Determination of metal–biomolecule interactions by relative mobility shift partial filling affinity capillary electrophoresis

Abstract

Metal ions and their interactions with biomolecules play an important role in human health. However, optical detectors commonly used for HPCE cannot directly detect metal ions without UV absorption. To make up for the shortcomings of existing HPCE detectors, a new universal HPCE detection system called an interface-induced current detector (IICRD) was constructed previously, with no need for derivatization procedures or complex instrumental modifications. Meanwhile, most of the reported studies on metal–biomolecule interactions only focused on the detection and analysis of biomolecules, commonly causing inaccurate or false-negative results, which is yet to be resolved. Here, the application of HPCE-IICRD realized the determination of metal–biomolecule interactions by directly measuring the electrophoretic parameters of metal ions for the first time, indicating that the interaction intensity can be measured more directly and accurately. Furthermore, an improved affinity capillary electrophoresis (ACE) method called relative mobility shift partial filling ACE-IICRD (rmsPF-ACE-IICRD) was originally developed to quantitatively analyze the binding strength. Binding behaviors between twelve free metal ions and three types of biomolecules (including two blood proteins, two enzyme proteins and two native DNAs) were investigated, and the values of the equilibrium dissociation constant (K_D) of metal–biomolecule complexes were calculated and evaluated by the nonlinear chromatography (NLC) method. The experimental results were basically consistent with the literature values. In particular, heavy metal ions showed stronger interactions with proteins and enzymes, while metal ions tended to show stronger binding with native DNAs than proteins and enzymes, which were in agreement with literature results. The combined use of HPCE-IICRD and rmsPF-ACE showed great advantages such as no need for pretreatment, low operating cost, good repeatability, simple operation and no interference from coexisting substances, which is hopeful to become an efficient metal ion detection method and also to expand the application scope of IICRD in the future.