SUPERCRITICAL-FLUID EXTRACTION WITH CARBON-DIOXIDE FOR THE CHARACTERIZATION OF CADMIUM, COPPER AND ZINC BOUND TO METALLOTHIONEINS

Supercritical fluid extraction with carbon dioxide (SC-CO2) was invest igated as a method for characterizing the cadmium, zinc and copper bou nd to metallothionein (MT) that had been isolated from rabbit liver. I n the presence of tetrabutylammonium dibutyldithiocarbamate (TBADBDTC) complexing reagent, Cd was recovered quantitatively if the MT had bee n dissolved in water but none was recovered if the solid protein was e xtracted 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 c onditions 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 w as apparently unchanged as judged by size exclusion chromatography. Va riations in the recoveries of protein-bound analytes under different e xtractor operating conditions suggested that firstly, even the two iso forms of the substrate protein were heterogeneous with respect to anal yte metal loadings and secondly, the low recoveries were principally t he result of pH induced changes in protein conformation that were reve rsible. The approach of supercritical fluid extraction (with or withou t added complexing reagent) coupled with on-line detection by atomic a bsorption spectrometry shows great promise as a speciation technique f or protein bound heavy metals.