Chemically Induced Unfolding of Bovine Serum Albumin by Urea and Sodium Dodecyl Sulfate: A Spectral Study with the Polarity‐Sensitive Charge‐Transfer Fluorescent Probe (E)‐3‐(4‐Methylaminophenyl)acrylic Acid Methyl Ester

Abstract

Sensitivity of the charge-transfer (CT) band of the fluorescence probe (E)-3-(4-methylaminophenyl)acrylic acid methyl ester (MAPAME) towards the polarity of its immediate environment is employed to investigate the binding interaction of the probe with bovine serum albumin (BSA) and uncoiling of BSA by the denaturants urea and sodium dodecyl sulfate micelles. Binding of the probe with BSA produces a blue shift and enhanced intensity of the CT emission band which clearly point toward a decrease in polarity of the immediate environment of MAPAME. This is expected, since binding with BSA moves the probe from a polar water environment to a much less polar, hydrophobic protein interior, where the CT band is expected to be blue-shifted. Higher intensity arises due to fewer non-radiative decay paths available to the probe in the hydrophobic protein environment. Chemically induced unfolding of BSA by urea and sodium dodecyl sulfate is tracked by monitoring the induced spectral changes of the protein-bound probe MAPAME. Red-edge excitation shift or REES, fluorescence resonance energy transfer (FRET) and anisotropy measurements are used to investigate and monitor these binding and unfolding processes.

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

“…total or partial loss of three dimensional structures. Proteins can be denatured by heat, pH or chemical denaturants such as urea, a well known chaotropic denaturant [30,[32][33][34][35]. After finding the binding interaction between BSA and PDOHBA, we intend to see the effect of chaotropes on this binding interaction.…”

“…Since an increase in rigidity of the surrounding environment of a fluorophore reflects in an increase in fluorescence anisotropy, this method can be successfully used in order to locate the probable position of the probe in complex molecular assembly including protein [30,[32][33][34][35]. From Fig.…”

“…(4) can be expressed in terms of fluorescence intensity. Assuming that the concentration of the probe-protein complex is very low compared to that of the free protein, the BenesiHildebrand relation for these types of complexation process can be written as follows [30,33,34], and discards the probability of 1:2 binding phenomenon (inset of Fig. 2 (b)).…”

“…Fluorescence lifetime serves as a sensitive parameter to cast further light on the local environment around a fluorophore in the proteinous media [30,[32][33][34][35]. Fluorescence decay curves are usually multiexponential in the case of probe-protein binding complexation.…”

“…Fluorescence spectroscopy serves as a feasible and powerful sensing tool for the investigation of local environment of the fluorophore yielding structural and dynamical information concerning the fluorophore environment [27][28][29]. Extrinsic fluorescence probe such as excited state charge, proton and electron transfer molecules can be used successfully for studying conformational changes of protein structure with variation of its solvent characteristics [30][31][32][33][34][35]. The molecule para-N,N-dimethylamino orthohydroxy benzaldehyde (PDOHBA) is one of the interesting organic molecule studied by our group which exhibits excited state coupled intramolecular charge transfer (CT) and proton transfer (PT) reaction [36].…”