Copper(II)-binding equilibria in human blood

It has been reported that Cu(II) ions in human blood are bound mainly to serum albumin (HSA), ceruloplasmin (CP), alpha-2-macroglobulin (alpha 2M) and His, however, data for alpha 2M are very limited and the thermodynamics and kinetics of the copper distribution are not known. We have applied a new LC-ICP MS-based approach for direct determination of Cu(II)-binding affinities of HSA, CP and alpha 2M in the presence of competing Cu(II)-binding reference ligands including His. The ligands affected both the rate of metal release from Cu center dot HSA complex and the value of K-D. Slow release and K-D = 0.90 pM was observed with nitrilotriacetic acid (NTA), whereas His showed fast release and substantially lower K-D = 34.7 fM (50 mM HEPES, 50 mM NaCl, pH 7.4), which was explained with formation of ternary His center dot Cu center dot HSA complex. High mM concentrations of EDTA were not able to elicit metal release from metallated CP at pH 7.4 and therefore it was impossible to determine the K-D value for CP. In contrast to earlier inconclusive evidence, we show that alpha 2M does not bind Cu(II) ions. In the human blood serum similar to 75% of Cu(II) ions are in a nonexchangeable manner bound to CP and the rest exchangeable copper is in an equilibrium between HSA (similar to 25%) and Cu(II)-His-Xaa ternary complexes (similar to 0.2%).