Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites |
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Authors: | Paolo Ascenzi Alessandra di Masi Massimo Coletta |
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Affiliation: | a Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University ‘Roma Tre’, Via della Vasca Navale 79, I-00146 Roma, Italy b National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Roma, Italy c Department of Molecular, Cellular and Animal Biology, University of Camerino, Via Filippo Camerini 2, I-62032 Camerino (MC), Italy d Department of Experimental Medicine and Biochemical Sciences, University of Roma ‘Tor Vergata’, Via Montpellier 1, I-00133 Roma, Italy e Department of Structural and Functional Biology, and Center of Neuroscience, University of Insubria, Via Alberto da Giussano 12, I-21052 Busto Arsizio (VA), Italy |
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Abstract: | 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. |
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Keywords: | Hb, hemoglobin HSA, human serum albumin HSA-heme, heme-albumin HSA-heme-Fe(II)-CO, ferrous carbonylated HSA-heme HSA-heme-Fe(II)-NO, ferrous nitrosylated HSA-heme IHP, inositol hexakisphosphate 1-MeIm, 1-methylimidazole |
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