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Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin |
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| Authors: | Paolo AscenziAlberto Bertollini Roberto Santucci Gino AmiconiMassimo ColettaAlessandro DesideriBruno GiardinaFrancesca PolizioRoberto Scatena |
| Affiliation: | Department of Pharmaceutical Chemistry and Technology, University of Turin Italy C.N.R., Center for Molecular Biology, Department of Biochemical Sciences “Alessandro Rossi Fanelli,” University of Rome “La Sapienza” Italy Department of Molecular, Cellular and Animal Biology, University of Camerino Italy Department of Organic and Biological Chemistry, University of Messina Italy C.N.R., Center for Chemistry of Receptors, Institute of Chemistry, Catholic University of Rome Italy Department of Biology, University of Rome “Tor Vergata” Italy Department of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata, Italy |
| Abstract: | The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10−1 M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10−1 M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb. |
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