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 for m of a well-characterized histidine (his) chelate has been adopted. This te chnique 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 invol ving alpha-NH2 terminal, two deprotonated peptide nitrogens (NH of Ala2, an d NH of His3), and the imidazole nitrogen of His3 in a square planar arrang ement. 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 o f [Cu(II)-DSA] complex at pH 7.3 confirmed the presence of two bonded nitro gens which substantiate the absence of strategically located His3. It has b een suggested that residues of non-nitrogen origin localized in the main bo dy of DSA may be involved in copper binding, which would explain the protec tion from the Sanger reaction.