Minimizing the impact of Joule heating as a prerequisite for the reliable analysis of metal-protein complexes by capillary electrophoresis.

Herein we report on a drastic release of metal ions from the Fe-bound transferrin, and Fe- or Mn-bound lactoferrin, observed upon the increase in the separation voltage during CE-based analysis. To verify whether this process is caused directly by electric field, we developed an Isothermal Voltage Increase approach (IVI), which is the extension of methods reported by Krylov et al. IVI ensures isothermal conditions while increasing separation voltage by a hydrodynamic pushing of the injected sample to the actively cooled capillary section, combined with a rationale choice of cooling temperature, dependent on the value of current. Interestingly, the application of IVI revealed that the previously observed effect was caused solely by the insufficient dissipation of Joule heating - the saturation of each protein remained unchanged despite a significant rise in the electric field. This outcome demonstrates how crucial is to ensure an effective temperature control for preventing systematic errors in the analysis of biomolecular complexes. IVI seems also to be a simple and useful tool for discovering new potential processes that may be stimulated directly by electric field.