Redox process is crucial for inhibitory properties of aurintricarboxylic acid against activity of YopH: virulence factor of Yersinia pestis

Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Niedzialkowski, Pawel; Gorska, Magdalena; Tuszynski, Jack A; Ossowski, Tadeusz; Wozniak, Michal

Bacterial recombinant YopH protein tyrosine phosphatase from Yersinia pestis was obtained from Millipore and YopH from Yersinia enterocolitica was obtained from Calbiochem. Human recombinant CD45 was obtained from Sigma-Aldrich. The solutions of the recombinant PTPs were prepared in 10 mM HEPES buffer pH 7.4. The final concentration of phosphatase in reaction samples was 0.8 μg/mL (10 nM). The YopHs and CD45 enzymes were untreated (control) or treated with solution of ATA and peroctanoic acid. The assay was performed in 96-well microplates, and the final volume of each sample was 200 μL. The enzymatic activities of YopHs and CD45 were measured using 1 mM chromogenic substrate paranitrophenyl phosphate (pNPP) in 10 mM HEPES buffer pH 7.4, at 37°C. Phosphatase hydrolyzed pNPP to paranitrophenol and inorganic phosphate. Para-nitrophenol is an intensely yellow colored soluble product under alkaline conditions. The increase in absorbance (due to para-nitrophenol formation) is linearly proportional to enzymatic activity concentration (with excessive substrate, i.e. zero-order kinetics) and was assessed at 405 nm on a microplate reader Jupiter (Biogenet) using DigiRead Communication Software (Asys Hitech GmbH).