Spectroscopic probe analysis for exploring probe-protein interaction: A mapping of native, unfolding and refolding of protein bovine serum albumin by extrinsic fluorescence probe

Steady state and dynamic fluorescence measurements have been used to investigate interaction between Bovine Serum Albumin (BSA) and fluorescence probe para-N,N-dimethylamino orthohydroxy benzaldehyde (PDOHBA), a structurally important molecule exhibiting excited state coupled proton transfer (PT) and charge transfer (CT) reaction. Fluorescence anisotropy, acrylamide quenching, and time resolved fluorescence measurements corroborate the binding nature of the probe with protein. The binding constant between BSA and PDOHBA has been determined by using Benesi-Hildebrand and Stern-Volmer equations. The negative value of ΔG indicates the spontaneity of this probe-protein complexation process. Observations from synchronous, three dimensional fluorescence spectra and circular dichroism spectra point toward the fact that the hydrophobicity as well as α-helix content of BSA are altered in presence of probe PDOHBA. The PT band of PDOHBA is found to be an excellent reporter for the mapping of destructive and protective behavior of SDS with variation of chaotrope concentration.

Graphical abstractpara-N,N-dimethylamino orthohydroxy benzaldehyde (PDOHBA), a structurally important molecule exhibiting excited state coupled proton transfer (PT) and charge transfer (CT) reaction has been used as an external fluorescence probe for studying folding, unfolding and refolding processes of protein bovine serum albumin.Download full-size imageResearch Highlights► Mapping of native, unfolding and refolding of protein BSA using extrinsic fluorescence probe. ► Binding of probe in the hydrophobic zone of the protein secondary structure. ► Unfolding of the constitutive polypeptides by probe-protein interaction. ► Utilization of extrinsic probe to study protective and destructive nature of SDS.