Supercritical fluid extraction with carbon dioxide for the characterization of cadmium, copper and zinc bound to metallothioneins

Abstract

Supercritical fluid extraction with carbon dioxide (SC-CO₂) was investigated as a method for characterizing the cadmium, zinc and copper bound to metallothionein (MT) that had been isolated from rabbit liver. In the presence of tetrabutylammonium dibutyldithiocarbamate (TBADBDTC) complexing reagent, Cd was recovered quantitatively if the MT had been dissolved in water but none was recovered if the solid protein was extracted directly. In the absence of complexing reagent, approximately 12, 13 and 15% of the MT bound Cd, Zn and Cu was mobilized as protein-bound analyte from 0.1 mol l^–1 NaHCO₃ solution. However, loss of Zn from the protein was extensive. By changing the extractor operating conditions 11% of the Cd–MT, 12% of the Cu–MT and 18% of the Zn–MT was recovered intact in the extractor eluate and the remaining substrate was apparently unchanged as judged by size exclusion chromatography. Variations in the recoveries of protein-bound analytes under different extractor operating conditions suggested that firstly, even the two isoforms of the substrate protein were heterogeneous with respect to analyte metal loadings and secondly, the low recoveries were principally the result of pH induced changes in protein conformation that were reversible. The approach of supercritical fluid extraction (with or without added complexing reagent) coupled with on-line detection by atomic absorption spectrometry shows great promise as a speciation technique for protein bound heavy metals.