Hydration study of proteins in solution by microwave dielectric analysis

We propose a method for evaluating protein hydration which enables one to evaluate the amount of restrained water and distinguish between ''weakly'' and ''strongly'' restrained water on proteins in an aqueous solution using microwave dielectric measurement. Measurements were taken with a precision microwave network analyzer and a thermostated glass cell at 20.0 +/- 0.01 degrees C with an open-end flat-surface coaxial probe. Examined proteins were cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and hemoglobin. The present method is based on the Wagner equation and the Hanai equation for emulsion analysis and assumes that there is ''weakly'' restrained water on proteins which has a simple Debye-type relaxation in the gigahertz region and that the dielectric constants of hydrated solutes at high-frequency limits are given by the electron polarizations of atom groups. This analysis enabled us to characterize more precisely the hydration state of proteins in water.