Sensing Dielectric Relaxation in Thermal Unfolding of Lipase

The dielectric relaxation illustrates the response of a dielectric material to the external electrical perturbation. There occurs a fractional time delay when the electrically perturbed system returns to the equilibrium state. The time delay is caused by the lag in the emergence and decay of the molecular polarization in the material with respect to the oscillating electric field. The dielectric relaxation time varies with the chemical composition, structure, and the local milieu of the material. The relaxation dynamic provides crucial clues into the structural changes of the complex protein molecule. Thermal unfolding and aggregation of protein manifest as changes in dipole moment, alteration in charge distribution, and the dielectric properties of local ambience near the protein molecules. Herein, we have examined the structural as well as the electrical property changes due to temperature-dependent unfolding through non-faradic impedance spectroscopy using a small quantity of protein (5 micro-liter). The relaxation time was calculated from the impedance spectra. The relaxation time of the protein molecules is found to be decreasing with cold and hot denaturation of lipase enzymatic protein. Henceforth, the decreasing relaxation time corroborates with the gradual functional inactivation of the enzyme due to temperature which was validated by para-Nitrophenol (pNPP) assay. The outcomes of these experiments would provide crucial insights into the structural dynamics of the protein unfolding.