The relative self-diffusion coefficients D/Do, of water in various solutions, in fresh barnacle muscle fibers, and in membrane-damaged fibers equilibrated with several media have been estimated from NMR relaxation rates in the presence of applied field gradients. A model has been developed to account for the contributions to the observed reduction in D/Do from small organic solutes, and from the hydration and obstruction effect of both soluble macromolecules and myofilament proteins. Intracellular ions do not affect D/Do, but all tested organic solutes do. Solute effects are additive. When artificially combined in the proportions found in barnacle muscle ultracentrifugate (measured D/Do = 0.77), organic acids, small nitrogenous solutes, and proteins give D/Do = 0.77. After correcting the D/Do measured in fibers for this value, we calculate the myofilament hydration, Hm, in fresh muscle to be 0.65 g H2O/g macromolecule. Only in membrane-damaged fibers, highly swollen by salt-rich media, was this significantly increased. Because our earlier NMR relaxation measurements indicate only 0.07 g H2O bound/g myofilament protein, we conclude that the "hydration" water measured by reduction of D/Do cannot be described by stationary layers of water molecules; instead, we propose that nonpolar groups on the proteins cause extensive, hydrophobically-induced interactions among a large fraction of solvent molecules, slowing their translational motion.

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Water in barnacle muscle has been studied using NMR techniques. Fresh fibers are compared with membrane-damaged fibers treated with solutes that greatly alter fixed charge and total water content. Both water (97%) and solute (3%) protons are visible in continuous wave spectra of oriented fresh fibers. No local field inhomogeneities were detected, nor are cell solutes significantly bound. In pulse experiments, all cell water is visible and exhibits a single exponential decay. In fresh fibers, T2 approximately or equal to 40 ms; faster decaying signals are assigned to immobile and mobile protons on macromolecules. T1 and T1p are frequency dependent. Using equations derived for a two-compartment model with fast exchange, we calculate the following: tau b, the correlation time for anisotropic rotational motion of bound water; Sb, its order parameter; tau ex, the correlation time for exchange between bound and free fractions; f, the fraction of water bound; and Hr, the grams of water bound per gram of macromolecule. Whereas f varies inversely with total water content, the other parameters are virtually constant, with values: tau b approximately or equal to 1.3 X 10(-8) S; tau ex approximately or equal to 8 X 10(-6) s; Sb approximately or equal to 0.06; and Hr approximately or equal to 0.1g H2O/g macromolecule. Thus, the NMR relaxation detectable properties of water bound to macromolecules are unaffected by solutes that greatly alter the macromolecular surface charge.

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