Relation between Human Serum Albumin Structure and Fluorescence Decay Parameters of Tryptophan Residue 214

Abstract

The purpose of this article is to study the relation between fluorescence decay parameters of tryptophan residue in human serum albumin (HSA) and the protein structure. HSA contains a single tryptophan residue, and thus, there will be no ambiguity on the data obtained. HSA comprises a single polypeptide of 585 amino acid residues with only one tryptophan residue. Tryptophan fluorescence is very sensitive to the local environment. Global form and structure of HSA are pH dependent. Thus, in this article, behavioral changes and fluorescence characteristics of tryptophan within HSA were studied at different pHs (2–12) and in the denatured state in the presence of guanidine hydrochloride solution. In an environment with a low pH (at pHs 2 and 3), tryptophan emits at a maximum of 330 nm. The peak position shifts to 340 nm at higher pH. Peak position values indicate no protein denaturation but a structural modification. The loss of the tertiary protein structure (complete denaturation) induces a shift in tryptophan fluorescence to 352 nm. At all pHs, tryptophan residue emits with three lifetimes. Lifetime measurements at different pHs along the emission wavelengths allowed us to differentiate the different forms of HSA. In the denatured state, tryptophan emission occurs also with three lifetimes. However, values and contributions of these lifetimes to the global emission decay differ between the native and denatured states. We have considered that fluorescence emission occurs from tryptophan substructures, each substructure is characterized by one lifetime along with its pre‐exponential. Populations of the substructures, characterized by the pre‐exponential values of the fluorescence lifetimes, are dependent on the microenvironment of the fluorophore and on the global protein structure.

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