Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (koff) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The koff value increases from (1.4 ± 0.2) × 10−4 s−1, in the absence of the drug, to (9.5 ± 1.2) × 10−3 s−1, in the presence of 1.0 × 10−2 M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of koff on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K1 = (3.1 ± 0.4) × 10−7 M, K2 = (1.7 ± 0.2) × 10−4 M, and K3 = (2.2 ± 0.2) × 10−3 M) were determined. The K3 value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10−3 M). Present data indicate that ibuprofen binds to the FA3–FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.