Molecular Mobility and Oxygen Permeability in Amorphous Bovine Serum Albumin Films

Abstract

We have used phosphorescence from the xanthene probe erythrosin B to characterize the molecular mobility and oxygen permeability as a function of temperature in amorphous solid bovine serum albumin (BSA) films. Analysis of the emission spectrum using a lognormal fitting function provided information on how temperature modulates the emission peak frequency and bandwidth (full width at half maximum). The peak frequency decreased gradually at low and more steeply at high temperature, whereas the bandwidth increased gradually at low and more steeply at high temperature, both changes indicating a softening of the protein matrix at ∼60°C. Phosphorescence intensity decay transients were well fit using a stretched exponential decay function at all temperatures. Lifetimes decreased gradually at low and more steeply at high temperature; Arrhenius analysis of the rate constant for nonradiative collisional quenching indicated an increase in quenching indicative of matrix softening at ∼70°C. The oxygen quenching rate was calculated from a comparison of emission lifetimes in the presence and absence of oxygen. This rate varied linearly with the collisional quenching rate over nearly three orders of magnitude, suggesting that the more global motions that control oxygen translational diffusion are modulated by more local motions that influence collisional quenching of erythrosin. The emission spectrum shifted to higher energy as a function of time following excitation, whereas the phosphorescence lifetime decreased with increasing emission wavelength; both behaviors provided strong evidence for distinct sites within the protein matrix varying in molecular mobility. These results enrich our molecular understanding of the intrinsic mobility of proteins within the amorphous solid phase, provide evidence for a dynamic transition within solid BSA, and provide insight into the molecular mechanisms controlling oxygen diffusion.