Bubuster

The amidolytic activity of arginine-specific (Rgp) and lysine-specific (Kgp) gingipains was assayed as described previously^{33 (link)}. P. gingivalis was cultured to mid-log phase and reacted for 18 h with pABA (1 mg/mL) or vehicle control. Cells were separated from the culture supernatant fraction by centrifugation (5,000 × g, 20 min), washed and resuspended in PBS to the original volume, or lysed by Bubuster (Millipore). The chromogenic p-nitroanillide substrates N-Benzoyl-L-Arginine-pNA or toluenesulphonyl-glycyl-prolyl-L-lysine pNA (Sigma, St. Louis, MO) were used to detect RgpA/B and Kgp, respectively. Samples (50 μl) were preincubated in 200 mM Tris HCl, 5 mM CaCl₂, 150 mM NaCl₂, supplemented with 10 mM cysteine in 96-well plates for 10 min at 37°C and assayed with 0.5 mM substrate. The rate of substrate hydrolysis and the accumulation of p-nitroanilide were monitored spectrophotometrically at 405 nm over time in a Spectramax M5 reader (Molecular Devices, Sunnyvale, CA), and the activity of enzyme is given as mOD/min/μl.