Abstract

Previously, we studied the physisorption of 2-(<i>p</i>-toluidinyl)naphthalene-6-sulfonate (TNS) to the hydrophobic sites or cavities of bovine serum albumin (BSA) and determined the effects of acrylamide (quencher) and urea (denaturant) on the complex using steady-state and dynamic fluorescence spectroscopy. In this paper, we extend this work to bovine β-lactoglobulin (β-Lg) and immunoglobulin G (IgG), and compare the complexes to the covalently attached TNS. These results show that the hydrophobic sites of different proteins can be probed by TNS with the use of steady-state and time-resolved fluorescence spectroscopy. The TNS binding site in B-Lg is the most hydrophobic in comparison to BSA and IgG. The excited-state decay kinetics for the TNS/protein complexes is best described by a distribution (Gaussian) plus a discrete decay model. TNS/protein adducts (covalent attachment of TNS), whereby the TNS sulfonate is converted to a sulfonamido group, strongly affects the probe photophysics. For example, a red shift is observed in the emission and excitation spectra due to covalent attachment. Acrylamide quenches the fluorescence of TNS/protein complexes, but does not quench 2-(<i>p</i>-toluidinyl)naphthalene-6-(N-propyl)-sulfonamide(TNS-PA) or the covalent TNS/protein adducts. Furthermore, the fluorescence decay kinetics of the covalent TNS/protein adducts is always best described by a double discrete decay model. In all cases studied, added urea does not affect the recovered lifetime values but does decrease the pre-exponential factor for the longer lifetime component. This result suggests a single-step urea denaturation mechanism involving two protein states—folded (native) and unfolded (denatured).

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