Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k(off)) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k(off) value increases from (1.4 +/- 0.2) x 10(-4) s(-1), in the absence of the drug, to (9.5 +/- 1.2) x 10(-3) s(-1), in the presence of 1.0 x 10(-2) M ibuprofen, at pH 7.0 and 10.0 degrees C. From the dependence of k(off) on the drug concentration, values of the dissociation equilibrium constant for ibuprofen binding to to HSA-heme-Fe(II)-NO (K-1 = (3.1 +/- 0.4) x 10(-7) M, K-2= (1.7 +/- 0.2) x 10(-4) M. and K-3 (2.2 +/- .2) x 10(-3) M) were determined. The K-3 value corresponds to the HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 +/- 0.3) x 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. (C) 2009 Elsevier Inc. All rights reserved.