The Absence of Specific Copper(II)-binding Site in Dog Albumin

Unlike human serum albumin (HSA), dog serum albumin (DSA) failed to exhibit the characteristics of a specific first binding site for copper(II). Equilibrium dialysis of DSA against Cu(II) at pH 7.5 indicated two preferred Cu(II)-binding sites of comparable affinities. Equilibrium dialysis of HSA and DSA against Cu(II), in the presence of l-histidine as competing ligand, demonstrated the absence in DSA of the first strong binding site apparent in HSA. The addition of 1 eq of Cu(II) to HSA almost quantitatively protected the terminal α-amino group from reacting with dinitrofluorobenzene at pH 11.0, whereas by the same treatment only 32% protection was afforded to DSA. The Cu(II)-binding properties of HSA and DSA were compared by spectrophotometric and proton displacement studies. HSA was found to have those binding features that have been found to characterize bovine serum albumin. Results with DSA in the presence of 1 and 2 eq of Cu(II) strongly suggested the partitioning of the first equivalent of Cu(II) between at least two sites. At basic pH one site would appear to involve the α-amino nitrogen, consistent with the partial protection of DSA in presence of Cu(II) from NH2-terminal modification by dinitrofluorobenzene. The partitioning of the first equivalent of Cu(II) between two sites seems most pronounced near neutral pH. At high pH the site involving the α-amino ligand is apparently somewhat preferred over the other possible loci. Possible explanations for the absence of the specific Cu(II)-binding site in DSA are discussed.