Phosphorescence from Single Tryptophan in Amorphous Solid Human Serum Albumin Exhibits Solvent-Protein Dynamics Slaving

Abstract

The physical properties of amorphous biomolecules are important to the texture and stability of low-moisture foods, the stability of pharmaceuticals, the permeability of edible films, and the viability of organisms during anhydrobiosis. Protein stability is often improved via the inclusion of small-molecule excipients during freeze-drying and organisms overproduce sugars such as sucrose or trehalose during anhydrobiosis. The effect on internal protein dynamics caused by substitution of a protein's surface water molecules with small sugar molecules is unclear. To explore this question, we have analyzed tryptophan phosphorescence decays of human serum albumin (HSA) in the dry amorphous solid state. Phosphorescence is an ideal approach, as the long-lived triplet state of tryptophan is sensitive to the long time-scale molecular motions of proteins in the dry state. Human serum albumin (HSA) was chosen because it contains a single, buried tryptophan residue and thus can provide information on the local dynamics of a specific site in the interior of the protein. Amorphous protein films were prepared by spreading concentrated solutions of HSA with and without sugar onto quartz slides, followed by rapid drying and extensive desiccation. Phosphorescence intensity decays were collected and fit with multiple exponential functions. From the average lifetime of these fits the rates of non-radiative decay (kNR) of the triplet state were calculated; kNR is dependent on the microviscosity of the site and is thus a measure of molecular mobility of the HSA tryptophan site. At all temperatures this measure of molecular mobility was lower in the films containing sucrose. Break-point analysis of a kNR Arrhenius plot revealed two temperature regimes with a transition occurring at the glass transition temperature of sucrose. Research supported in part by the National Research Initiative of USDA-CSREES.