Show simple item record

dc.contributor.authorKrenzel, Eileen Susanen_US
dc.date.accessioned2015-08-04T20:29:43Z
dc.date.available2015-08-04T20:29:43Z
dc.date.issued2012en_US
dc.date.submitted2012en_US
dc.identifier.other(ALMA)contempen_US
dc.identifier.urihttps://hdl.handle.net/2144/12457
dc.descriptionThesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.en_US
dc.description.abstractIn circulation human serum albumin (HSA) is the principal carrier for endogenous lipophilic compounds, primarily non-esterified long chain fatty acids (FA). Since FA bind multiple binding sites with varying affinities, it would be useful to probe the relative affinities for FA binding sites with a method that distinguishes individual binding sites. NMR spectroscopy is a powerful approach for studying interactions of FA with HSA and FA-competitive drugs. In the physiologically relevant solution state, 2D-NMR provides a unique view of HSA-ligand binding in a site-specific manner. Here we show nine, well-resolved peaks in 1H-13C-NMR spectra of 18-13C-oleic acid (OA)/HSA complexes. Different NMR signals arise, representing FA bound at different sites throughout the protein, with the varying intensities corresponding to the different relative affinities of HSA for OA. This investigation probes the site-specific FA binding sites with four approaches: (i) observation of order of filling with increasing FA molar ratios to HSA; (ii) addition of FA acceptors to observe the dissociation of FA from HSA; (iii) addition of drugs known to bind to low affinity FA sites; and (iv) development and use of HSA mutants that disrupt the binding of FA at high affinity sites. From the order of filling, the three highest affinity-binding sites are clearly differentiated from the six lower/medium affinity-binding sites at the physiologically relevant FA:HSA molar ratio- 4:1 . Methyl-β-cyclodextrin (MβCD) extracted FA from individual sites, in a concentration dependent manner, with the highest concentrations removing FA from the highest affinity sites. Relative affinities determined as above were consistent with the binding of drugs to previously defined primary drug binding sites, which displaced bound FA from specific lower affinity sites. HSA mutants were successfully expressed and purified, but FA binding to these proteins did not yield interpretable data. The other aim of this study was to identify the binding location of bilirubin on HSA, for it remains elusive despite intensive study. Unlabeled bilirubin competition suggested binding at primary drug binding sites, while directly probing the bilirubin-binding site with 13C-bilirubin analogs suggested binding in subdomain IB. These studies provide a methodological approach for further analysis of site-specific binding on HSA.en_US
dc.language.isoen_USen_US
dc.publisherBoston Universityen_US
dc.titleIdentification of fatty acid and bilirubin binding sites on human serum albumin by 2D-NMR spectroscopyen_US
dc.typeThesis/Dissertationen_US
etd.degree.nameDoctor of Philosophyen_US
etd.degree.leveldoctoralen_US
etd.degree.disciplinePharmacologyen_US
etd.degree.grantorBoston Universityen_US


This item appears in the following Collection(s)

Show simple item record