Hydration study of globular proteins by microwave dielectric spectroscopy

A microwave dielectric measurement was performed to study the hydration properties of proteins in solution with a precision network analyzer with high reproducibility within the errors of 0.02 in relative dielectric constant over 2 to 10 GHz. A measurement was carried out for catalase, chymotrypsinogen A, cytochrome C, hemoglobin, peroxidase, lysozyme, myoglobin, ovalbumin, and bovine serum albumin at 20.0 +/- 0.01 degreesC. The hydration properties of protein molecules were evaluated based on the Wagner mixture theory combined with single Debye approximation to the complex dielectric constant of hydrated solutes in the above frequency range. This was used to evaluate the loosely bound water number N-w from the single Debye fitting, which gives the relaxation frequency (f(c)) of the hydration shell loosely bound and tightly bound water number N, with lower relaxation frequencies than f(c). Those hydration numbers were compared with the monolayer water numbers accessible to hydrophobic and hydrophilic exposed atoms, respectively, obtained from the calculation of accessible surface area of each protein structure based on the protein database. The result showed a good agreement both in the ratio and numbers.