NMR TRIPLE-QUANTUM FILTERED RELAXATION ANALYSIS OF O-17-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 O-17-wat er was used to study the properties of water in insulin solutions at d ifferent Zn2+ concentrations and pH values. it was established that st rongly bound water molecules are already present in Zn-free insulin. O n the assumption that the effective correlation time of a strongly bou nd water molecule, tau(sb). is 10 ns, the apparent number of strongly bound water molecules was similar to 3 to 4 per insulin monomer. Addit ion of Zn2+ equivalent to similar to 2 g-atoms per hexamer did not pro duce substantial increases in the overall O-17-water TTQF signal inten sity and apparent fraction of bound water. The dramatic enhancement of the TTQF signals observed for samples with a Zn2+/hexamer ratio great er than similar to 2:1 could be attributed to the increase in correlat ion time of the strongly bound water, due to the formation of higher-o rder oligomers of the protein. (C) 1998 Elsevier Science B.V.