Uncapping the N-terminus of a ubiquitous His-tag peptide enhances its Cu2+ binding affinity.

Metal complexes with an N-terminally free and N-terminally acetylated polyhistidine region of Echis ocellatus venom, with an interesting His-rich motif present in numerous metal binding proteins from all kingdoms of life (DHDHDHHHHHHPGSSV-NH2 and Ac-DHDHDHHHHHHPGSSV-NH2) show the role of the free amino group in the thermodynamic enhancement of Cu2+, Ni2+ and Zn2+ binding. In the studied sequences, Cu2+ can be coordinated by different sets of imidazole rings, and a 3-10 helix is detected in close proximity of Cu2+ binding sites. The complexes are more stable than those with a typical His6-tag, despite a similar copper(ii) coordination mode in both cases.

General aspects of metal toxicity.

This review is focused on the general mechanisms of metal toxicity in humans. The possible and mainly confirmed mechanisms of their action are discussed. The metals are divided into four groups due to their toxic effects. First group comprises of metal ions acting as Fenton reaction catalyst mainly iron and copper. These types of metal ions participate in generation of the reactive oxygen species. Metals such as nickel, cadmium and chromium are considered as carcinogenic agents. Aluminum, lead and tin are involved in neurotoxicity. The representative of the last group is mercury, which may be considered as a generally toxic metal. Fenton reaction is a naturally occurring process producing most active oxygen species, hydroxyl radical: Fe(2+) + He2O2 ↔ Fe(3+) + OH(-) + OH(•) It is able to oxidize most of the biomolecules including DNA, proteins, lipids etc. The effect of toxicity depends on the damage of molecules i.e. production site of the hydroxyl radical. Chromium toxicity depends critically on its oxidation state. The most hazardous seems to be Cr(6+) (chromates) which are one of the strongest inorganic carcinogenic agents. Cr(6+) species act also as oxidative agents damaging among other nucleic acids. Redox inactive Al(3+), Cd(2+) or Hg(2+) may interfere with biology of other metal ions e.g. by occupying metal binding sites in biomolecules. All these aspects will be discussed in the review.

Metal transport and homeostasis within the human body: toxicity associated with transport abnormalities.

In this work, latest reports about metal toxicity, transport and homeostasis have been thoroughly described and discussed. Although diseases associated with transport and homeostasis abnormalities are those of great interest, still a variety of the phenomena associated with these processes are under debate. In this paper, we try to summarize the newest theses on this topic, presenting contradictory points of view. We focus on toxic and essential metal pathways crossing and try to follow the exact metal binding molecules within the body and provide insight into the transport mechanism. Special attention is given to the mechanism of action of lately investigated metal transporters.

Gonadoliberin (GnRH) and its copper complex (Cu-GnRH) enzymatic degradation in hypothalamic and pituitary tissue in vitro.

The amount of decapeptide decapeptide gonadoliberin (GnRH) that reaches pituitary gland depends not only on transcriptional, translational and posttranslatonal processes but also on the extent of degradation exerted by specific proteolytic enzymes. The copper-gonadoliberin (Cu-GnRH) complex preserves the native GnRH amino acid sequence but contains Cu(2+) ion bound to the nitrogen atom at the imidazole ring of the His(2). The aim of this study was to determine whether GnRH and Cu-GnRH molecules differ in their susceptibility to proteolysis in male rat hypothalamic and pituitary tissue in vitro. RIA was applied for a time-dependent study based on 0-90 min incubations at 30°C of exogenous peptide (2.5 µg GnRH or Cu-GnRH) in respective hypothalamic/pituitary supernatant and pellet fractions. To compare the protective effect of bacitracin, a competitive PEP inhibitor, incubations were made with (125 µg/sample) or without an inhibitor. In the second experiment 100 µg of GnRH or Cu-GnRH were incubated for 5 h at 37°C in hypothalamic and pituitary tissue in vitro and then HPLC analysis was applied both to characterize the elution pattern of GnRH and Cu-GnRH degradation products as well as to determine their AA composition. In both tissues, Cu-GnRH remained more resistant to enzymatic degradation and fully protected in the presence of bacitracin. In conclusion, the obtained data suggest that copper ion changed GnRH conformation and significantly modified its physiological properties due to a hindered endopeptidases access to specific AA bonds. Therefore, the Cu-GnRH complex might be considered as GnRH analog potentially able to prolong the occupation of a GnRH receptor at the gonadotrope cells.

Nickel(II) binding to Cap43 protein fragments.

Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein.

Molecular mechanism of hydrogen peroxide conversion and activation by Cu(II)-amikacin complexes.

The interactions between Cu(II)-amikacin complexes [Cu(II)-Ami] and hydrogen peroxide were studied by spectroscopy (EPR, UV-vis, CD, XAS) and cyclic voltammetry. A monomer-dimer equilibrium was detected at complex concentrations above 5 mM (log K(dim) = 1.84 +/- 0.03). The dimeric complex undergoes easy, although irreversible oxidation (ca. 0.5-0.6 V) to a Cu(III) species on platinum electrode. However, the monomeric complexes are able to catalyze hydrogen peroxide disproportionation reaction at pH 7.4 in a multistep process, mediated by hydroxyl radicals and involving both Cu(I)/Cu(II) and Cu(II)/Cu(III) redox pairs.

Impact of Cu(II) and Ni(II) on a structure of chiral peptide nucleic acids having four, six and eight thymines in a peptide side chain.

The studies on binding ability of longer chiral peptide nucleic acids (having four, six and eight thymines in a peptide side chain) have shown that the interactions between the nucleic base rings within a ligand molecule have a critical impact on the complex stability. Thymines inserted in the peptide side chain interact with each other as well as with peptide back-bone increasing the structural organization of the cPNA molecule. The metal ion coordination to cPNA, on the other hand, induces a very specific ligand structure, which may have a basic impact on the cPNA self-recognition processes.

Ni(II) and Cu(II) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR.

The tetradecapeptide containing the 10 aminoacid repeated sequence on the C-terminus of the Ni(II)-induced Cap43 protein, was analyzed for Ni(II) and Cu(II) binding. A combined pH-metric and spectroscopic UV-VIS, EPR, CD and NMR study of Ni(II) and Cu(II) binding to the blocked CH3CO-Thr-Arg-Ser-Arg-Ser-His-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-NH2 (Ac-TRSRSHTSEGTRSR-Am) peptide, modeling a part of the C-terminal sequence of the Cap43 protein, revealed the formation of octahedral complexes involving imidazole nitrogen of histidine, at pH 5.5 and pH 7 for Cu(II) and Ni(II), respectively; a major square planar 4N-Ni(II) complex (about 100% at pH 9, log K* = -28.16) involving imidazole nitrogen of histidine and three deprotonated amide nitrogens of the backbone of the peptide was revealed; a 3N-Cu(II) complex (maximum about 70% at pH 7, log K*=-13.91) and a series of 4N-Cu(II) complexes starting at pH 5.5 (maximum about 90% at pH 8.7, log K* = -21.39 for CuH(-3)L), were revealed. This work supports the existence of a metal binding site at the COOH-terminal part of the Cap43 peptide.

Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant.

D,L-Dithiothreitol (DTT), known also as Cleland reagent, is a thiol group protectant, used commonly in peptide and protein chemistry. Therefore, it is often added at high concentrations in preparations of proteins relevant to heavy metal biochemistry. The coordination of five of these metal ions, Zn(II), Cd(II), Pb(II), Ni(II) and Cu(I) to DTT was studied by means of potentiometric titrations, and UV-Vis and NMR spectroscopies. It was found that DTT forms specific and very stable polymeric and monomeric complexes with all of these metal ions, using both of its sulfur donors. The quantitative description of these complexes in solution and the solid state provides the basis for predictions of interference from DTT in studies of metal ion binding of thiol-containing biomolecules.

Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity.

Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance.