DETECTION OF INSULIN AGGREGATES WITH PULSED-FIELD GRADIENT NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Self-association of metal-free human insulin in aqueous solution at pH 9.4 has been studied using pulsed-field gradient (PFG) NMR spectrosco py. The diffusion coefficients were measured for two different insulin concentrations, 0.72 mM in which the dimer is the predominant species and 3 mM which is a mixture of dimer and tetramer. In the more concen trated solution, exchange between the two aggregates is slow on the NM R chemical shift time scale. As a result, the dimer and tetramer give rise to separated resonances in the aromatic region of the H-1 NMR spe ctrum. The diffusion coefficient determined for the tetramer by PFG NM R is 1.07 x 10(-6) cm(2) s(-1) at 298 K, while that for the dimer is 1 .38 x 10(-6) cm(2) s(-1). The hydrodynamic diameters calculated for th e tetramer and dimer are 46 and 36 Angstrom, respectively, which are i n good agreement with those measured by dynamic light scattering. The advantage of PFG NMR for the measurement of insulin aggregation in the se solutions results from the chemical shift selectivity which allows diffusion coefficients to be directly calculated for the different agg regates. The T-1 and T-2 relaxation times of the dimer and tetramer ar omatic protons were also measured in order to optimize the parameters of the PFG NMR experiment and correctly interpret the results of the m easured spectra. (C) 1995 Academic Press, Inc.