NMR triple-quantum filtered relaxation analysis of ¹⁷O-water in insulin solutions: An insight into the aggregation of insulin and the properties of its bound water

Transverse triple-quantum filtered NMR spectroscopy (TTQF) of ¹⁷O- water was used to study the properties of water in insulin solutions at different Zn²⁺ concentrations and pH values. It was established that strongly bound water molecules are already present in Zn-free insulin. On the assumption that the effective correlation time of a strongly bound ware molecule, τ(sb), is 10 ns, the apparent number of strongly bound water molecules was ~ 3 to 4 per insulin monomer. Addition of Zn²⁺ equivalent to ~ 2 g-atoms per hexamer did not produce substantial increases in the overall ¹⁷O-water TTQF signal intensity and apparent fraction of bound water. The dramatic enhancement of the TTQF signals observed for samples with a Zn²⁺/hexamer ratio greater than ~ 2:1 could be attributed to the increase in correlation time of the strongly bound water, due to the formation of higher-order oligomers of the protein.