-Gly coupled to the fluorogenic reporter molecule 7-amino-4-methylcoumarin (AMC). Upon cleavage by a specific enzyme, the AMC group is released, resulting in a measurable fluorescent signal.
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4 protocols using arg leu arg gly gly amc
1
SARS-CoV-2 PLpro Inhibition Assay
2
MERS-CoV PL(pro) Enzyme Activity Assay
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The enzyme activity of MERS-CoV PLpro was measured by modifying the SARS-CoV PLpro assay that we previously described15 (link). The fluorogenic peptide Arg-Leu-Arg-Gly-Gly-AMC (ENZO Life Sciences, Farmingdale, NY) and 300 nM purified MERS-CoV PLpro in 20 mM sodium acetate buffer (pH 5.5) were used as the substrate and enzyme, respectively. For inhibition studies, 300 nM MERS-CoV PLpro and 0–200 μM of the individual compounds were mixed with the substrate (50 μM) at 37 °C. The fluorescence intensity was monitored at excitation and emission wavelengths of 360 and 460 nm, respectively, on a SpectraMax M2e multimode microplate reader (Molecular Devices, Sunnyvale, CA).
3
Inhibition of SARS-CoV-2 PLpro by Biobran
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40 μL of 142 nM PLpro in buffer A (50 mM HEPES, pH 7.5; 0.1 mg/mL bovine serum albumin (BSA), and 5 mM Dithiothreitol (DTT)) was dispensed in a 96-well plate and then incubated with 100 μL of different concentrations of Biobran for 5 min. Reactions were triggered by adding the fluorogenic substrate Arg-Leu-Arg-Gly-Gly-AMC (Enzo Biochem, USA) (RLRGG-AMC, 10 μL of 250 μM) to buffer A, forcefully shaking for 30 s, and incubating for 6 min. Next, 10 μL acetic acid (0.5 M) was used to halt the reactions, the solution was shaken for 30 s, and the fluorescence emission intensity was measured (wavelength of excitation: 360 nm; wavelength of emission: 460 nm). This allowed the measurement of the inhibition percentage (%).
4
SARS-CoV-2 Protease Inhibition Assay
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SARS-CoV PL pro activity was measured by the method previously described by our group 9, 10, 22 . The inhibition assay was optimized in a 96-well plate to establish suitable assay conditions and incubation times. The fluorogenic peptide, Arg-Leu-Arg-Gly-Gly-AMC (ENZO Life Sciences, Farmingdale, NY) and 208 nM purified PL pro in 20 mM Tris-HCl buffer (pH 6.8) were used as the substrate and the enzyme, respectively. For the inhibition studies, 54 nM PL pro and 0-200 mM of the individual compounds were mixed with the substrate (50 mM) at 37 C, and the fluorescence intensity was monitored at excitation and emission wavelengths of 360 and 460 nm on a SpectraMax M 2e Multimode Reader (Molecular Devices Co., Sunnyvale, CA).
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