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

摘要

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.