1. Studies of copper(II) binding to glycylglycyl-L-tyrosine-N-methyl amide, a peptide mimicking the NH2-terminal copper(II)-binding site of dog serum albumin by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy
D Muller, B Sarkar, B Decock-Le Révérend J Inorg Biochem . 1984 Jul;21(3):215-26. doi: 10.1016/0162-0134(84)83005-6.
Unlike human serum albumin (HSA), dog serum albumin (DSA) does not possess the characteristics of the specific first binding site for Cu(II). In DSA, the important histidine residue in the third position, responsible for the Cu(II)-binding specificity in HSA, is replaced by a tyrosine residue. In order to study the influence of the tyrosine residue in the third position of DSA, a simple model of the NH2-terminal native sequence tripeptide of DSA, glycylglycyl-L-tyrosine-N-methylamide (GGTNMA) was synthesized and its Cu(II)-binding properties studied by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy. The species analysis indicated the existence of five mono-complexes at different protonation states: MHA, MA, MH-1A, MH-2A, MH-3A, and only one bis-complex MH-2A-2. The complexing ability of GGTNMA to Cu(II) was found to be weaker than that of the Cu(II) binding peptide models of HSA. The visible absorption spectra of Cu(II)-GGTNMA complexes are similar to those observed in the case of DSA-Cu(II) complexes. The weaker binding and the spectral properties of Cu(II)-GGTNMA complexes are consistent with less specific Cu(II)-binding properties of the peptide of this sequence similar to what was noted with DSA. CD results are in excellent agreement with species analysis and visible spectra where it is clearly evident that Cu(II) binds to GGTNMA starting from the alpha-NH2 group and step by step to deprotonated amide nitrogens as the pH is raised. The absence of any charge transfer band around 400 nm strongly indicates that Cu(II) does not bind to the phenolate group. Furthermore, NMR results are consistent with the noninvolvement of the tyrosine residue of GGTNMA in Cu(II) complexation. Thus, it is clear that the low Cu(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the NH2-terminal region of the protein.
2. Nickel(II) binding to glycylglycyl-L-tyrosine-N-methyl amide, a peptide mimicking the NH2-terminal nickel(II)-binding site of dog serum albumin: a 1H- and 13C-nuclear magnetic resonance investigation
B Sarkar, J D Glennon, D W Hughes J Inorg Biochem . 1983 Dec;19(4):281-9. doi: 10.1016/0162-0134(83)80001-4.
The nonspecificity of dog serum albumin (DSA) for Ni(II) is mimicked by the simplest tripeptide, glycylglycyl-L-tyrosine-N-methyl amide, which forms a planar complex at high pH. In this study, the 1H and 13C nuclear magnetic resonance (nmr) spectra of the free and complexed peptide are reported. As the pH is increased for the free peptide, the deprotonation of the terminal amino group (pKa = 7.94) is reflected most strongly by the chemical shift changes of the NH2-terminal -CH2CO- unit. Large upfield and downfield shifts for the tyrosine C xi, C epsilon and C gamma carbon resonances occur on the ionization of the phenolic hydroxyl group. The planar Ni(II) complex is in slow exchange on the nmr time scale and is of 1:1 stoichiometry. The greater chemical shift changes on Ni(II) coordination are observed from the protons nearest the peptide and amino nitrogens:amide CH3 (-0.704), Tyr(3) alpha-CH (-0.667), Gly(1) alpha-CH2 (-0.382), and Gly(2) alpha-CH2 (-0.519, -0.487). In the 13C spectrum, the Gly(1) C alpha (+7.58) is most affected. The Ni(II) ion is therefore at the center of four coordinating nitrogens. Changes in the coupling constants for the Tyr(3) -CH-CH2- moiety suggests a mainly gauche conformation with the tyrosyl ring positioned above the plane of coordination and a weak bonding interaction with the Ni(II) ion is indicated. These results provide structural information regarding the reduced affinity of DSA for Ni(II).
3. The non-specificity of dog serum albumin and the N-terminal model peptide glycylglycyl-L-tyrosine N-methylamide for nickel is due to the lack of histidine in the third position
B Sarkar, J D Glennon Biochem J . 1982 Apr 1;203(1):25-31. doi: 10.1042/bj2030025.
Equilibrium dialysis of dog serum albumin (DSA) against Ni(II) in 0.1 M-N-ethylmorpholine/HCl, pH 7.53, demonstrates the absence of a specific Ni(II)-binding site in DSA. To evaluate at the molecular level the influence of the genetic substitution of L-tyrosine for L-histidine at the N-terminal of DSA, a simple model tripeptide of the N-terminal residues, glycylglycyl-L-tyrosine N-methylamide, was synthesized and its Ni(II)-binding properties studied. A comparison of the visible absorption characteristics of Ni(II)-DSA with those of Ni(II)-glycylglycyl-L-tyrosine N-methylamide reveals a similar change from octahedral to planar co-ordination as the pH is increased. Both systems exhibit a low Ni(II)-binding affinity at physiological pH, with DSA binding a greater percentage of Ni(II) owing to the availability of at least two binding sites of similar affinities. The complex equilibria between Ni(II) and glycylglycyl-L-tyrosine N-methylamide were studied by analytical potentiometry (0.15 M-NaCl, 25 degrees C). Four major complex species, MHA, MH-1A2, MH-2A2 and MH-3A [where M and A represent Ni(II) ion and anionic peptide respectively], were detected, MHA being the single species at physiological pH. There is no evidence for the involvement of the phenolic hydroxy group in the octahedral MHA complex, or within the plane of co-ordination in the high-pH species. The results provide direct evidence that the low Ni(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the N-terminal region of the protein.
