Protein hydration. Nuclear magnetic resonance relaxation studies of the state of water in native bovine serum albumin solutions

Abstract

N.m.r. relaxation times have been measured for protein and water protons in aqueous solution of native bovine serum albumin (BSA), as a function of protein concentration and temperature. Estimates of protein hydration were obtained from n.m.r. studies on frozen protein solutions. Unambiguous evidence is obtained that most of the water in native BSA solutions has a mobility equal to that of pure water at the same temperature. This exchanges rapidly with a small fraction of water which is bound largely to polar groups of the protein amino acid side chains and which has an average mobility about a hundred-fold lower than that in pure water. The marked increase in water proton relaxation rates and in the rigidity of the protein chains observed at high (>10 %) BSA concentrations is attributed to association of protein molecules. It is shown that the increase in relaxation rates results from an increase in protein hydration on association due to (i) a reversible unfolding of the protein molecule which exposes new sites, and (ii) bridging of water molecules between adjacent protein molecules.