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 õ60-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 õ70-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.
The properties of amorphous solid proteins influence the texture and stability of low-moisture foods, the shelf-life of pharmaceuticals, and the viability of seeds and spores. We have investigated the relationship between molecular mobility and oxygen permeability in dry food protein films-bovine α-lactalbumin (α-La), bovine β-lactoblobulin (β-Lg), bovine serum albumin (BSA), soy 11S globulin, and porcine gelatin-using phosphorescence from the triplet probe erythrosin B. Measurements of the phosphorescence decay in the absence (nitrogen) and presence (air) of oxygen versus temperature provide estimates of the non-radiative decay rate for matrixinduced quenching (k TS0 ) and oxygen quenching (k Q [O 2 ]) of the triplet state. Since the oxygen quenching constant is the product of the oxygen solubility ([O 2 ]) and a term (k Q ) proportional to the oxygen diffusion coefficient, it is a measure of the oxygen permeability through the films. For all proteins except gelatin, Arrhenius plots of k TS0 reveal a gradual increase of apparent activation energy across a broad temperature range starting at ∼50°C; this suggests that there is a steady increase in the available modes of molecular motion with increasing temperature within the protein matrix. Arrhenius plots for k Q [O 2 ] were linear for all proteins with activation energies ranging from 24 to 29 kJ/mol. The magnitude of the oxygen quenching constants varied in the different proteins; the rates were approximately 10-fold higher in α-La, β-Lg, and BSA than in 11S glycinin and gelatin. Although the rate of oxygen permeability was not directly affected by the increased mobility of the protein matrix, plots of k Q [O 2 ] versus k TS0 were linear over nearly three orders of magnitude in the protein films, suggesting that the matrix mobility plays a specific role in modulating oxygen permeability. This effect may reflect differences in matrix-free volume that directly influence both mobility and oxygen solubility.
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