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Potatoes ( Solanum tuberosum L.) from the high Tiber valley area (TVA; Tuscany, Italy), have been sampled and analyzed for selected mineral content (Na, Mg, K, Ca, and Mn, Fe, Cu and Zn) and vitamin C, the year 2012; some samples from 2011 and 2013 crops were also collected and analyzed. The varieties were Daytona (DAY), Kennebec (KEN), Sifra (SIF) and Volare (VOL). Control samples consisted of mixed commercial varieties from the local market, namely C1, C2 and C3. The low content of sodium, especially for KEN (46 ± 3 mg/kg FD (freeze dry), year 2012) and SIF (47 ± 3) (VOL (55 ± 3) and DAY (61 ± 3) have a little higher values) is worth of note and in agreement with the scarce concentration of Na in the soil (291 ± 12 mg/kg DM). Magnesium was abundant in KEN (1434 ± 75 mg/kg FD, year 2012) and VOL (1334 ± 70). The content of K for DAY and KEN (13,147 ± 900 and 13,185 ± 900 mg/kg FD) was higher than for VOL and SIF; whereas Ca was in the range 340 ± 16–490 ± 28 mg/kg FD. The contents of Cu and Zn were higher in KEN (8.1 ± 0.3 and 25 ± 1 mg/kg FD) when compared to the other varieties and controls. The content of vitamin C is high for KEN and SIF and decreased significantly upon cooking (50% for KEN).

Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(II) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(II)–viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(II)–viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme.

Teicoplanin, a member of the “last chance” antibiotic family has a similar structure and the same mechanism of action as parent drug vancomycin, which is proved to be an effective binder of Cu(II) ions. However, the potentiometric and spectroscopic studies (UV–visible, CD, NMR) have shown that the modification of the N-terminal structure of the peptide backbone in teicoplanin affects considerably the binding ability towards Cu(II) ions. While vancomycin forms almost instantly the stable 3 N complex species involving the N-terminal and two amide nitrogen donors, in case of teicoplanin only two nitrogen donors derived from the N-terminal amino group and adjacent peptide bond are coordinated to Cu(II) ion within the whole pH range studied. The major factor influencing the binding mode is most likely the structure of the N-terminus of the peptide unit in the antibiotic ligand.

The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).

Ferulic acid (FA) is a highly effective antioxidant and photo-protective agent, already approved in Japan as a sunscreen, but it is poorly suited for cosmetic application because of its low physicochemical stability. We prepared the inclusion complex of FA with alpha-cyclodextrin by co-precipitation from an aqueous solution, and used (1)H NMR and molecular dynamics to investigate the most probable structure of the inclusion complex. In rotating frame nuclear Overhouser effect spectroscopy (ROESY) experiments FA penetrated the alpha-CD hydrophobic cavity with the alpha,beta-unsaturated part of the molecule and some of its aromatic skeleton. In proton chemical shift measurements of FA and alpha-cyclodextrins we determined the stoichiometry of the association complex (1:1) by Job's method, and its stability constant (K(1:1) 1162+/-140 M(-1)) and described the molecular dynamics of the complex on the basis of theoretical studies. Encapsulation with alpha-cyclodextrin improves (i) the chemical stability of FA against UVB stress (10 MED [Minimal Erythemal Dose: 1 MED=25 mJ/cm(2) for skin phototype II: 30]), since no degradation products are formed after irradiation, and (ii) the bioavailability of FA on the skin, slowing its delivery (Strainer cell model).

Colistin and transition metal ions are commonly used as feed additives for livestock animals. This work presents the results of an analysis of combined potentiometric and spectroscopic (UV–vis, EPR, CD, NMR) data which lead to conclude that colistin is able to effectively chelate copper(II) ions. In cell-free system the oxidative activity of the complex manifests itself in the plasmid DNA destruction with simultaneous generation of reactive OH species, when accompanied by hydrogen peroxide or ascorbic acid. The degradation of RNA occurs most likely via a hydrolytic mechanism not only for complexed compound but also colistin alone. Therefore, huge amounts of the used antibiotic for nontherapeutic purposes might have a potential influence on livestock health.

The use of the antiepileptic drug, 4-aminobutyrate transaminase (GABA-T) inhibitor vigabatrin (VIGA), has been recently cautioned because it is associated to irreversible field defects from damage of the retina. Since novel GABA-T inhibitors might prove useful in epilepsy or other CNS pathologies as VIGA substitutes, the aim of the present investigation was to characterize the biochemical properties of some taurine analogues (TA) previously shown to act as GABA-T inhibitors. These include (+/-)piperidine-3-sulfonic acid (PSA), 2-aminoethylphosphonic acid (AEP), (+/-)2-acetylaminocyclohexane sulfonic acid (ATAHS) and 2-aminobenzenesulfonate (ANSA). Kinetic analysis of the activity of partially purified rabbit brain GABA-T in the presence of VIGA and TA showed that PSA and AEP caused a linear, mixed-type inhibition (Ki values 364 and 1010 microM, respectively), whereas VIGA, ANSA and ATAHS behaved like competitive inhibitors (Ki values 320, 434 and 598 microM, respectively). Among the compounds studied, only VIGA exerted a time-dependent, irreversible inhibition of the enzyme, with Ki and k(inact) values of 773 microM and 0.14 min(-1), respectively. Furthermore, the ability of VIGA and TA to enhance GABA-ergic transmission was assessed in rabbit brain cortical slices by NMR quantitative analysis. The results demonstrate that VIGA as well as all TA promoted a significant increase of GABA content. In conclusion, PSA, ANSA and ATAHS, reversible GABA-T inhibitors with Ki values close to that of VIGA, represent a new class of compounds, susceptible of therapeutic exploitation in many disorders associated with low levels of GABA in brain tissues.

Protonation and copper(II) coordination properties of neomycin B were studied in solution by potentiometry, NMR, UV/Vis, CD, and EPR spectroscopy, XAS and mass spectrometry. Mono- and dinuclear complexes were found depending on the metal-to-ligand molar ratio. Neomycin B anchors CuII ions above pH 5.0 with an NH2 group from ring B. Simultaneously, the second amino group of the same ring and the hydroxyl group of ring A complete the binding set of donors. With an increase in pH the remaining –NH3+ functional groups in the neomycin B molecule are deprotonated without affecting the complexation pattern. However, these groups, particularly the ones located in the D-ring of the antibiotic, may coordinate the second copper(II) ion when the metal is present in excess. We have proved this process with the use of potentiometry, CD and especially mass spectrometry. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The products of hexavalent chromium [Cr(VI)] reduction by glutathione (GSH) alone or in the presence of equimolar quantities of aspartate (Asp) and/or glutamate (Glu) and a chromium-containing material extracted from bovine liver were studied by ultraviolet-visible spectrum (UV-vis) studies, electrospray mass spectrometry (ES-MS), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR). Reduction of chromate by GSH was followed by UV-vis and NMR, revealing the formation of a paramagnetic complex in which GSH acts as a ligand. ES-MS and EPR measurements provided unequivocal evidence of a dimeric Cr(V)(2)GSH(2) species in which the two metal ions are bridged by the Gamma-Glu carboxylate. The analysis of the (1)H and (13)C shifts experienced by GSH protons and the values of paramagnetic contributions to proton spin-lattice relaxation rates provided a set of constraints for structural determination. The same experiments were repeated in the presence of an equimolar concentration of Asp, revealing the formation of a dimeric Cr(V) paramagnetic complex in which the two metals are now bridged by Asp. Nuclear magnetic resonance dispersion profiles show that water is not displaced by Asp and that the correlation time of this complex is slowed by the increased complexity. When Glu is also included in the solution in equimolar concentration to GSH and Asp, data are consistent with the formation of many mono- and dinuclear species, with the three ligands competing with each other. Finally, the spectroscopic investigation of the chromium-containing material extracted from bovine liver revealed the presence of a complicate mixture of Cr(IV) or Cr(V) complexes, among which some Cr(V)-GSH species are present alone or with other ligands in the metal coordination sphere.

Potentiometric, spectroscopic and theoretical studies have shown that the α-hydroxymethylserine (HmS) residue is a very specific amino acid residue when inserted into a peptide sequence. The theoretical calculations as well as evaluated deprotonation microconstants indicated that in the HmS-HmS-His tripeptide the N-terminal ammonium group is more acidic than the imidazole nitrogen. The hydrogen bond formation between the N-terminal amino group and imidazole nitrogen stabilizes the cyclic conformation of the metal-free peptide. The unusual gain in the 4N complex stability in the copper(II) and nickel(II) complexes with HmS-HmS-His ligands seems to derive from the enhancement of the π-electron contribution to the metal–amide nitrogen bond.

The copper (II) complex of amikacin in water solution at pH 5.5 was investigated by 13 C-NMR. The temperature dependence of spin–lattice relaxation rates was measured and fast exchange conditions were shown to apply. The motional correlation time of the complex was approximated by the pseudo-isotropic rotational correlation time of free amikacin in water solution ( τ c =0.17 ns at 300 K). Formation of a pseudo-tetrahedral 1:1 complex was demonstrated by relaxation rates analysis and also by UV–Vis spectrophotometry. Two amino nitrogens of amikacin, together with the amide nitrogen and the hydroxyl in the hydroxyl–aminopropyl carbonyl side chain, were assigned as the copper-binding sites and a model of the complex was built by using copper–carbon distances obtained by NMR analysis as input parameters.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV–visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50 μM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The k obs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Capreomycin is an important therapeutic agent having intriguing and diverse molecular features. Its polypeptidic structure rich in nitrogen donors makes the drug a promising chelating agent for a number of transition metal ions, especially for copper(II). The results of the model investigational studies suggest that capreomycin anchors Cu(2+) ion with an amino function of the α,β-diaminopropionic acid residue at pH around 5. At physiological pH copper(II) ion is coordinated by two deprotonated amide nitrogen atoms of the α,β-diaminopropionic acid, the serine residue as well as the amino function deriving from the β-lysine. Above that pH value we observe a rearrangement within the coordination sphere leading to movement of Cu(2+) to the center of the peptide ring with concurrent coordination of four nitrogen donors. Spin-lattice relaxation enhancements and potentiometric measurements clearly indicate that deprotonated amide nitrogen atom from the β-ureidodehydroalanine moiety is the fourth donor atom.

The interaction between the antimalarial drug chloroquine and red blood cells was investigated by measuring 1H NMR spin-lattice relaxation rates upon double-selective excitation of dipolarly connected spin pairs within the ligand molecule. Geometric and dynamic features were inferred, for the free as well as for the bound ligand, by the cross-relaxation terms between protons at fixed and time-dependent distances, respectively. A change in orientation of the side chain in respect of the aromatic moiety was shown to result from the binding interaction.

A method was developed for delineating the structure of paramagnetic metal complexes. The selective disappearance of cross-peaks in proton-carbon shift correlated 2D NMR maps was shown to uniquely depend upon the scalar and/or dipolar interaction between ligand nuclei and the unpaired electron(s), thus providing a means of identifying binding sites. Copper(II) was shown to form metal complexes with both Penicillin (PNC) and Pilocarpine (PLC) and the structure of the two 1:2 complexes in water solution at physiological pH were determined.

The copper(II) complex of amitraz, a formamidine pesticide, was investigated by measuring 1 H NMR spin-lattice relaxation rate enhancements (PRRE) in [ 2 H 6 ]-DMSO solution. The occurrence of fast exchange conditions was demonstrated by the temperature dependence of the PRRE. The PRRE was interpreted by the Solomon-Bloembergen-Morgan approach; the number of solvent molecules in the coordination shell was calculated at q = 2; the reorientational correlation time in the 1 : 1 metal complex was assumed to be the same as in the free ligand and calculated at τ c = 69 ps. The metal ion was shown to stabilize a cisoid conformation of the ligand.

Dynamic and geometric features of pilocarpine in water solution were delineated by interpreting 13C and 1H NMR spin-lattice relaxation rates and nuclear Overhauser effects. The motional correlation time was evaluated at 53 ± 5 ps at 298 K. 1H—1H distances, as calculated from double-selective 1H spin-lattice relaxation rates, and 13C—1H distances, as calculated from selective 13C–{1H} Overhauser effects, gave evidence of a C7 puckered conformation of the furanone ring and of an angle of ca. 50° between the two rings.

The reaction of the ruthenium(II) complex fac-[Ru(CO)(3)Cl(2)(N(1)-thz)] (I hereafter; thz = 1,3-thiazole) with human beta-amyloid peptide 1-28 (Abeta(28)) and the resulting {Ru(CO)(3)}(2+) peptide adduct was investigated by a variety of biophysical methods. (1)H NMR titrations highlighted a selective interaction of {Ru(CO)(3)}(2+) with Abeta(28) histidine residues; circular dichroism revealed the occurrence of a substantial conformational rearrangement of Abeta(28); electrospray ionization mass spectrometry (ESI-MS) suggested a prevalent 1:1 metal/peptide stoichiometry and disclosed the nature of peptide-bound metallic fragments. Notably, very similar ESI-MS results were obtained when I was reacted with Abeta(42). The implications of the above findings for a possible use of ruthenium compounds in Alzheimer's disease are discussed.

Ferulic acid (Fer), a natural anti-oxidant and chemo-protector, is able to suppress experimental carcinogenesis in the forestomach, lungs, skin, tongue and colon. Several Fer derivatives have been suggested as promising candidates for cancer prevention, being the biological activity related also to the capacity of partitioning between aqueous and lipid phases. In the present work, pulsed field gradient (PFG) NMR diffusion measurement and NMR relaxation rates have been adopted for investigating the interaction of three Fer derivatives (Fer-C11, Fer-C12 and Fer-C13) with human erythrocytes. Binding to the erythrocyte membrane has been shown for all derivatives, which displayed a similar interaction mode such that the aromatic moiety and the terminal part of the alkyl chain were the most affected. Quantitative analysis of the diffusion coefficients was used to show that Fer-C12 and Fer-C13 display higher affinity for the cell membrane when compared with Fer-C11. These findings agree with the higher anti-oxidant activity of the two derivatives.

This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations of metal effects upon peptide–receptor interactions with the use of NMR methods. Cerium(III) is herein used for assessing metal effects upon the interaction between angiotensin II and a fragment from the AT1Areceptor. Angiotensin II forms a complex with cerium(III) in water while the fCT300–320receptor fragment is poorly affected by cerium(III). However, the addition of the fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for the receptor and probing the peptide residues involved in receptor binding.

The complexes formed by kanamycin A at three different pH values (5.5, 7.4 and 12.0) were investigated by NMR and EPR spectroscopy. Paramagnetic relaxation contributions to proton relaxation rates were measured using a combination of the TOCSY sequence with the inversion recovery experiment in order to gain signal resolution in the bulk region. Measured contributions were converted into distances and used for structural determination by restrained simulated annealing where all possible chair and boat conformations of the rings were taken into account. The interaction of the Cu(II) ion with the nitrogen of the C ring is apparent at all pH values. At higher pH also the amino group of ring A starts to be involved in the metal coordination sphere. This is accompanied by a switch in conformation of ring C. Structures are consistent with the involvement in the coordination sphere either of the 2' or 4' hydroxyl oxygens at pH 5.5 and the 5 and the 6' hydroxyl oxygens (or the ring oxygen) at pH 12.0.

Interaction between angiotensin II (Ang II) and the fragment peptide 300-320 (fCT300-320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9Lys(fCT) and 6His(ang), 10Phe(fCT) and 8Phe(ang), HN proton of 3Tyr(fCT) and Halpha of 4Tyr(ang), 5Phe(fCT)Hdelta and Halpha of 4Tyr(ang) indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300-320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an alpha-helix backbone structure to fCT300-320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment.

Angiotensin II is shown by nuclear magnetic resonance (NMR) to form a complex in water at pH 4.0 with cerium(III), the ideal paramagnetic probe for Ca(2+). Paramagnetic shifts induced by the metal were used for the determination of dissociation constant and complex stoichiometry. ROESY cross-peaks and 3 J(HN-H)(alpha) coupling constants were converted into distance and angular constraints to determine the structure of the complex by molecular dynamics using the simulated annealing protocol. The complex is kinetically labile and involves the Asp-1 side chain and the Phe-8 terminal carboxylates as binding groups resembling a hairpin which has been suggested as a possible biologically active structure.

The copper(II) complex of lincomycin in water solution at pH = 7.15 was characterized by 1 H and 1 3 C NMR and UV-vis spectroscopy. A 1:1 complex is formed in these conditions. The temperature dependence of spin-lattice relaxation rates was measured, showing that all protons behave in a similar fashion and slow exchange conditions prevail. The spin-lattice relaxation rate enhancements were interpreted by the Solomon-Bloembergen-Morgan theory. Reorientational dynamics of the complex was approximated by evaluating the motional correlation time of free lincomycin in water solution. The observed proton and carbon relaxation rate enhancements allowed us to calculate copper-proton and copper-carbon distances that were used for building a molecular model of the complex. The obtained data provide an interpretation of the relatively high stability constant.

Potentiometric and spectroscopic data, including NMR, CD and EPR results, as well as theoretical calculations showed that the insertion of a nucleic base into a peptide chain results in formation of very effective ligands (chiral peptide nucleic acids, C-PNAs) for CuII and NiII. The most effective ligand was that containing thymine base. The thymine moiety interacts with metal ion in the apical position via the O6 oxygen and forms a set of effective hydrogen bonds with the N-terminal amino group. In the case of adenine C-PNA, two nucleic bases may form stacking interactions, which also increases the complex stability. These interactions of nucleic bases mean that metal ion binding to peptide backbone nitrogens of PNAs leads to formation of complexes which are several orders of magnitude stronger than those obtained with oligoglycine. Metal ion binding to C-PNAs, on the other hand, induces specific conformations of the side chain groups, which would affect critically the self-recognition interactions between the C-PNA strands.

An NMR investigation has been carried out on amikacin in water solution in the physiological pH range. Two-dimenstional heterocorrelated maps provide 1H NMR chemical shifts from the unambiguous assignment of the 13C NMR spectrum. Reorientational dynamics at the molecular level are interpreted in terms of a pseudoisotropic motion with a correlation time of 0.17 ns at 300 K. The pH and temperature dependences of 13C NMR chemical shifts are interpreted to delineate protonation equilibria (all pKs are determined) and to assess the occurrence of two intermolecular hydrogen bonds, which are confirmed by molecular modelling.

The conformation of cytochalasin D in solution has been delineated by measuring 13C and 1H NMR spin–lattice relaxation rates and NOESY spectra. The motional correlation time was evaluated at 0.26 ns at 300 K. A comparison with the structure of cytochalasin B in the same solvent discloses several similarities, except for a small distortion of the five-membered ring. The difference in activity is therefore ascribed to the different hydrophobicity of substituents within the macrocycle.

Successful application of the potentiometric method together with NMR, EPR, CD and absorption spectroscopy yielded accurate data concerning the stabilities of the complexes formed and their binding modes between CuII and squash trypsin inhibitor. The major residue involved in the metal ion co-ordination is the His-25 imidazole side chain, which acts as an anchoring donor and is bound to metal ion over the whole pH range (3–11.5) studied. The 3N complex with {Nimid, N−His25, N−Glu24} binding mode dominates at physiological pH. The data obtained indicate that the protein after a particular mutation could be useful to model metal centres of large proteins.

The effect of copper(II) ions on the probabilities of existence of the four detectable conformers of the tetrapeptide Tyr-Pro-Phe-Pro (beta-casomorphin 4) in [2H6]DMSO was investigated by 1H NMR spectroscopy. Integration of the Phe-NH signals provided the relative populations in the free state as tt/tc/ct/cc=28:34:29:9 at 293 K (c=cis, t=trans). Copper(II) was shown to bind to all four isomers, yielding complexes with two different structures, depending on the conformation of Pro(2). The interpretation of paramagnetic relaxation rates of Pro(2)-Halpha signals provided the corresponding isomeric probabilities in the metal-bound state as 13:36:20:31. The observed stabilization of the conformation with the lowest probability of existence (cc) may be relevant for the biological role of copper and other metal ions.

The structure of the copper complex of melanostatin was investigated by NMR relaxation rate analysis and ESR spectral simulation. A 1:1 complex is formed at any ligand:metal ratio. The assumption that the motional correlation time equals that for the peptide free in solution allowed us to calculate several copper-carbon and copper-proton distances that were used to build up a molecular model of the metal complex. The metal ion is in a distorted tetrahedral arrangement and is coordinated by four nitrogen atoms.

A low-molecular weight chromium-containing fraction of the material resulting from dichromate reduction by bovine liver homogenate was investigated by NMR and ES-MS. The ES-MS spectrum showed a readily detectable peak at m/z = 786.1. The same molecular weight reasonably agreed with the relatively low diffusion coefficient measured by NMR-DOSY experiments on the main species observed in the1H NMR spectrum. At least two downfield shifted and broad paramagnetic signals were apparent in the1H NMR spectrum. Temperature dependence of chemical shift was exploited in order to estimate the diamagnetic shift of the signals in the diamagnetic region of the spectrum. 2D TOCSY, NOESY, COSY and1H-3C HMQC spectra revealed the presence of aromatic protons (which were assigned as His residues), Gly and some other short chain amino-acids. Combinations of the molecular masses of such components together with acetate (which is present in the solution) and chromium atoms allowed a tentative proposal of a model for the compound.