The k and n value represent the rate constant per minute, the heterogenicity parameter. The predicted amino acid sequence similarity between the two recombinant toxins TRH and TDH used in this study was 67.3%. From 2 L of culture supernatant of JM109 (DE3) harboring the recombinant plasmid, a final amount

of 6 mg signal peptide-deleted TRH was purified using a series of column chromatography procedures. Purified TRH showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular size of both purified TRH and purified TDH was 23 kDa. This molecular size of TRH is consistent with that estimated in a previous study on the purification of native TRH from V. parahaemolyticus clinical isolates (Honda et al., 1988). TDH forms tetramer in solution (Fukui et al., 2005; Hamada Autophagy activity inhibition et al., 2007). We performed size-exclusion chromatography to investigate the association state HDAC inhibitor of TRH in solution. The elution volume of TRH corresponded with that of tetrameric TDH, indicating that TRH is organized into a tetrameric structure (Fig. 1a). We investigated

the association and equilibrium state of TRH by analytical ultracentrifugation (Fig. 1b). Sedimentation equilibrium showed that the molecular mass of TRH was 75 000 ± 200 Da, which is similar to the molecular mass of tetrameric TDH of 75 000 ± 700 Da as determined by sedimentation equilibrium analysis (Hamada et al., 2007). The molecular mass Selleck Metformin of monomeric TRH was calculated as 18 600 Da. Therefore, TRH also exists as a tetramer in solution. Further, transmission electron microscopy of negatively stained samples showed tetrameric oligomerization of TRH (Fig. 1c). To investigate differences in structure and function

between TRH and TDH at the atomic level, we built the homology model of TRH and compared the three-dimensional structures of TRH and TDH. The structure of TRH exhibited a good fit to that of TDH (Fig. 1d). Furthermore, the three-dimensional position of R46, E138, and Y140, which may participate in π-cation interactions and maintain TDH tetrameric structures, was also conserved in TRH, suggesting that they may be important factors for tetrameric structure and hemolysis for these toxins. To compare the hemolytic activities of TRH and TDH, we measured their activities in human erythrocytes (Fig. 2a). At 1 μM, the hemolytic activity of TRH was higher than that of TDH. However, this difference was not observed when the concentration was >4 μM. To investigate whether TRH shows an Arrhenius effect, the hemolytic activity of TRH was measured after various heat treatments (Fig. 2b). TDH lost its hemolytic activity after heating at 60 °C for 30 min (TDH, fibril); however, the activity was recovered after rapid cooling from the denatured state at 90 °C (TDH, refold; the Arrhenius effect).