Single-Tryptophan Phosphorescence of Human Serum Albumin Embedded in Sugar Matrices: Protein Dynamics on the Millisecond to Second Timescale

Abstract

Long-term stability of amorphous biomolecules is often improved via the inclusion of small-molecule excipients. The effect on slow protein dynamics caused by substitution of a protein’s surface water molecules with small sugar molecules is unclear. To explore this question, we have conducted a series of luminescence studies on human serum albumin (HSA) in the dry amorphous solid state. Tryptophan phosphorescence is an ideal approach, as the long-lived triplet state is sensitive to the long timescale molecular motions of the protein in the dry state. The experiments provide evidence that slow protein dynamics are coupled to those of the embedding sugar matrix. HSA also binds small molecular probes which may report a variety of information at a site on the protein’s surface. The water-sensitive charge-transfer probe 8-hydroxypyrene 1,3,6-trisulfonate (pyranine) was bound to HSA and its fluorescence spectra were measured to extract information on the amount of the water in the protein’s hydration shell. Dry films from sugars doped with both magnesium ion and glycerol were also made to explore the effect of osmolyte and plasticizer. Magnesium sequestered water from the protein’s hydration shell and drastically slowed protein dynamics. Glycerol, which is known to have a rigidifying effect on the ps timescale, acts as a plasticizer of protein dynamics on the ms to s timescale.