Dynamic properties of bound water studied through macroscopic water relaxations in concentrated protein solutions

Magnetization transfer experiments using an off-resonance irradiation technique were performed on bovine serum albumin solutions by varying the irradiation frequency and concentration. Observed macroscopic magnetizations of water protons were well expressed by the rate equations of populations for spins, which gave the tumbling time of protein protons and the intrinsic relaxation rates of water and protein protons. These parameters conformed to a model of rapid-exchange water system with bound water molecules in the interior of the protein that interact with protein protons. Analysis of the data enabled the separation of relaxation rates into the respective contributions by the interior bound water and water in the hydration layer at the protein surface, and determined the amounts and the average correlation times of these water fractions. The average residence time of the interior bound water with respect to exchange with the bulk water was found to be (5 +/- 2).10(-6) s. The estimation of the hydration layer showed excellent agreement with the amount measured by a thermodynamical method.