Tryptophan fluorescence intensity and anisotropy measurements reveal a unique unfolding intermediate in human serum albumin

Abstract

Human serum albumin (HSA) has only one tryptophan residue located in the center of its hydrophobic interior adjacent to a hinge region. Thus, HSA is an excellent protein for modeling protein denaturation and refolding using trp fluorescence spectroscopy, particularly since HSA is widely available at low cost in a highly purified form. To determine the nature of unfolding of HSA around the trp, steady-state fluorescence intensity and fluorescence polarization anisotropy were measured in HSA equilibrated into increasing concentrations of guanidine. The fluorescence intensity of trp is highly susceptible to solvent effects, and thus, trp-bearing proteins typically loose trp fluorescence intensity on unfolding as the hydrophobic interior is exposed to the aqueous solvent. HSA fluorescence intensity measurements in increasing guanidine suggest complete exposure of the trp to an aqueous environment by 3M guanidine. However, high trp polarization anisotropy, suggestive of low rotational freedom of the fluorophore, was measured at the same concentration of guanidine, but fell to its lowest levels only by 4M gaunidine. These findings suggest that there is a unique guanidine-induced unfolding intermediate in HSA, occurring around 3M guanidine, in which the microenvironment around the trp is both highly restricted (high anisotropy) and very polar (low fluorescence intensity). This combination is unusual and worthy of further investigation. We report results further exploring the nature of this unique guanidine-mediated unfolding intermediate with iodide fluorescence quenching analysis, and report similar results using other denaturants, including urea. The suitability of using projects related to these findings in an undergraduate teaching lab is also discussed.