Hydrogen bonding plays a significant role in the binding of coomassie brilliant blue-R to hemoglobin: FT-IR, fluorescence and molecular dynamics studies

An analog of coomassie brilliant blue-R (CBB-R) was recently found to act as an antagonist to ATP-sensitive purinergic receptors (P2X7R) and has potential to be used in medicine. With the aim of understanding its transportation and distribution through blood, in this investigation, we measured the binding parameters of CBB-R with bovine hemoglobin (BHG). The molecule specifically bound to a single binding site of the protein with a stoichiometric ratio of 1 : 1 and the observed binding constant K-a was 3.5, 2.5, 2.0 and 1.5 x 10(5) M-1 at 20 degrees C, 27 degrees C, 37 degrees C and 45 degrees C, respectively. The measured respective Delta G(0) values of the binding at four temperatures were -30.45, -22.44, -18.04 and -11.95 kJ mol(-1). The Delta H-0 (change in enthalpy) and Delta S-0 (change in entropy) values were -23.6 kJ mol(-1) and -70.66 J mol(-1) respectively in the binding process. The negative value of Delta H-0 and Delta S-0 indicated that the binding of the molecule was thermodynamically favorable. The best energy structure in the molecular docking analysis revealed that CBB-R preferred to be intercalated in the cavity among the alpha 2, beta 1 and beta 2 subunits and the binding location was 7.4 angstrom away from T37 in the beta 2 subunit. The binding of the molecule with the protein was stabilized by hydrogen bonds involving the side chain of two amino acid residues. The residues were Lys104 and Glu101 in the beta 2 subunit. The binding was further stabilized via hydrogen bond formation between the amide group of the peptide backbone (residue Tyr145 of the beta 1 subunit) and CBB-R. A shift of the amide I (-C=O stretching) band frequency of similar to 8 cm(-1) to low energy was ascribed to the hydrogen bond interaction involving the polypeptide carbonyl of the protein and the CBB-R molecule. In addition, two pi-cation interactions between Lys99 of the alpha 2 subunit and Lys104 of the beta 2 subunit and CBB-R contributed favorably in the binding processes. No substantial change in the soret and Q absorption bands of BHG could be observed in the presence of CBB-R. It indicated that the oxygen binding domain or the heme proximity was not blocked or substantially perturbed due to the binding of CBB-R. The circular dichroism and the molecular dynamics analysis further established that the binding interaction caused no significant alteration in the protein long range secondary structure.