13Carbon-nuclear magnetic resonance investigation of the Cu(II)-binding to the native sequence peptide representing the Cu(II)-transport site of human albumin. Evidence for the involvement of the beta-carboxyl side chain of aspartyl residue.

A "C nuclear magnetic resonance investigation was undertaken to elucidate the structure of the Cu(I1)transport site of human serum albumin.The studies were carried out with the peptide, L-aspartyl-L-alanylthistidine-N-methyl amide which represents the Cu(II)-transport site of human serum albumin.Information concerning the complexation site was obtained by measuring both the spin-lattice (TI) and spin-spin (T2) relaxation times.For all of the carbon atoms which are affected by Cu(II), the ratio of T1JTzP > > > 7/6; in this condition, the broadening is determined predominantly by a scalar mechanism so that interpretations based on an r " ' dependence are invalid, but it is possible to interpret the line-broadening data in terms of the actual binding of paramagnetic metal.The system is found ta be in fast exchange (pH 7) verified by the observation that from 20-67 "C, TG has a negative temperature dependence, Le. linewidths decreased with increasing temperature.This is also consistent with the fact that linewidths are not affected at 25.16 and 90.54MHz.The curves of the 2 ' ; : versus pH plots were obtained for the I3C resonance of each functional group.The T;; values for Asp COO-, Asp CO, His-C2,4,6, Asp C,, Asp Cg, and His C p show a sharp maximum between pH 3 and 7.But in the wide range of pH 1 to 10, Ala CO, His CO, His C,, Ala C,, NHCH3, and Ala CHa are not affected.The maximum of Asp COO-, Asp Cg, and Asp CO, His-C2,4,E, His C , linewidths were encountered at about pH 4 and pH 6.This is the region where the main Cu(KI) species is formed.It was found that linewidths of Asp CB, serves as a sensitive probe to study the interaction between Asp COOand the paramagnetic metal ion.On the basis of these results, it is proposed that in addition to the four nitrogen ligands (one amino, two peptides, and one imidazole nitrogen) at the Cu(lI)-transport site, there is the involvement of the side chain carboxyl group in a pentacoordinated structure.