Brigitte Decock-Le Reverend

Specific interactions of the β-carboxylate group of the aspartic acid residue in oligopeptides containing one, two or three such residues with copper(II) ions. A potentiometric and spectroscopic study

Results are reported of a study of the influence of Asp and Glu residues in a peptide chain on the ability of the peptide to co-ordinate to copper(II) ions. The tetrapeptides Asp-Ala-Ala-Ala, Ala-Asp-Ala-Ala, Ala-Ala-Ala-Asp, Asp-Ala-Ala-Asp, Ala-Asp-Asp-Ala, Ala-Asp-Ala-Asp, Ala-Ala-Asp-Asp, Glu-Ala-Ala-Ala, Ala-Glu-Ala-Ala and Ala-Ala-Glu-Ala and the tripeptide Asp-Asp-Asp have been synthesised and their complexes with H+ and Cu2+ ions studied using potentiometry and spectroscopy (visible, CD and ESR). Results show that while the Glu residue has little influence on co-ordination equilibria, an N-terminal Asp residue stabilizes significantly the copper(II) complex with only one nitrogen atom co-ordinated (1N) as a result of chelation through the β-carboxylate group rather than the peptide CO oxygen. This stabilization is much greater than with aspartic acid itself. Aspartic acid residues in the second or third positions of the peptide sequence stabilize 2N and 3N complexed species respectively, delaying significantly and, in some cases preventing completely, formation of 4N complexes. An Asp residue in the fourth position has a much smaller effect.

Circular dichroism study

SummaryN methacryloyl-S-methyl-L-cysteine (SMC) homopolymer along with a copolymer with methacrylic acid were prepared in the purpose of studying their complexation with Pd(II). The circular dichroism study showed that the binding of SMC residue to a polymeric chain does not change the coordination donor set of Pd complexation, i.e. ¦N, s ¦. The number of side chains per metal ion involved in the complexation depends on the molar fraction of SMC in the case of the copolymer and on the SMC/Pd ratio for both homopolymer anc copolymer. The variation of viscosity when adding Pd(II) suggests the formation of intramolecular loops.

NMR and CD studies of palladium(II) complexes with S-methyl-L-cysteine containing peptides

Abstract The studies on Pd(II) complexes with Gly-SMC, SMC-Gly and SMC-SMC have shown that S-methyl-L-cysteine as a residue in dipeptide ligands binds palladium ions by {N,S} donor set, creating a new chirality center on sulfur atom. The conformation of SMC in the formed complexes seems to be different when it acts as a dipeptide residue, as compared to that found in PdSMC complex. The presence of vicinal N-Terminal residue ( e.g. Gly in PdGly-SMC) stablizes the δ conformer of the SMC chelate ring, as well as changing the kinetics of the sulfur inversion in palladium complexes. The formation of the metal thioether sulfur bond enriches considerably the UV region of the CD spectra where the S → Pd(II) charge transfer (as well as intra sulfur transition) could be observed.

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

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.

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

The NH2-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 alpha-NH2, 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 NH2 and step by step to three deprotonated amide nitrogens. There is no major conformational change of the peptide fragment upon complexation.

A potentiometric and spectroscopic study of the interaction of the N terminal tetrapeptide fragment of fibrinopeptide A with CuII and NiII

The syntheses are reported of the amino-terminal tetrapeptide fragment of human fibrinopeptide A (Ala-Asp-Ser-Gly) and derivative tetrapeptides with the β-carboxylate of the aspartate residue and the hydroxy group of the serine residue blocked to prevent co-ordination to metal ions. Complexes with hydrogen ion and copper(II), and in the case of Ala-Asp-Ser-Gly with nickel(II) also, have been studied by a combined approach of potentiometric and spectroscopic [absorption, circular dichroism, e.s.r. and, with nickel(II), 1H n.m.r.] techniques. Stability constants of copper(II) and nickel(II) complexes of ‘tetra-alanine’(Ala-Ala-Ala-Ala) have also been measured. The β-carboxylate group has been shown to co-ordinate strongly to CuII over the pH range 4–9 to give an unusually stable [CuH–1L] species (overall charge omitted). Fibrinopeptide A, which is present in locally high concentrations near the sites of bodily injury, would similarly be expected to co-ordinate strongly to CuII.

The interaction of Cu(II) with Phe-Phe-Ser-Asp-Lys and other peptides containing the Phe-Phe sub-unit

Abstract The synthesis of Phe-Phe-Ser-Asp-Lys is reported together with the results of a potentiometric and spectroscopic (ESR, CD and UV-Vis) study of its complexes with H + and Cu 2+ , together with complexes of Phe-Phe and Phe-Phe-Phe. Evidence is presented for axial coordination of the β-carboxyl group of the Asp residue.

A potentiometric and spectroscopic study of the interaction of angiotensin II and some of its peptide fragments with copper(II)

The synthesis is reported of the peptide fragments of angiotensin II, Asp-Arg-Val-Tyr and MeCO-Tyr-IIe-His, together with the results of a potentiometric and spectroscopic (visible, circular dichroism, and e.s.r.) study of the interaction with CuII of angiotensin II itself and the peptides. Results show that, in the pH range 6–8, CuII appears to co-ordinate to the imidazole nitrogen of the histidyl side-chain together with neighbouring deprotonated peptide nitrogens. When the pH is raised above 8 the co-ordination centre moves to give a complex resembling that found in simple peptides, i.e. through the terminal amino nitrogen and its neighbouring peptide nitrogens.

Studies of peptide analogues of the copper(II)-transport site of dog serum albumin

Abstract The transport protein for Cu(II) in serum is albumin. Unlike human serum albumin (HSA), dog serum albumin (DSA) does not possess the characteristics of the specific first binding site for Cu(II) [1]. Results with DSA in the presence of 1 Cu(II) strongly suggest the partitioning of the first Cu(II) between two sites. However, the NH 2 -terminal site of DSA still seems to be the preferred site. Copper(II) bound to this site appears to be the transport form of Cu(II) in dog serum. The amino acid sequence analysis at the NH 2 -terminal region of DSA showed that the important histidine residue in the third position, responsible for the Cu(II)-binding specificity in HSA, is replaced by a tyrosine residue in DSA [2]. In order to study the influence of the tyrosine residue in the third position of DSA, Cu(II)-binding studies are carried out with glycylglycyl-L-tyrosine-N-methyl amide (GGTNMA) using CD and 13 C-NMR spectroscopy. Furthermore, the 24-residue peptide fragment from the NH 2 -terminal (P 24 ) of DSA has been obtained in pure form to study the nature of the Cu(II)-transport site of DSA. Experimental The peptide glycylglycyl-L-tyrosine-N-methyl amide was synthesized according to the previously published procedure [3]. The CD spectra were recorded on a JASCO JV1A spectrometer using a Cu(II) concentration of 5 × 10 −3 M (Cu(II):Peptide = 1:2). The 13 C-NMR spectra were obtained on a Nicolet 360 at 90.54 MHz and on a Bruker WP80SY at 20.15 MHz using a Cu(II):peptide ratio of 1:500 and Cu(II) concentration of 10 −1 M . The P 24 was obtained by controlled peptic digestion and purified by Sephadex G-25 fractionation and Celex D-ion exchange chromatography. Results and Discussion A variation was observed in the d–d transition band energy as the pH of the Cu(II)-GGTNMA solution was raised indicating the progressive involvement of the nitrogens around the Cu(II) nucleus. No charge transfer transition O − Cu(II) was observed suggesting that no phenolic oxygen is involved in the binding. In the 13 C-NMR investigation of Cu(II)-binding to GGTNMA at pH 7.9, the temperature variation and corresponding T 2 measurements established that the fast exchange limit was obtained. At pH 7.9, broadening of the first CO and CH 2 of both glycine residues was observed. This would imply the Cu(II) coordinated to the α-NH 2 and the first peptide nitrogen. No line broadening of the tyrosine ring carbons was observed which is consistent with our earlier observation that tyrosine group is not involved in the Cu(II) binding to DSA. The chemical shifts of the side chains of the amino acid residues of P 24 have been assigned by 13 C- and 1 H-NMR experiments and the Cu(II)-binding studies are currently underway.