An evaluation of the hydration of lysozyme by an NMR titration method.

In this study a new titration method is proposed to study the motional properties of water molecules in conjunction with globular proteins using proton NMR relaxation measurements. The method was applied to the study of the interaction of water with lysozyme and allowed identification of four water fractions-superbound water, polar-bound water, structured water and bulk water - in exchanged equilibrium. The titration demonstrated that 193 water molecules are hydrogen bonded directly to the lysozyme molecule. The combination of structured and bound water extends to 1.4 g H2O per g lysozyme and approx. two to three layers from the surface of the macromolecule. It is proposed that this structured water is related to non-isotropic water rotation in conjunction with hydrophobic patches and directly related to 'hydrophobic bonding' changes. Water amounts greater than 1.4 g H2O per g lysozyme are sufficiently distant from the macromolecule for motion to revert to that typical of water in bulk. The typical correlation times for water motion in the four fraction are: over 10(-6) s (superbound); 10(-9) s (polar bound); 10(-11) s (structured) and 10(-12) s (bulk). These results correlate well with results from other measurement techniques found in the literature.