Determination of Protein Denaturation and Glass Transition Temperatures Using High-Frequency Time Domain Reflectometry

Hydrated proteins exhibit a broad dielectric loss extending over the frequency range from 1 MHz to 10 GHz which can be decomposed into a number of constituent dispersions. One of these dispersions with a relaxation time of ∼18 ns has been attributed to the relaxation of protein backbone peptide groups in the protein interior. In the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. In the low temperature region, the temperature-dependence of the dispersion magnitude showed a marked increase in gradient at a critical temperature indicating a transition from a relatively rigid to a more mobile protein structure. At higher temperatures, the response increased rapidly, reaching a maximum value at a second critical temperature. Glucose oxidase samples raised above this temperature showed significantly reduced enzyme activity. Both critical temperatures decreased with increasing protein water content. This is consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. Both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals.