Mca avlqsgfr lys dnp lys nh2

Manufactured by GL Biochem

Sourced in China

MCA-AVLQSGFR-Lys(DNP)-Lys-NH2 is a synthetic peptide used in various research applications. It serves as a substrate for enzymatic assays and can be utilized in the development and testing of analytical methods.

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Lab products found in correlation

Fluoroskan ascent, by Thermo Fisher Scientific (1 mentions) Gc376, by Targetmol (1 mentions)

5 protocols using mca avlqsgfr lys dnp lys nh2

Measuring SARS-CoV-2 3CL Protease Activity

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The in vitro biochemical activity of the SARS-CoV-2 3CL protease was measured as previously described^{5 (link)}. The fluorogenic peptide MCA-AVLQSGFR-Lys(DNP)-Lys-NH2, corresponding to the nsp4/nsp5 cleavage site in the virus, was synthesized (GL Biochem), then resuspended in DMSO to use as the substrate. Different concentrations of this substrate, ranging from 5 μM to 100 μM, were prepared in the assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA) in a 96 well-plate. The protease was then added to each well at a concentration of 0.2 μM, and then fluorescence was continuously measured on a plate reader for 3 min. The catalytic efficiency of the protease was then calculated by generating a double-reciprocal plot.

Iketani S., Forouhar F., Liu H., Hong S.J., Lin F.Y., Nair M.S., Zask A., Huang Y., Xing L., Stockwell B.R., Chavez A, & Ho D.D. (2020). Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors. bioRxiv.

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SARS-CoV-2 Protease Activity Assays

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The enzyme activity assays was performed as described previously [32 (link)]. Briefly, the protease activity of 3CLpro of SARS-CoV-2 virus was measured by a continuous kinetic assay, with the substrate MCA-AVLQSGFR-Lys (Dnp)-Lys-NH2 (GL Biochem, Shanghai), using wavelengths of 320 nm and 405 nm for excitation and emission, respectively. Similarly, the protease activity of PLpro of SARS-CoV-2 virus was also measured with the substrate Ubiquitin-AMC.

Lin C., Tsai F.J., Hsu Y.M., Ho T.J., Wang G.K., Chiu Y.J., Ha H.A, & Yang J.S. (2021). Study of Baicalin toward COVID-19 Treatment: In silico Target Analysis and in vitro Inhibitory Effects on SARS-CoV-2 Proteases. Biomedicine Hub, 6(3), 122-137.

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Enzymatic Assay of CoV M^pro Activity

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Enzymatic assay was performed using a fluorogenic substrate with consensus sequence of CoV M^pro, MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 (>95% purity, GL Biochem Shanghai Ltd., Shanghai, China), as previously reported48 (link)49 (link)53 (link). Fluorescence intensity was monitored using a Fluoroskan Ascent instrument (Thermo Scientific, USA) with excitation and emission wavelengths of 320 nm and 405 nm, respectively. The assay was performed in a buffer solution consisted of 50 mM Tris-HCl (pH 7.3) and 1 mM EDTA at 30 °C. Kinetic parameters, including K_m and k_cat of apo HCoV-NL63 M^pro and K_i and k₃ of inhibitor N3, were determined using methods described in detail in our previous work49 (link).

Wang F., Chen C., Tan W., Yang K, & Yang H. (2016). Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design. Scientific Reports, 6, 22677.

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Kinetic Analysis of SARS-CoV-2 3CL Protease

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The in vitro biochemical activity of the SARS-CoV-2 3CL protease was measured as previously described⁵. The fluorogenic peptide MCA-AVLQSGFR-Lys(DNP)-Lys-NH2, corresponding to the nsp4/nsp5 cleavage site in the virus, was synthesized (GL Biochem, Shanghai, China), then resuspended in DMSO to use as the substrate. Different concentrations of this substrate, ranging from 5 to 100 µM, were prepared in the assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA) in a 96 well-plate. The protease was then added to each well at a concentration of 0.2 µM, and then fluorescence was continuously measured on a plate reader for 3 min. The catalytic efficiency of the protease was then calculated by nonlinear regression (GraphPad Prism, GraphPad Software, San Diego, CA, USA). For calculations, a 100% active enzyme was assumed.

Iketani S., Forouhar F., Liu H., Hong S.J., Lin F.Y., Nair M.S., Zask A., Huang Y., Xing L., Stockwell B.R., Chavez A, & Ho D.D. (2021). Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors. Nature Communications, 12, 2016.

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Protocol for Inhibitor Evaluation

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Geraniin, nirmatrelvir (PF-07321332, PF-332), and GC-376 were purchased from TargetMol (Shanghai, China). The FRET substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2), MCA-AVLQ peptide, and FP probe (FITC-AVLQSGFRKK-Biotin) were chemically synthesized (GL Biochem Shanghai Co. Ltd., Shanghai, China) , and the purity was more than 95.0%.

Zhang R., Yan H., Zhou J., Liu X, & Chen Y. (2024). Invalidation of geraniin as a potential inhibitor against SARS-CoV-2 main protease. Natural product research, 38(12).

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