An evaluation of the HPLC-Gel chromatographic method for analyzing metallothioneins in aquatic organisms

The use of high-performance liquid chromatography to determine the concentrations of metal-binding proteins (MBP) in freshwater mussels has been evaluated. Initial use of dilute buffers (e.g., 1OmM TRIS, pH 7 or 8), to minimize competition between the buffer and the metal-cytosolic ligand complexes, proved unacceptable; high losses of low molecular weight marker proteins occurred during chromatographic separation, presumably as a result of adsorption to the gel-permeation column packing. Losses were more dependent on salt than pH; satisfactory recoveries were obtained in the pH range 6-8 with 1OmM buffer solutions and 100mM added electrolyte (NACl; KCl). Competition experiments performed with commercial metallothionein (pre-labelled with (109)Cd) and fresh mussel cytosol extract demonstrated that no appreciable metal exchange occurred during the 20-min pre-equilibration or the subsequent chromatographic separation step. With (203)Hg-labelled metallothionein occasional losses were noted, however, appreciable loss of the radioisotope ranging from 50 to 58% did occur with (65)Zn-labelled metallothionein during the chromatographic separation step. These results, particularly for Zn indicate that the recovery of metals after separation using this chromatographic method may vary, even for metals sharing similar chemical properties.