Characterization of Chloride Ion Binding to Human Serum Albumin Using Cl NMR Null Point Spectral Analysis

Cl NMR null point spectra have been applied to study the binding of chloride ions to human serum albumin. The density matrix formalism was used to simulate the null point spectra. We found that (1) the results were consistent with a weak and a strong class of binding sites; (2) relaxation at the weak sites could be modeled using a single correlation time for the fluctuation of the electric field gradient at the bound site, whereas binding at the strong site was modeled using a two-step model; (3) for the weak sites the enthalpy of dissociation was 20.2 kJ mol⁻¹, and at 310 K the correlation time was 3.4 ns; and (4) for the strong site at 310 K the slow correlation time was 35 ns and the upper limit for the fast correlation time was 0.5 ns, and the dissociation constant for the strong site was determined to be 0.1 M. This study shows that analysis of the fine spectral structure at the null point provides a more detailed source of motional information than the normally used measurements of longitudinal and transverse relaxation. This method also allows more accurate determination of correlation times and enables the interpretation of quadrupolar ion binding data when the relaxation is too nonexponential to allow the use of usual methods.