Abstract
Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II).
Highlights
IntroductionHuman serum albumin (HSA), the most abundant protein in human plasma (with a bloodstream concentration of about 0.7 mM), binds a wide variety of endogenous ligands including non-esterified fatty acids, bilirubin, hemin and hormones
Human serum albumin (HSA), the most abundant protein in human plasma, binds a wide variety of endogenous ligands including non-esterified fatty acids, bilirubin, hemin and hormones
Kinetics of WF and carbon monoxide (CO) binding to HSA-heme-Fe(II) Kinetic progress curves for CO binding to HSA-heme-Fe(II), in the presence of WF, and for WF binding to HSA-heme-Fe(II), in the absence and presence of CO, have been obtained employing a SX.18MV stopped-flow apparatus provided with the diode array accessory for transient spectra collection (Applied Photophysics, Salisbury, UK)
Summary
Human serum albumin (HSA), the most abundant protein in human plasma (with a bloodstream concentration of about 0.7 mM), binds a wide variety of endogenous ligands including non-esterified fatty acids, bilirubin, hemin and hormones. The heme binds with high affinity (Kd , 161028 M) [19,20] within a narrow oblate hydrophobic cavity in the HSA subdomain IB [12,14,21], which is called fatty acid binding site 1 (FA1; see Fig. 1A) and represents the ‘‘heme binding cleft’’ This cavity is limited by Tyr138 and Tyr161 residues that provide p-p stacking interactions with the porphyrin and supply a donor oxygen (from Tyr161) to the heme-Fe(III)atom for the formation of human serum heme-albumin (HSAheme) [3,12,14,19,21,22,23]. The heme-binding cleft (i.e., FA1) and Sudlow’s site I (i.e., FA7) are allosterically coupled (Fig. 1B), since the heme-Fe(III) affinity for HSA decreases by about one order of magnitude upon WF binding; heme-Fe(III) binding to HSA decreases the affinity of ligands (e.g., drugs) for Sudlow’s site I by the same extent [3,17,19,20,21,30,31,32,33,34,35]
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