Analysis of spectrometry and thermodynamics of the metallothionein in freshwater crab Sinopotamon henanense for its binding ability with different metals

The metal binding nature of heterologously expressed metallothionein of Sinopotamon henanense (ShMT) had been demonstrated previously. In this study, we analysed the stoichiometry of ShMT yielded in vivo and exchange reactions of the Zn-ShMT with Cd2+, Pb2+ and Cu2+ in vitro via electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), and isothermal titration calorimetry (ITC). The results of ESI-TOF-MS analyses showed that metal-ShMT synthesized in vivo had three major forms, namely Zn15-, Cd9-, and Pb5-ShMT. The ITC analyses of exchange reactions demonstrated that Zn-ShMT exhibited up to 6, 6, and 7 binding sites for Cd2+, Pb2+ and Cu2+. By the analyses of the UV and CD spectra in the substitution experiments showed that the geometric structural stability of metal-ShMT could be influenced when excess of over 6, 6, or 7 equivalents of Cd2+, Pb2+, or Cu2+ were added into Zn-ShMT. Although both the reconstructed apo-ShMT and substituted Zn-ShMT with three metal ions fitted the same M6Ⅱ- and M7Ⅰ-ShMT binding models for divalent and monovalent metals, the differences in their thermodynamic data suggested that discrepancies exit in their physiological functions. These results suggested that ShMT yielded in vivo had a higher storage capability for Zn2+ and a uptake ability for Cd2+, and Zn-ShMT was more easy to release Zn2+ as well as to uptake Cd2+, Cu2+, or Pb2+. The results presented in this work will be very valuable to understand the function(s) of ShMT not only in a normal physiological condition but also in the presence of non-essential metals in crabs.