Luminescence from Extrinsic and Intrinsic Probes in Solid State Amorphous Human Serum Albumin Demonstrates Solvent-Protein Slaving

Abstract

The physical properties of amorphous biomolecules are important to the stability of low-moisture foods and pharmaceuticals. Freeze-dried proteins are often stabilized via inclusion of excipients. The effect on protein dynamics of substitution of surface water with sugars is unclear. To explore this question, we have conducted luminescence studies on human serum albumin (HSA) in the dry amorphous state using both extrinsic probes and intrinsic tryptophan. Phosphorescence is an ideal approach, as the long-lived triplet state of molecular probes is sensitive to the long time-scale motions of dry proteins. HSA binds luminescence probes that report on the protein's surface; it contains a single, buried tryptophan that reports on the interior of the protein. Amorphous protein-sugar films were prepared by spreading concentrated solutions of sugar + HSA with bound probe onto quartz slides, followed by rapid drying and extensive desiccation. We have bound the water-sensitive probe pyranine to HSA and measured its fluorescence spectra to extract information on the amount of the water in the protein's hydration shell. We have also made dry films from sugars + HSA bound with erythrosin B or vanillin and collected these probe's phosphorescence decays as a function of temperature. These measurements are compared to those of HSA's buried tryptophan residue. Intensity decays were fit with multi-exponential functions and the rates of non-radiative decay (kNR) were calculated from the average lifetime; kNR is dependent on the microviscosity of the site and is thus a measure of local molecular mobility. The degree to which the fits are non-exponential reveals dynamic heterogeneity experienced by the probe. Analysis of kNR Arrhenius plots reveal relations between the mobility in the bulk solid solvent with that at the surface and in the interior of the protein.