Isolation and two-dimensional 1H-NMR of peptide [1–24] of dog serum albumin and studies of its complexation with copper and nickel by NMR and CD spectroscopy

Abstract

The NH 2-terminal peptide fragment [1–24] of dog serum albumin was obtained by controlled peptic digestion of the protein. The peptide was purified to homogeneity by gel filtration and ion-exchange chromatography. The NMR assignments of the protons of the individual amino acid residues were made by using two-dimensional correlation matrix, spin-decoupling experiments and analysis of the titration curves. The polypeptide itself has a random-coil conformation. There is a conformational change as a function of pH, but it does not arise from any direct involvement of the amino acid side chains. Complexation of the peptide fragment with Ni(II) and Cu(II) has been investigated by NMR and CD. The Ni(II) complex is in slow exchange with the free ligand on the NMR time scale. The complexation involves the α-NH 2, three deprotonated amide nitrogens of Ala-2, Tyr-3 and Lys-4 residues. The phenolate oxygen of Tyr-3 is not involved in the metal binding; however, an interaction between the aromatic ring and the metal ion is likely. The CD results of Cu(II)-binding to this peptide suggest that the complexation takes place from the terminal NH 2 and step by step to three deprotonated amide nitrogens. There is no major conformational change of the peptide fragment upon complexation.