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dc.contributor.authorTogashi, Denisio M.
dc.contributor.authorRyder, Alan G.
dc.contributor.authorO'Shaughnessy, Domhnall
dc.date.accessioned2016-01-29T16:32:17Z
dc.date.available2016-01-29T16:32:17Z
dc.date.issued2010-03
dc.identifier.citationTogashi, D.M.; Ryder, A.G.;O'Shaughnessy, D.; (2010) 'Monitoring Local Unfolding of Bovine Serum Albumin During Denaturation Using Steady-State and Time-Resolved Fluorescence Spectroscopy'. Journal Of Fluorescence, 20 :441-452.en_IE
dc.identifier.issn1573-4994
dc.identifier.urihttp://hdl.handle.net/10379/5503
dc.description.abstractIn a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropiclly induced denaturation of Bovine Serum Albumin (BSA) using the fluorescence decay kinetics at different stages in the denaturation of BSA by guanidinium hydrochloride (GuHCl). In this work, we gain a more detailed insight into the BSA denaturation process by investigating the thermodynamics of the process. Structural changes were monitored spectrophotometrically via the intrinsic protein fluorescence from tryptophan residues, and the extrinsic fluorescence from 1,8-anilinonaphthalene sulphonate (ANS). ANS tends to locate in a variety of binding sites in BSA which are located in different domains, and these can be selectively populated using different, 1:1 and 1:10 molar ratios of BSA to ANS. The data from steady-state and time-resolved fluorescence spectroscopy were analyzed using thermodynamic two-state and three-state models and the lifetime data clearly indicated the presence of an intermediate state during denaturation. A global analysis using non-linear regression gave a Delta G(H2O,D)(0) = 6.7 kcal.mol(-1) for the complete unfolding of the BSA-ANS complexes, and a Delta G(H2O,I)(0) = 0.9 kcal.mol(-1) for the first step to the intermediate. Therefore, the unfolding energy of the intermediate, which appears mostly at intermediate GuHCl concentrations (1.0 to 1.5 M), to the denatured state, is 5.8 kcal.mol(-1). The lifetime analysis of the BSA-ANS complexes also shows clearly that there are differences in stability of the BSA domains, with domain III unfolding first at low GuHCl concentrations (en_IE
dc.description.sponsorshipScience Foundation Ireland (Principal Investigator grant number 02/ IN.1/M231 to AGR), National Biophotonics Imaging Platform (an Irish Higher Education Authority Programme for Research in Third Level Institutions)en_IE
dc.formatapplication/pdfen_IE
dc.language.isoenen_IE
dc.publisherSpringeren_IE
dc.relation.ispartofJournal Of Fluorescenceen
dc.subjectProteinen_IE
dc.subjectBovine serum albuminen_IE
dc.subjectFluorescenceen_IE
dc.subjectANSen_IE
dc.subjectGaussian lifetime distributionen_IE
dc.subjectThree-state transition modelen_IE
dc.subjectTryptophan Fluorescenceen_IE
dc.subjectGuanidine-hydrochlorideen_IE
dc.subjectAlpha-chymotrypsinen_IE
dc.subjectEnergy-transferen_IE
dc.subjectProteinen_IE
dc.subjectBindingen_IE
dc.subjectProbesen_IE
dc.subjectSiteen_IE
dc.subjectStabilityen_IE
dc.subjectMechanismen_IE
dc.subjectChemistryen_IE
dc.titleMonitoring local unfolding of bovine serum albumin during denaturation using steady-state and time-resolved fluorescence spectroscopyen_IE
dc.typeArticleen_IE
dc.date.updated2015-12-22T16:32:58Z
dc.identifier.doiDOI 10.1007/s10895-009-0566-8
dc.local.publishedsourcehttp://dx.doi.org/10.1007/s10895-009-0566-8en_IE
dc.description.peer-reviewedpeer-reviewed
dc.contributor.funder|~|1267882|~|1267883|~|
dc.internal.rssid1338049
dc.local.contactAlan Ryder, School Of Chemistry, Room 213, Arts/Science Building, Nui Galway. 2943 Email: alan.ryder@nuigalway.ie
dc.local.copyrightcheckedNo
dc.local.versionACCEPTED
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