ESI-MS measurements for the equilibrium constants of copper(II)-insulin complexes

Abstract

Trace elements regulate many biological reactions in the body. Copper(II) is known as one of trace elements and capable of binding to proteins. Insulin is a blood glucose-lowering peptide hormone and it is secreted by the pancreatic B-cells. In this study, Cu(II)-insulin complexes were investigated by using ESI-MS method. Insulin molecule gives ESI-MS peaks at +4, +5, +6 and +7 charged states. Cu(II)-insulin complexes can be monitored and quantified on the ESI-MS spectra as the shifted peaks according to insulin peaks. The solutions of Cu(II)-insulin complexes at different pHs and mole ratios of Cu(II) ions to insulin molecule were measured on the ESI-MS. The highest complex formation ratio for Cu(II)-insulin were found at pH 7. The multiple bindings of Cu(II) ions to insulin molecule was observed. The formation equilibrium constants of Cu(II)-insulin complexes were calculated as Kf(1): 334 x 10(4), Kf(2): 2.99 x 10(4), Kf(3): 7.00 x 10(3) and Kf(4):2.86 x 10(3). The specific binding property of Cu(II) ions was controlled by using different spray ion sources including electrospray and nano-electrospray. The binding property of Cu(II) also investigated by MS/MS fragmentation. It was conduded from the ESI-MS measurements that Cu(II) ion has a high affinity to insulin molecules to form stable complexes. (C) 2018 Elsevier B.V. All rights reserved.