Peptides showing a slope ≥1% were considered to be HABPs. Numbers shown in the first column correspond to our institute’s serial numbering system. Superscripts Luminespib mouse at the beginning and end of the sequence indicate the peptide amino acid position within the protein. (B) Saturation binding curves for HABPs 30979 and 30987 binding with high activity to U937 cells. Saturation curves were

obtained by plotting the specifically bound 125I-HABP concentration versus free 125I-HABP. Affinity constants and the maximum number of binding sites per cell were obtained from these curves. Inset: the abscissa is log F in the Hill plot and the ordinate is log [B/(B m - B)], where B m is the maximum amount of bound peptide, B is the amount of bound peptide and F is the amount of free peptide. Rv0679c HABPs 30979 and 30987 were assessed by means of a saturation assay using concentrations of radiolabeled peptide larger than the ones used in conventional binding assays in order to determine dissociation constants (K d), Hill coefficients (n H) and approximate number Citarinostat order of binding sites per cell (Figure 4b). The results showed that binding of these

HABPs to surface receptors of U937 cells was saturable and of cooperative nature (n H = 1.50 for HABP 30979 and n H = 1.12 for HABP 30987). A dissociation constant of 1,100 nM and about 1.0 × 106 binding sites per cell were identified for HABP 30979, while HABP 30987 showed a dissociation constant of 600 nM and about 1.8 × 106 binding sites per cell. Secondary structure analyses of Rv0679c peptides by circular dichroism CD spectra of Rv0679c peptides Montelukast Sodium obtained in 30% TFE are shown in Figure 5. The spectra of peptides 30982 and 30987 showed random coil SCH772984 order structures, while the spectra of peptides 30979, 30981 and 30985 were consistent with α-helical structures. The remaining peptides of Rv0679c (30980, 30983, 30984 and 30986) displayed θλ values not related to any defined structures. Figure 5 CD spectra of Rv0679c peptides. HABPS

spectra were grouped in order to enable scale appreciation. Spectra were obtained by averaging three scans taken at 0.1 nm intervals from 260-190 nm at 20°C. [Θ] is the mean residue ellipticity per amino acid residue in the peptide. CD resolution: 0.1 millidegree (at ± 2.000 mdeg). Inhibition of M. tuberculosis H37Rv invasion into A549 and U937 cells The ability of Rv0679c HABPs to block mycobacterial entrance into A549 and U937 cells was evaluated using a flow-cytometry-based assay. Rv0679c peptides analyzed in such assay included peptides 30979 and 30987, which had been identified as HABPs for both cell lines, peptides 30985 and 30986 which had been identified as HABPs for A549 cells, and a low activity binding peptide (30982) which was used as negative control. Invasion of U937 cells was significantly inhibited by HABPs 30985 and 30986, but neither of these two HABPs showed a clear dose-dependent inhibitory behavior.