High-Affinity Binding Site for Copper(II) in Human and Dog Serum Albumins (an EPR Study)

Spectroscopic studies have been performed to investigate the high-affinity binding site for copper in human serum albumin (HSA) and dog serum albumin (DSA). A new approach based on exposure to albumin of the copper in the form of a well-characterized histidine (his) chelate has been adopted. This technique has been shown to minimize interaction at the lower affinity sites. The analysis of the S-band EPR spectrum of [Cu(his) 2 ] at pH 7.3 revealed the major component is a complex formed with two histidines in a histamine-like coordination. Detailed analysis of S-band and X-band EPR and optical spectra of [Cu(II)−HSA] revealed that copper forms a complex with HSA involving α -NH 2 terminal, two deprotonated peptide nitrogens (NH of Ala2, and NH of His3), and the imidazole nitrogen of His3 in a square planar arrangement. The spectral data were found to be independent of pH in the range 4.5−9.0 and did not confirm axial Asp1 carboxylate chelation. The EPR study of [Cu(II)−DSA] complex at pH 7.3 confirmed the presence of two bonded nitrogens which substantiate the absence of strategically located His3. It has been suggested that residues of non-nitrogen origin localized in the main body of DSA may be involved in copper binding, which would explain the protection from the Sanger reaction.