QUANTITATIVE-DETERMINATION OF METALLOTHIONEIN-LIKE PROTEINS IN MUSSELS - METHODOLOGICAL APPROACH AND FIELD-EVALUATION

Electrochemical quantitation of metallothionein-like proteins (MLP) in mussels was based on the determination of their constituent cysteinyl residues according to Brdicka's catalytic reaction. Calibration was performed by an internal MLP standard isolated from the digestive gland of Mytilus galloprovincialis for which protein concentration had been estimated by Bradford's spectrophotometric method. For that purpose three metal-binding proteins [MLP-I, MLP-II and Cu-BP (binding protein)] were separated by DEAE-Sephadex A-25 chromatography from the digestive gland of mussels previously exposed to Cd. The most negatively charged MLP-II fraction was characterized by the fact that it contained the largest amount of both total metal and sulphydryl (-SH) content per mass of protein, although this was approximately two times lower than the -SH level of commercially available MT from rabbit liver. Exposure of mussels to a relatively low level of cadmium (0.2 mug Cd l-1) added into the seawater either by itself or as a mixture with other metals (2 mug Cu l-1 and 1.6 mug Pb l-1) resulted in a measurable level of MLP induction within 14 d in comparison to the control specimens. The effect of the metal mixture on MLP synthesis appears to be less than additive, suggesting a competitive interaction between metal ions for uptake and binding sites as well as differing potentials for MLP induction. Variations in the MLP content observed in the digestive gland of mussels seasonally collected from the same location are in the range 2.1 +/- 0.4 mg g-1 on a wet weight basis. The methodological and conceptual aspects of the application of MLP induction in the Mytilus sp. as a biomarker in seawater trace metal monitoring are critically evaluated.