Protein-bound water molecule counting by resolution of H-1 spin-lattice relaxation mechanisms

Water proton spin-lattice relaxation is studied in dilute solutions of bovi ne serum albumin as a function of magnetic field strength, oxygen concentra tion, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurat ely Lorentzian with an effective correlation time of 41 +/- 3 ns when measu red at low proton and low protein concentrations to minimize protein aggreg ation. Elimination of oxygen flattens the relaxation dispersion profile abo ve the rotational inflection frequency, nearly eliminating the high field t ail previously attributed to a distribution of exchange timer; for either w hole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a t ime scale of order one ns. Measurements as a function of isotope compositio n permit separation of intramolecular and intermolecular relaxation contrib utions. The magnitude of the intramolecular proton-proton relaxation rate c onstant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correla tion time of 42 ns. This number of bound water molecules neglects the possi bility of focal motions of the water in the binding site; inclusion of thes e effects may increase the number of bound water molecules by 50%.