Investigation of the Hydration of Bovine Serum Albumin by an NMR Titration Method

Abstract

In this study, NMR titration method was used to determine the dynamics of hydration water. Solutions were made by dehydration procedures and relaxation measurements were carried out on a Vnmr (Varian NMR) spectrometer operating at 300 MHz for proton and relaxation rates (1/ T₁) were plotted versus M_s/M_w. It was seen that the NMR titration data was separated into 2 line segments. The intercepts of the first line segment with lower mass of solute/mass of water (M_s/ M_w) ratio and the second line segment with higher M_s/M_w are 2.15 and 2.57, respectively. The correlation times for free and structured water were obtained as 5.56 × 10^-12 s and 1.5 × 10^-11 s, respectively. The 1/T₁ and 1/T₂ relaxation rates increase as the protein concentration increases. This implies that the protein is reducing the motion of water molecules. The correlation times (τ_c) were calculated by using the experimental data and relaxation theory related. These results suggest that the relaxation times of water layers surrounding a protein could be determined by T₁ measured versus M_p/M_w. Present results were correlated well with the results from other measurements techniques. The data also suggest that the relaxation mechanism of bovine serum albumin (or serum proteins) can be explained in terms of fast chemical exchange of protons between bulk water and water bound to proteins.