Tos gpk pna

Manufactured by Merck Group

Tos-GPK-pNA is a substrate for the quantitative determination of trypsin-like proteases. It is a chromogenic peptide that, upon cleavage by the target protease, releases a colored para-nitroaniline (pNA) group which can be measured spectrophotometrically.

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Lab products found in correlation

L bapna, by Merck Group (2 mentions) Kyt 1, by Peptide Institute (1 mentions) Kyt 36, by Peptide Institute (1 mentions)

2 protocols using tos gpk pna

Purification and Activation of Gingipains

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Arg-X gingipains (RgpA and RgpB) and the Lys-X gingipain (Kgp) were purified from spent growth media of P. gingivalis HG66, as described previously [76 (link), 77 (link)]. The concentrations of active Rgp and Kgp gingipains were determined by active site titration using the gingipain-specific inhibitors Kyt-1 and Kyt-36, respectively (Peptide Institute, Japan) [32 (link)]. The purified enzymes were activated by 15 min incubation at 37°C in 100 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, and 20 mM cysteine (pH 7.5), and then diluted to the required concentrations in culture medium supplemented with 10 mM cysteine. Gingipain activity was inhibited by incubating cells with Kyt-1 and/or Kyt-36 (1 μM) for 15 min at 37°C. The efficiency of enzyme inhibition was verified using L-BApNA (Sigma-Aldrich) as a substrate for Arg-X gingipains and Tos-GPK-pNA (Sigma-Aldrich) for the Lys-X gingipain.

Bryzek D., Ciaston I., Dobosz E., Gasiorek A., Makarska A., Sarna M., Eick S., Puklo M., Lech M., Potempa B., Potempa J, & Koziel J. (2019). Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits. PLoS Pathogens, 15(5), e1007773.

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Quantification of Gingipain and PPAD Activities

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Gingipain activity was measured by the spectrophotometric method with specific chromogenic substrates N-(p-Tosyl)-Gly-Pro-Lys-pNA (Tos-GPK-pNA) and N_α-benzoyl-Arg-pNA (L-BApNA, Sigma). Briefly, P. gingivalis strains were grown overnight under anaerobic conditions. Cultures were adjusted to OD₆₀₀ = 1.0 and cells were collected by centrifugation (10,000 × g for 15 min). Supernatants served as cell-free culture medium samples. The detailed protocol was described previously (Pomowski et al., 2017 (link)). PPAD activity was measured in PBS-washed bacterial cells using a modified Boyde method with N-acetyl-L-arginine substrate (Bereta et al., 2019 (link); Boyde & Rahmatullah, 1980 (link)).

Luo M., He C., Toth K.S., Zhang C.X, & Lipton H.L. (1992). Three-dimensional structure of Theiler murine encephalomyelitis virus (BeAn strain).. Proceedings of the National Academy of Sciences of the United States of America, 89(6), 2409-2413.

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