Interaction of human serum albumin with charge transfer probeethyl ester of N,N-dimethylamino naphthyl acrylic acid: An extrinsic fluorescence probe for studying protein micro-environment

Abstract

The charge transfer (CT) probe ethyl ester of N,N-dimethylamino naphthyl acrylic acid (EDMANA) bound to Human Serum Albumin (HSA) serves as an efficient reporter of the polarity and conformational changes of protein in aqueous buffer (Tris-HCl buffer, pH=7.03) and in presence of denaturant, quencher and reverse micelles. The change in fluorescence intensity and the position of emission maxima of EDMANA in presence of HSA well reflect the nature of binding and location of the probe inside the proteinous environment. The increase in steady state anisotropy values with increase of protein concentration indicate restriction imposed on the mobility of the probe molecules in the proteinous medium. The results of fluorescence quenching of EDMANA by acrylamide, Fluorescence Resonance Energy Transfer (FRET) and Red Edge Excitation Shift (REES) studies throw light on the accessibility to the probe bound to HSA and hence indicate the probable location of the probe within the hydrophobic cavity of HSA. The complicated nature of protein unfolding in presence of urea is well studied by change in the fluorescence properties of EDMANA bound to HSA protein.

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

“…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.…”

“…The measurement of excitation anisotropy is a wavelength sensitive tool which provides a more vivid picture of the surrounding atmosphere of the probe while emitting from the excited state [30,42]. At a particular protein concentration, if the anisotropy increases with the shifting of the excitation wavelength towards red end, then the probe is expected to exist in a motionally restricted environment.…”

“…(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].…”