Human serum albumin coordinates Cu(II) at its N-terminal binding site with 1 pM affinity

The conditional stability constant at pH 7.4 for Cu(II) binding at the N-terminal site (NTS) of human serum albumin (HSA) was determined directly by competitive UV–vis spectroscopy titrations using nitrilotriacetic acid (NTA) as the competitor in 100 mM NaCl and 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes). The log KNTSc value of 12.0 ± 0.1 was determined for HSA dissolved in 100 mM NaCl. A false log log KNTSc value of 11.4 ± 0.1 was obtained in the 100 mM Hepes buffer, owing to the formation of a ternary Cu(NTA)(Hepes) complex. The impact of the picomolar affinity of HSA for Cu(II) on the availability of these ions in neurodegenerative disorders is briefly discussed.