Fig 1.
Effect of Ole on the primary association constants (K) of DNSA (A, Closed circles) and ibuprofen (B; Open circles) at pH 6.5 and 25°C.
Number of primary binding sites (n) was 1 for each system. The concentration of HSA was 40μM. The results are the mean ± S.D. for at least three observations.
Fig 2.
Effects of Ole on the binding of DNSA (4–24μM) to HSA (40μM) in the presence of ibuprofen at pH 6.5 and 25°C.
The concentration ratio of Ole to HSA are 0 (A), 1 (B) and 3 (C). Closed circles are the experimental values for DNSA binding in the presence of ibuprofen (A, B; 20μM, C; 24μM). Solid line represents theoretical curves assuming the independent binding of the two ligands. Broken line represents theoretical curves assuming competitive binding between DNSA and ibuprofen. Dotted line represents theoretical curves assuming anti-cooperative (allosteric) interaction between DNSA and ibuprofen. All theoretical curves were constructed using the association constant for each ligand (A; DNSA 1.2×105 M-1, ibuprofen 37.1×105 M-1, B; DNSA 1.3×105 M-1, ibuprofen 26.7×105 M-1, C; DNSA 1.7×105 M-1, ibuprofen 6.8×105 M-1).
Fig 3.
Effects of Ole on the binding of ibuprofen (4–24μM) to HSA (40μM) in the presence of DNSA at pH 6.5 and 25°C.
The concentration ratio of Ole to HSA are 0 (A), 1 (B) and 3 (C). Open circles are the experimental values for ibuprofen binding in the presence of DNSA (24μM). Solid line represents theoretical curves assuming the independent binding of the two ligands. Broken line represents theoretical curves assuming competitive binding between ibuprofen and DNSA. Dotted line represents theoretical curves assuming anti-cooperative (allosteric) interaction between ibuprofen and DNSA. All theoretical curves were constructed using the association constant for each ligand (A; DNSA 1.2×105 M-1, ibuprofen 37.1×105 M-1, B; DNSA 1.3×105 M-1, ibuprofen 26.7×105 M-1, C; DNSA 1.7×105 M-1, ibuprofen 6.8×105 M-1).
Fig 4.
Effects of Ole on coupling constant (χ) for interactions between DNSA and ibuprofen.
Closed circles are coupling constants calculated from the binding of DNSA in the presence of ibuprofen. Open circles are coupling constants calculated from the binding of ibuprofen in the presence of DNSA. The results are the mean ± S.D. for at least four determination.
Fig 5.
Effect of ibuprofen on CD spectra of the DNSA-HSA system in the absence (A) and presence (B) of Ole at pH6.5.
Concentrations of HSA, DNSA, Ole and ibuprofen were 40μM, 40μM, 120μM, 10–120μM, respectively.
Fig 6.
Molecule of ibuprofen at site II in the absence (A) and presence of DNSA and/or Ole (B and C).
The optimized docking poses for ibuprofen are represented in green sticks. Hydrogen bonds were highlighted by bright-blue colored broken lines. Right-blue and yellow letters represent residues that interact with ibuprofen by hydrogen bonding and hydrophobic interactions, respectively.
Table 1.
Docking data of ligands-HSA complexes.