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 mea surements are carried out using a coaxial line dipped in the liquid, a nd 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 prot ein 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 assumption s about the protein volume. However, when we use a crystallographicall y derived value for the volume of myoglobin, we find the density of th e hydration water to be larger than that of the free water. The absenc e of a bound water dispersion above 1 GHz indicates that water molecul es remain rotationally ''frozen'' during their residence time in the h ydration shell. Measurements on pH 4.0 solutions of metMb are consiste nt with a reduced excluded volume in the partially unfolded structure, In conjunction with changes in the solution density, this suggests th e direct detection of a decrease in the protein volume. On the basis o f the success of these measurements, we have also performed a Sensitiv e in situ measurement in an attempt to observe a hydration change prop osed to occur when deoxyHb is oxygenated.