PROTEIN HYDRATION INVESTIGATIONS WITH HIGH-FREQUENCY DIELECTRIC-SPECTROSCOPY

We utilize dielectric spectroscopic measurements to study hydration in aqueous solutions of myoglobin, hemoglobin, and cytochrome c. The measurements are carried out using a coaxial line dipped in the liquid, and they yield the complex dielectric function epsilon(omega) = epsilon'(omega) - i epsilon''(omega) between 45 MHz and 20 GHz, which can be used to determine the dielectric relaxation spectra of solute-modified free water in solutions. When combined with accurate density and protein concentration measurements, the dielectric excluded volume yields absolute values for the hydration number per protein molecule, N-hyd = 340 +/- 60 on average for metMb, N-hyd = 1030 +/- 70 for HbO(2), and N-hyd = 180 +/- 40 for Cc(3+), which are independent of any assumptions about the protein volume. However, when we use a crystallographically derived value for the volume of myoglobin, we find the density of the hydration water to be larger than that of the free water. The absence of a bound water dispersion above 1 GHz indicates that water molecules remain rotationally ''frozen'' during their residence time in the hydration shell. Measurements on pH 4.0 solutions of metMb are consistent with a reduced excluded volume in the partially unfolded structure, In conjunction with changes in the solution density, this suggests the direct detection of a decrease in the protein volume. On the basis of the success of these measurements, we have also performed a Sensitive in situ measurement in an attempt to observe a hydration change proposed to occur when deoxyHb is oxygenated.