Mass Spectroscopic Evidence on Inhibiting Effect of Maltosyl–β‐Cyclodextrin on Insulin Self‐association

The effect of maltosyl–β-cyclodextrin (G2-β-CyD) on self-association of insulin was investigated by electrospray ionization mass spectrometry (ESI-MS) coupled with measurement of hydrogen/deuterium (H/D) exchange rates of the peptide. Bovine insulin (MW 5734) gave three signals at 956, 1147 and 1433 corresponding to the [M + 6H]6+, [M + 5H]5+ and [M + 4H]4+ ions, respectively. By the addition of G2-β-CyD, a new signal was observed at 1438 which corresponds to the 1: 1 adduct of penta-ionized insulin with G2-β-CyD, [M + G2-β-CyD + 5H]5+. The H/D exchange rate of insulin was fast in 30% v/v acetic acid solution where the peptide is predominantly in a monomer state, and the rate was unchanged by the addition of G2-β-CyD. However, the exchange rate significantly slowed down in pH 2.0 solution where insulin is predominantly in a dimer state, and the rate increased with increasing G2-β-CyD concentration, indicating that G2-β-CyD shifts the monomer-dimer equilibrium of insulin in favour of the dissociated form. These results suggest that G2-β-CyD inhibits the self-association of insulin through the interaction with hydrophobic amino acid residues of the peptide, and mass spectroscopic technique is a useful means for analysis of protein-CyD interaction.