Copper-binding proteins in human erythrocytes: Searching for potential biomarkers of copper over-exposure

The recognition that copper is essential but also potentially toxic to humans has prompted the search for biomarkers of copper excess. The experimental approach followed here involves the isolation and subsequent characterization of copper-binding molecules (CuBP) from human erythrocytes. Incubation (0–60 min) of freshly obtained erythrocytes in the presence of increasing concentrations of copper (10–50 M; as ⁶⁴Cu-histidine) led to time- and concentration-dependent uptake of the radioisotope. A near-maximal incorporation was attained after 20 min, with 45–55% of the radioactivity being recovered in 20,000×g hemolysate supernatants (S-20). ⁶⁴CuBP from S-20 were separated by size exclusion and metal-affinity chromatography. Most radioactivity loaded into a Sephadex G-75 column was recovered in association with molecules of MMr greater than 60 KDa (largely accounted for by hemoglobin; Hb). Only negligible amounts of radioactive Cu were associated with metallothionein. With further purification, the higher MMr ⁶⁴Cu-binding fractions were resolved by Sephadex G-200 into two major peaks. The cpm/g protein ratios of the first peak (high MMr) were proportional to the concentrations of copper presented to the erythrocytes. The second one contained mostly Hb molecules. Proteins from the first peak were concentrated in an affinity chromatography mini-column, suited to trap CuBP. The higher-affinity CuBP were eluted as a single peak which comprised around 60% of the load. An SDS-PAGE analysis of such peak reveals the presence of three bands, of which two are non-hemoglobin Cu-binding proteins. The latter, whose identity remains to be established, had MMr of approximately 30 and 40 KDa, respectively. Preliminary data indicate that the two bands bind ⁶⁴Cu within a range of concentrations, relevant to those expected to occur during copper over-exposure conditions.