Stoichiometry between Humate Unit Molecules and Metal Ions in Supramolecular Assembly Induced by Cu2+ and Tb3+ Measured by Gel Electrophoresis Techniques.

Humic acid (HA), a fraction of humic substances, can strongly complex with metal ions to form a supramolecular assembly via coordination binding and other intermolecular forces. However, determining the supramolecular size distribution and stoichiometry between small HA unit molecules constituting HA supramolecules and metal ions has proven to be challenging. Here, we investigated the changes in the size distributions of HAs induced by Cu2+ and Tb3+ ions using unique PAGE for the separation and quantification of HA complexes and metal ions bound, followed by UV-vis spectroscopy and excitation-emission matrix-parallel factor analysis. By determining the concentrations of HA and metal ions, it was possible to estimate the stoichiometry of the HA unit molecule to metal ions in supramolecular complexes. It was found that the supramolecular behaviors of Cu2+ and Tb3+ complexes with HA collected from peat (PAHA) and deep groundwater (HHA) differed. For example, two HHA unit molecules form a supramolecule via cross-linking by a Cu2+ ion in the case of Cu2+-HHA. Our results suggest that this supramolecular stoichiometry is related to the abundance of sulfur atoms in the elemental composition of HHA. Our experimental results and analysis provide new insights into HA supramolecules formed via metal complexation.

Advanced Gel Electrophoresis Techniques Reveal Heterogeneity of Humic Acids Based on Molecular Weight Distributions of Kinetically Inert Cu2+-Humate Complexes.

Humic acids (HAs) play important roles for the fate of metal ions in the environment. Most chemical speciation models involving HAs assume heterogeneous metal ion binding. However, these models also assume that the binding affinities of metal ions with HAs are the same regardless of the molecular weight (MW) ranges of the HAs involved. Here, we develop new polyacrylamide gel electrophoresis (PAGE) techniques to investigate the MW distributions of HAs with strongly complexed Cu2+ ions. By combining contaminant metal-free and high-resolution PAGE for HAs, this work was able to provide accurate MW distributions for the complexed metal ions. The MW distribution of Cu2+ binding ability per quantity of HA indicates that strong metal-binding moieties in HAs are heterogeneous in terms of MW. Coupling of the PAGE techniques with UV-vis and excitation-emission matrix (EEM) spectrometry-parallel factor analysis (PARAFAC) methods revealed new insights into kinetically inert interactions between HAs and Cu2+ ions. By this method, we found that the protein-like fluorescence components in the high- and low-MW regions cooperatively responded through Cu2+ binding. Thus, the advanced gel electrophoresis techniques developed herein are able to shed new light on the heterogeneity of metal binding affinities of HAs in terms of MW.