P. gingivalis cells were washed in TC150 buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl2, pH 8.0,) supplemented with cysteine hydrochloride (10 mM), suspended in an equivalent culture volume of the same solution and designated whole-cell fractions. Culture supernatants were ultracentrifuged at 103,900 g, 4°C for 40 min and designated supernatant fractions. Fractions were analysed for Arg- and Lys-specific proteinase activities as previously described [91 (link)] using chromogenic substrates N-α-benzoyl-L-Arg-p-nitroanilide (BApNA) and Kgp substrate was N-Tosylglycyl-L-prolyl-L-lysine4-nitroanilide acetate salt (LpNA). The Arg- and Lys-specific assay mixtures contained either whole-cell fractions (3.8 x 107 and 5 x 107 cells respectively) or supernatant fractions (45 μL and 60 μL respectively), cysteine hydrochloride (10 mM), 2 mM BApNA or LpNA and 5% (v/v) dimethyl sulfoxide made up to 200 μL in TC150 buffer. The release of pNA was monitored using a microtitre plate reader (Wallac VICTOR™ 3 Multilabel Counter, PerkinElmer™ Pty. Ltd.) by the change in OD at 405 nm over time.
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