The inner and outer membrane fractions were recovered as a supernatant and a pellet, respectively, by ultracentrifugation at 100,000 ICG-001 × g for 60 min at 4°C [34]. In-gel digestion of proteins and Peptide Mass Fingerprinting To identify the 27-kDa protein, P. gingivalis KDP161 cells were harvested, and the cell pellets were dissolved with RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris-HCl, pH 8.0) and then immunoprecipitated by EZview red protein A affinity gel (Sigma) with anti-HBP35 polyclonal antibody, followed by SDS-PAGE analysis with CBB staining and immunoblot analysis. Protein bands from the SDS-PAGE gel were

excised and subjected to in-gel tryptic digestion as described previously [8, 9]. Gel pieces were washed in 50 mM NH4HCO3-ethanol (1:1, vol/vol), reduced, alkylated with dithiothreitol and iodoacetamide, respectively, and digested with sequencing-grade modified trypsin (10 ng/μl) (Promega) overnight at 37°C. Each digest (0.5 μl) was then analyzed by mass spectrometry using an Ultraflex Proteasome purification TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) in positive-ion and reflectron mode. A saturated solution of α-cyano-4-hydroxycinnamic acid was prepared in 97:3 (vol/vol) acetone-0.1% aqueous trifluoroacetic acid (TFA). A thin layer of matrix was prepared by pipetting and immediately transferring 2 μl of this solution onto 600-μm anchors of an AnchorChip target plate (Bruker Daltonics). The tryptic

digest sample (0.5 μl) was then deposited onto the thin layers with 2.5 μl of 0.1% (vol/vol) TFA for 1 min. Mass spectra were calibrated by external calibration using a standard peptide mix (Bruker Daltonics). Proteins were identified by PMF against the P. gingivalis database (available at The Institute for Genomic Research [TIGR] website [http://​www.​tigr.​org]) not using an in-house Mascot search engine (Matrix Science Ltd., London, United Kingdom) and BioTools 2.2 software (Bruker Daltonics) and by comparison with tryptic

peptide mass lists generated by using General Protein Mass Analysis for Windows software (Lighthouse Data, Odense, Denmark). Northern blot analysis Total RNA extraction and Northern blot analysis of mRNA were carried out as described previously [35] with some modifications. The 0.96-kb DNA fragment coding for Q22 to P344 of HBP35 and the 0.80-kb DNA fragment coding for M1 to S266 of ErmF were obtained by PCR that were used as the radiolabelled hbp35 and ermF probes, respectively. To label the DNA probes, [α-32P]dCTP and the ready-to-go DNA labeling beads kit (GE Healthcare) was used. The radiolabelled products were analyzed with a fluoro-image analyzer buy Adavosertib FLA-5100 (Fujifilm). Hemin binding assay Hemin binding to rHBP35 proteins was assayed using the catalytic property of hemoprotein, which has peroxidase activity in the presence of H2O2, by the method of Shibata et al. [7] with some modifications. Ten microliters of protein solution (2 μg) was treated with 1.5 μl of 1.