Faculty of Baiotechnology

Glutathione, ),-Glu-Cys-Gly, is one of the most abundant small molecules in biosphere. Its main form is the reduced monomer (GSH), serving to detoxicate xenobiotics and heavy metals, reduce protein thiols, maintain cellular membranes and deactivate free radicals. Its oxidized dimer (GSSG) controls metal content of metallothionein. The results presented provided a quantitative and structural description of Zn(II)glutathione complexes, including a novel ternary Zn(II)-GSH-His complex. A solution structure for this complex was obtained using 2D-NMR. The Complexes studied may contribute to both zinc and glutathione physiology. In the case of Ni(ll) complexes an interesting dependence of coordination modes on the ratios of reactants was found. At high GSH excess a Ni(GSH)2 complex is formed, with Ni(ll) bonded through S and N and/or O donor atoms. This complex may exist as a high-or low-spin species. Another goal of the studies presented was to describe the catalytic properties of Ni(II) ions towards GSH oxidation, which appeared to be an important step in nickel carcinogenesis. The pH dependence of oxidation rates allowed to determine the Ni(GSH)2 complex as the most active among the toxicologically relevant species. Protonation and oxidation of metal-free GSH and its analogues were also studied in detail. The monoprotonated form HL 2 of GSH is the one most susceptible to oxidation, due to a salt bridge between Sand NH3 groups, which activates the thiol.

Free zinc ions are potent eVectors of proteins. Their tightly controlled Xuctuations ("zinc signals") in the picomolar range of concentrations modulate cellular signaling pathways. Sulfur (cysteine) donors generate redox-active coordination environments in proteins for the redox-inert zinc ion and make it possible for redox signals to induce zinc signals. Amplitudes of zinc signals are determined by the cellular zinc buVering capacity, which itself is redox-sensitive. In part by interfering with zinc and redox buVering, reactive species, drugs, toxins, and metal ions can elicit zinc signals that initiate physiological and pathobiochemical changes or lead to cellular injury when free zinc ions are sustained at higher concentrations. These interactions establish redox-inert zinc as an important factor in redox signaling. At the center of zinc/redox signaling are the zinc/thiolate clusters of metallothionein. They can transduce zinc and redox signals and thereby attenuate or amplify these signals. (W. Maret).

Cerebrocortical neurons that store and release zinc synaptically are widely recognized as critical in maintenance of cortical excitability and in certain forms of brain injury and disease. Through the last 20 years, this synaptic release has been observed directly or indirectly and reported in more than a score of publications from over a dozen laboratories in eight countries. However, the concentration of zinc released synaptically has not been established with final certainty. In the present work we have considered six aspects of the methods for studying release that can affect the magnitude of zinc release, the imaging of the release, and the calculated concentration of released zinc. We present original data on four of the issues and review published data on two others. We show that common errors can cause up to a 3000-fold underestimation of the concentration of released zinc. The results should help bring consistency to the study of synaptic release of zinc.

“Free Zn2+” (rapidly exchangeable Zn2+) is stored along with glutamate in the presynaptic terminals of specific specialized (gluzinergic) cerebrocortical neurons. This synaptically releasable Zn2+ has been recognized as a potent modulator of glutamatergic transmission and as a key toxin in excitotoxic neuronal injury. Surprisingly (despite abundant work on bound zinc), neither the baseline concentration of free Zn2+ in the brain nor the presumed co-release of free Zn2+ and glutamate has ever been directly observed in the intact brain in vivo. Here, we show for the first time in dialysates of rat and rabbit brain and human CSF samples from lumbar punctures that: (i) the resting or “tonic” level of free Zn2+ signal in the extracellular fluid of the rat, rabbit and human being is approximately 19 nM (95% range: 5–25 nM). This concentration is 15,000-fold lower than the “300 μM” concentration which is often used as the “physiological” concentration of free zinc for stimulating neural tissue. (ii) During ischemia and reperfusion in the rabbit, free zinc and glutamate are (as has often been presumed) released together into the extracellular fluid. (iii) Unexpectedly, Zn2+ is also released alone (without glutamate) at a variable concentration for several hours during the reperfusion aftermath following ischemia. The source(s) of this latter prolonged release of Zn2+ is/are presumed to be non-synaptic and is/are now under investigation. We conclude that both Zn2+ and glutamate signaling occur in excitotoxicity, perhaps by two (or more) different release mechanisms.

Fundamental issues in zinc biology are how proteins control the concentrations of free Zn(II) ions and how tightly they interact with them. Since, basically, the Zn(II) stability constants of only two cytosolic zinc enzymes, carbonic anhydrase and superoxide dismutase, have been reported, the affinity for Zn(II) of another zinc enzyme, sorbitol dehydrogenase (SDH), was determined. Its log K is 11.2 ± 0.1, which is similar to the log K values of carbonic anhydrase and superoxide dismutase despite considerable differences in the coordination environments of Zn(II) in these enzymes. Protein tyrosine phosphatase 1B (PTP 1B), on the other hand, is not classified as a zinc enzyme but is strongly inhibited by Zn(II), with log K = 7.8 ± 0.1. In order to test whether or not metallothionein (MT) can serve as a source for Zn(II) ions, it was used to control free Zn(II) ion concentrations. MT makes Zn(II) available for both PTP 1B and the apoform of SDH. However, whether or not Zn(II) ions are indeed available for interaction with these enzymes depends on the thionein (T) to MT ratio and the redox poise. At ratios [T/(MT + T) = 0.08-0.31] prevailing in tissues and cells, picomolar concentrations of free Zn(II) are available from MT for reconstituting apoenzymes with Zn(II). Under conditions of decreased ratios, nanomolar concentrations of free Zn(II) become available and affect enzymes that are not zinc metalloenzymes. The match between the Zn(II) buffering capacity of MT and the Zn(II) affinity of proteins suggests a function of MT in controlling cellular Zn(II) availability.

The results are presented of measurements of protonation constants (potentiometry and NMR), UV spectroscopic properties and redox potentials of GSH and its five analogues, which are modified at the C-terminal glycine residue (γGlu-Cys-X, X = Gly, Gly-NH 2 , Gly-OEt, Ala, Glu, Ser). Strong linear correlations were found between various properties of the thiol and other functions of these peptides. These results allow discussion of the relationships between the structures and properties in glutathione and its analogues, and provide a novel chemical background for the issue of control of GSH reactivity.

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.

The hexapeptides AcThrAlaSerHisHisLysNH 2 , AcThrGluAlaHisHisLysNH 2 , AcThrGluSerAlaHisLysNH 2 and AcThrGluSer-HisAlaLysNH 2 which represent modifications of the 120 Á/125 sequence of histone H2A were synthesized and their interactions with Cu(II) ions were studied with potentiometric and spectroscopic (UV Á/Vis, EPR and CD spectroscopy) studies. All peptides coordinate Cu(II) efficiently. At physiological pH, AcThrAlaSerHisHisLysNH 2 forms dimeric species while the rest of the peptides form monomers. The dimer is formed when Cu(II) ions coordinate equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues, plus the imidazole nitrogen of the His-5 residue of a second peptide molecule after deprotonation. At higher pH peptides AcThrAlaSerHisHisLysNH 2 and AcThrGluAlaHisHisLysNH 2 are using the second histidine residue for coordination at the apical position while Cu(II) ions coordinate equatorially with the imidazole nitrogen of His-5 or His-4 and three amido nitrogens with the peptides AcThrGluSerAlaHisLysNH 2 and AcThrGluSerHisAlaLysNH 2 .

GSH and L-His are abundant biomolecules and likely biological ligands for Zn(II) under certain conditions. Potentiometric titrations provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favour of L-His (ca. 1 log unit of stability constant). The solution structure of the ZnH(GSH)(L-His)(H 2 O) complex at pH 6.8, determined by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calculations of competitivity of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(II) carrier.

Acid−base properties and metal-binding abilities of tris(2-carboxyethyl)phosphine (TCEP), a newly introduced thiol group protectant, were studied in solution, using potentiometry, 1 H and 31 P NMR, and UV−vis spectroscopy, and also in the solid state by X-ray diffraction. Stability constants of complexes of the P-oxide of TCEP (TCEPO) were established by potentiometry. The list of metal ions studied included Ni(II), Cu(II), Zn(II), Cd(II), and Pb(II). Cu(II) catalyzed oxidation of TCEP to TCEPO. For all other systems ML complexes were found as major species at neutral pH with TCEP and TCEPO. Monoprotonated MHL species were also detected in weakly acidic conditions for all TCEP complexes and for the Pb(II) complex of TCEPO, while hydrolytic MH -1 L complexes were found for TCEP at the weakly alkaline pH range. The NiL 4 complex was found to form at excess of TCEP. Overall, the complexes were found to be rather weak, with log ML values around 3−5 for TCEP and 1.5−2.5 for TCEPO. The phosphorus pK a value for TCEP, 7.68, suggests that it can be a good buffer for studies at physiological pH. Figure 9. The comparison of pH-dependent metal-binding abilities of TCEP and DTT, using logarithmic competition plots, calculated for 1 mmol dm -3 TCEP, 1 mmol dm -3 DTT, and 0.1 mmol dm -3 metal ions, using DTT stability constants published previously: 4 (0) M 2+ ; (4) M-DTT; (b) M-TCEP. Krȩz 3 el et al.

The hexapeptides AcThrAlaSerHisHisLysNH2, AcThrGluAlaHisHisLysNH2, AcThrGluSerAlaHisLysNH2 and AcThrGluSerHisAlaLysNH2 which represent modifications of the 120–125 sequence of histone H2A were synthesized and their interactions with Cu(II) ions were studied with potentiometric and spectroscopic (UV–Vis, EPR and CD spectroscopy) studies. All peptides coordinate Cu(II) efficiently. At physiological pH, AcThrAlaSerHisHisLysNH2 forms dimeric species while the rest of the peptides form monomers. The dimer is formed when Cu(II) ions coordinate equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues, plus the imidazole nitrogen of the His-5 residue of a second peptide molecule after deprotonation. At higher pH peptides AcThrAlaSerHisHisLysNH2 and AcThrGluAlaHisHisLysNH2 are using the second histidine residue for coordination at the apical position while Cu(II) ions coordinate equatorially with the imidazole nitrogen of His-5 or His-4 and three amido nitrogens with the peptides AcThrGluSerAlaHisLysNH2 and AcThrGluSerHisAlaLysNH2.Four hexapeptides which represent modifications of the 120–125 sequence of histone H2A were synthesized and their interactions with Cu(II) ions were studied. At physiological pH, -TASHHK- forms a dimer while the rest of the peptides (-TEAHHK-, -TESAHK-, -TESHAK-) form monomers. The structure and stability of the complexes formed are discussed based on spectroscopic data.

A comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, respectively) and short peptide analogues was performed using potentiometry and spectroscopic techniques. It was found that while qualitative aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quantitative parameters (stability and rate constants) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modelling of Cu(II) and Ni(II) transport by albumins.

The interactions of Zn(ll) ions with the blocked hexapeptide models -TESHHK-, -TASHHKand -TEAHHKof the -ESHHmotif of the C-terminal of historic H2A were studied by using potentiometric and IH-NMR techniques. The first step of these studies was to compare the pKa values of the two His residues inside each hexapeptide calculated by potentiometric or H-NMR titrations. Hereafter, the potentiometric titrations in the pH range 5 11 suggest the formation of several monomeric Zn(ll) complexes. It was found that all hexapeptides bind to Zn(ll) ions initially through both imidazole nitrogens in weakly acidic and neutral solutions forming slightly distorted octahedral complexes. At higher pH values, the combination of potentiometric titrations and one and two dimensional NMR suggested no amide coordination in the coordination sphere of Zn(II) ions. Obviously, these studies support that the -ESHHsequence of histone H2A is a potential binding site for Zn(II) ions similarly with the Cu(II) and Ni(ll) ions, presented in previous papers.

The coordination properties of Ni(II), Cu(II) and Zn(II) ions towards the terminally blocked (CH 3 CONH-and -CONH 2 ) hexapeptides -TESHHK-, -TASHHK-, -TEAHHK-, -TESAHK-and -TESHAK-, which are all models of the C-terminal btailQ (-ESHH-) of histone H2A, were studied by potentiometric and several spectroscopic techniques (UV/Vis, CD, NMR, EPR). The peptides were chosen in such a way as to compare the effect of Glu, Ser and His residues on the stability, the coordination and hydrolytic abilities of the complexes formed. It was found that all peptides bind to the metal ions initially through one or two imidazole nitrogens in weakly acidic and neutral solutions forming slightly distorted octahedral complexes.

A linear correlation between pH-meter readings in equivalent D2O and H2O solutions, determined experimentally, leads to a novel equation, which allows for a direct recalculation of pKa values measured in D2O into a H2O equivalent: . The comparison of this equation with the previously used approach is discussed.

Previously we demonstrated that Ni(II) complexes of Ac-Thr-Glu-Ser-His-His-Lys-NH 2 hexapeptide, representing residues 120-125 of human histone H2A, and some of its analogs undergo E-S peptide bond hydrolysis. In this work we demonstrate a similar coordination and reactivity pattern in Ni(II) complexes of Ac-Thr-Glu-Thr-His-His-Lys-NH 2 , its threonine analogue, studied using potentiometry, electronic absorption spectroscopy and HPLC. For the first time we present the detailed temperature and pH dependence of such Ni(II)-dependent hydrolysis reactions. The temperature dependence of the rate of hydrolysis yielded activation energy E a = 92.0 kJ mol −1 and activation entropy ∆S ≠ = 208 J mol −1 K −1 . The pH profile of the reaction rate coincided with the formation of the four-nitrogen square-planar Ni(II) complex of Ac-Thr-Glu-Thr-His-His-Lys-NH 2 . These results expand the range of protein sequences susceptible to Ni(II) dependent cleavage by those containing threonine residues and permit predictions of the course of this reaction at various temperatures and pH values.

The coordination properties of Ni() ions towards the terminally blocked (CH 3 CONH-and -CONH 2 ) hexapeptides -TESHHK-, -TASHHK-, -TEAHHK-, -TESAHK-and -TESHAK-were studied by using potentiometric and spectroscopic techniques (UV/Vis, CD, NMR). The peptides were chosen in such a way as to compare the effect of Glu, Ser and His residues on the stability, the coordination and hydrolytic abilities of the complexes formed. All peptides bind to Ni() ions initially through one or two imidazole nitrogens in weakly acidic and neutral solutions forming slightly distorted octahedral complexes. At higher pH values, a series of square-planar complexes are formed, where Ni() ions bind simultaneously through an imidazole and three amide nitrogens in an equatorial plane. This proposed conformation includes the participation of only one imidazole nitrogen, in the case of all peptides, in the coordination sphere of Ni() ions. In basic solutions, the peptides -TASHHK-and -TESAHKwere hydrolyzed in a Ni()-assisted fashion. No hydrolytic processes were noticed in peptides -TEAHHK-and -TESHAK-where the Ser or His-5 residues are replaced with the Ala residue. The Ni()-assisted hydrolysis of the analogues of -TESHHK-may provide an insight into the novel mechanism of genotoxicity, combining the damage to the nucleosome with the generation of further toxic Ni() species.

CH 3 CO-Thr-Glu-Ser-His-His-Lys-NH 2 , a hexapeptide representing the 120-125 sequence of histone H2A, coordinates Cu(II) ions efficiently. Monomeric complexes are formed. In the major complex at physiological pH, CuH -1 L, Cu(II) is coordinated equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues, and axially through the imidazole nitrogen of the His-5 residue. This complex reacts with H 2 O 2 and the resulting reactive oxygen intermediate efficiently oxidizes 2′-deoxyguanosine. The underlying mechanism involves the formation of Cu(III) and a metal-bound hydroxyl radical species.

Previously we demonstrated that Ni(II) complexes of Ac-Thr-Glu-Ser-His-His-Lys-NH 2 hexapeptide, representing residues 120-125 of human histone H2A, and some of its analogs undergo E-S peptide bond hydrolysis. In this work we demonstrate a similar coordination and reactivity pattern in Ni(II) complexes of Ac-Thr-Glu-Thr-His-His-Lys-NH 2 , its threonine analogue, studied using potentiometry, electronic absorption spectroscopy and HPLC. For the first time we present the detailed temperature and pH dependence of such Ni(II)-dependent hydrolysis reactions. The temperature dependence of the rate of hydrolysis yielded activation energy E a = 92.0 kJ mol −1 and activation entropy ∆S ≠ = 208 J mol −1 K −1 . The pH profile of the reaction rate coincided with the formation of the four-nitrogen square-planar Ni(II) complex of Ac-Thr-Glu-Thr-His-His-Lys-NH 2 . These results expand the range of protein sequences susceptible to Ni(II) dependent cleavage by those containing threonine residues and permit predictions of the course of this reaction at various temperatures and pH values.