Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants.
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ABSTRACT:
SUBMITTER: Watanabe H
PROVIDER: S-EPMC1221951 | biostudies-other | 2001 Jul
REPOSITORIES: biostudies-other
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Project description:Radioactive isotopes are used as drugs or contrast agents in the medical field after being conjugated with chelates such as DOTA, NOTA, DTPA, TETA, CyDTA, TRITA, and DPDP. The N-terminal sequence of human serum albumin (HSA) is known as a metal binding site, such as for Co2+, Cu2+, and Ni2+. For this study, we designed and synthesized wAlb12 peptide from the N-terminal region of HSA, which can bind to cobalt, to develop a peptide-based chelate. The wAlb12 with a random coil structure tightly binds to the Co(II) ion. Moreover, the binding property of wAlb12 toward Co(II) was confirmed using various spectroscopic experiments. To identify the binding site of wAlb12, the analogs were synthesized by alanine scanning mutagenesis. Among them, H3A and Ac-wAlb12 did not bind to Co(II). The analysis of the binding regions confirmed that the His3 and α-amino group of the N-terminal region are important for Co(II) binding. The wAlb12 bound to Co(II) with Kd of 75 μM determined by isothermal titration calorimetry when analyzed by a single-site binding model. For the use of wAlb12 as a chelate in humans, its cytotoxicity and stability were investigated. Trypsin stability showed that the wAlb12 - Co(II) complex was more stable than wAlb12 alone. Furthermore, the cell viability analysis showed wAlb12 and wAlb12 + Co(II) to be non-toxic to the Raw 264.7 and HEK 293T cell lines. Therefore, a hot radioactive isotope such as cobalt-57 will have the same effect as a stable isotope cobalt. Accordingly, we expect wAlb12 to be used as a peptide chelate that binds with radioactive isotopes.
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Project description: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).
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Project description:Human serum albumin (HSA) binds a variety of xenobiotics, including flavonoids and warfarin. The binding of another ligand to the IIA binding site on HSA can cause warfarin displacement and potentially the elevation of its free concentration in blood. Studies dealing with flavonoid-induced warfarin displacement from HSA provided controversial results: estimated risk of displacement ranged from none to serious. To resolve these controversies, in vitro study of simultaneous binding of warfarin and eight different flavonoid aglycons and glycosides to HSA was carried out by fluorescence spectroscopy as well as molecular docking. Results show that warfarin and flavonoids do not share the same binding region in binding to HSA. Interactions were only observed at high warfarin concentrations not attainable under recommended dosing regimes. Docking experiments show that flavonoid aglycons and glycosides do not bind at warfarin high affinity sites, but rather to different regions within the IIA HSA subdomain. Thus, the risk of clinically significant warfarin-flavonoid interaction in binding to HSA should be regarded as negligible.
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