Hydration of apomyoglobin in native, molten globule, and unfolded states by using microwave dielectric spectroscopy.

The high resolution dielectric spectra of semidilute solutions of apomyoglobin in native (N, pH = 5), acid-induced molten globule (A, pH = 4), and unfolded (U(A), pH = 3) states have been measured in the range from 0.2 to 20 GHz. Based on a two-component mixture theory, we obtained the following hydration numbers per protein molecule: 590 +/- 65 for N, 630 +/- 73 for A, and 1110 +/- 67 for U(A). There was no clear difference between N and A states in contrast to the 25% reduction of helix content and the 50% reduction of heat capacity change upon unfolding. This suggests that the association of hydrophobic moieties might follow the disruption of secondary structures from N to A states. The measured hydration number of U(A) was close to that of the accessible water number (1340) of a protein molecule calculated for a fully extended structure, indicating that the structure of U(A) is extended but somewhat more compact than that of a fully extended state.