Monitoring Local Unfolding of Bovine Serum Albumin During Denaturation Using Steady-State and Time-Resolved Fluorescence Spectroscopy

Abstract

In 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 formation of an intermediate state during denaturation. A global analysis using non-linear regression gave a . 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 (<1.5M).

Structural Transformation of Bovine Serum Albumin Induced by Dimethyl Sulfoxide and Probed by Fluorescence Correlation Spectroscopy and Additional Methods

“…[60,68] In this kind of situation, it is possible to find out whether the change in the spectral position is actually due to conformational change of the protein by following the procedure of Secundo et al, [69] which requires separate monitoring of the fluorescence of l-tryptophan and BSA as a function of the added solvent. This can result from two factors: 1) Trp134 of domain I, which is located on the surface of the protein and exposed to water, experiences a less polar environment with increasing DMSO concentration; and/or 2) rearrangement of domain II of BSA in the presence of DMSO, which makes the microenvironment of Trp213 more hydrophobic.…”

Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

“…The magnitudes of G • u in solution suggest that domain stabilities are I > II > III, although experimental uncertainty prevents definite ordering. The literature differs over which domain is most stable in solution but agrees that Domain III is probably least stable and unfolds first in chemical denaturation [38][39][40][41]. The order of domain stability of adsorbed BSA is the same but the Gibbs energy of unfolding is significantly decreased, as measured by G • u .…”

Study of the Binding of Thiazolyazoresorcinol and Thiazolyazocresol Dyes with BSA and Adenine by Spectral, Electrochemical and Molecular Docking Methods

“…The thermodynamic parameter, free energy change (ΔG 0 ) of interaction system is important to interpret the binding mode and it can be simply derived from the following equation; ΔG 0 = -RT ln Ka (4) where Ka is the binding constant, R is the gas constant and T is the experimental temperature. The values of ΔG 0 for BSA and adenine systems are listed in Table 1.…”

“…Such studies can provide useful information about characteristics of binding sites and the nature of the interaction. Therefore, these investigations attracted many researchers to employ fluorescence probes to understand these proteins-ligands interactions [2][3][4][5][6]. Such studies also give crucial information about ligand-protein interactions, which will have significant importance in pharmacology and drug discovery.…”