Tracking Zone-wise perturbation during unfolding of some globular proteins using Eu(III) complex of Tetracycline as a probe exhibiting Stark splitting

Abstract

Enhanced ‘Antenna effect’ of a suitably designed ternary complex of Eu(III), Tetracycline hydrochloride (TC) and globular proteins viz bovine serum albumin (BSA), human serum albumin (HSA) and β-lactoglobulin A (BLGA) in aqueous medium is employed to characterize the different partially unfolded states along with investigation of the micro- heterogeneous environment of the proteins during their stepwise unfolding. The zone-wise perturbation for the proteins upon denaturation by Urea and Guanidine hydrochloride (Gdn. HCl) is followed by the emission of Eu(III) through ‘Antenna Effect’ and that of the tryptophan (Trp) residues of the proteins as a function of denaturants both by steady state and time resolved emission study. With Gdn. HCl as denaturant, both BSA and BLGA show quenching of Eu(III) emission compared to pure protein while HSA exhibits an enhancement of antenna effect during unfolding as compared to that in its absence. In the presence of Urea, HSA and BSA show enhancement of antenna effect accompanied by Stark splitting of the 5D0→7F2 transition of Eu(III) although BLGA follows the similar pattern of quenching of Eu(III) emission as observed with Gdn. HCl without any Stark splitting. The proteins exhibit a two state transition with ΔGD values of ~ 2–3 kcal mol−1. Thus the use of Eu(III) emission as an efficient probe is advocating here to rationalize the microenvironment of the proteins during their stepwise unfolding.