Sodium-23 NMR studies of cation-DNA interactions

Sodium-23 NMR has been used to study the extent to which monovalent cations associate with double stranded DNA in aqueous solution (28°C, pH = 7.5). On the basis of the two site model for rapid exchange the ²³Na linewidth can be related to the fraction of sodium ions associated with DNA. To test the applicability to this system of the condensation model for the association of small counterions with polyelectrolytes, the concentration dependence of the sodium linewidth has been determined by making additions of NaCl to solutions of tetraethyl or tetrabutylammonium DNA. ([P], the DNA phosphate concentration was about 0.02M). The resulting titration curves extend over a wide range of the ratio [Na]/[P] (0.3–30). When [Na]/[P] ? 3 only sodium is associated, and the extent to which it compensates the charges on DNA does not vary with the addition of salt, at least until [Na]/[P] ≈ 30, the highest concentration examined. When [Na]/[P] ? 3 the tetraalkylammonium species is also associated with DNA; an equation has been derived to account for the effect on the ²³Na linewidth of the competition between sodium and another monovalent cation. Based on the assumption that the fraction of uncompensated charge remaining on DNA after the condensation of both species is constant, this equation fits all the linewidth data if the charge fraction is in the range 0.25 ± 0.10. The value required by the condensation model for DNA in the presence of monovalent counterions is ξ^?1 = 0.24. The reasonable agreement between experimental and theoretical values of the charge fraction and its invariance with respect to large variations in the concentration of added salt indicate that even in moderately concentrated solutions of DNA, the association of sodium can usefully be described in terms of the condensation model. If the theoretical value of the charge fraction is assumed, it follows from fitting the titration curves that the approximate relative affinities for DNA of Na⁺, Et₄N⁺, and Bu₄N⁺ are in the ratio 20:5:1, and the transverse relaxation rate of condensed sodium is 180 ± 10 s^?1.     

23Na NMR relaxation study of the effects of conformation and base composition on the interactions of counterions with double-helical DNA

NMR relaxation rates (T1(-1) and T2(-1)) have been determined for 23Na in aqueous salt solutions containing various types of helical double-stranded deoxyribonucleic acids. These measurements were performed on three synthetic polynucleotides having different overall conformations, poly-(dA-dT).poly(dA-dT) (alternating B-DNA), poly(dG-dC).poly(dG-dC) at low salt (B-DNA), and Br-poly(dG-dC).Br-poly(dG-dC) (left-handed Z-DNA), and on four types of natural DNA differing in base composition, Clostridium perfringens (26% GC), calf thymus (40% GC), Escherichia coli (50% GC), and Micrococcus lysodeikticus (72% GC). For all types of DNA investigated, except poly(dA-dT).poly(dA-dT), the 23Na NMR spectra measured at 21 degrees C and an applied field of 4.7 T are non-Lorentzian. These non-Lorentzian spectra were analyzed on the basis of the two-state model and the standard theory of nonexponential quadrupolar relaxation processes in order to obtain estimates of the correlation times (tau c) characteristic of the sodium nuclei associated with the various nucleic acids. All of the correlation times estimated in this way are in the range of nanoseconds. The magnitudes of these correlation times show a significant dependence on the overall conformation of the nucleic acid (B vs. Z) but not on its base composition. To investigate the concentration dependence of tau c, sodium or magnesium salts were added to solutions of Br-poly(dG-dC).Br-poly(dG-dC) (Z-DNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

23Na NMR study of DNA thermal transconformation in presence of cysteamine radioprotector

DNA thermal transconformation is studied in absence and in presence of the cysteamine radioprotector, by observing the delta nu 1/2 variation of 23Na NMR peaks. The sodium state (Free or Bound) is discussed with the help of a two states model with RF and RB relaxation rates. The delta nu 1/2 behaviour during the DNA transconformation shows clearly the electrostatic interaction with cysteamine which is accompanied by an Na+ ejection out of phosphate sites. The temperature dependence of delta nu 1/2 in all cases leads to the conclusion that RBc (the average relaxation rate of sodium nuclei that remain bound in the coil state of DNA) tends to zero.     

Induction of B-A transitions of deoxyoligonucleotides by multivalent cations in dilute aqueous solution.

Circular dichroism (CD) spectra of d(CCCCGGGG) in the presence of Co(NH3)6(3+) are very similar to spectra of r(CCCCGGGG). In contrast, B-form characteristics are observed for d(CCCCGGGG) in the presence of Na+ and Mg2+, even at high salt concentrations. Spermidine induces modest changes of the CD of d(CCCCGGGG). The NMR chemical shifts of the nonexchangeable protons of d(CCCCGGGG) in the absence and presence of Co(NH3)6(3+) were assigned by proton two-dimensional (2D) NOESY and COSY measurements. The chemical shifts of the GH8 protons of d(CCCCGGGG) move upfield upon titration with Co(NH3)6Cl3. The sums of the sugar H1' coupling constants decrease with added Co(NH3)6Cl3. Cross peak intensities in the 2D proton NOESY spectra show a transformation from B-DNA to A-DNA characteristics upon the addition of Co(NH3)6Cl3. The temperature-dependent 59Co transverse and longitudinal relaxation rates demonstrate that Co(NH3)6(3+) is site-bound to the oligomer. Such localization is not a general feature of Co(NH3)6(3+) binding to oligonucleotides. 59Co NMR relaxation and CD measurements demonstrate chiral discrimination by d(CCCCGGGG) for the two stereoisomers of Co(en)3(3+). Both stereoisomers bind tightly as judged by 59Co NMR, and both cause large (but nonequivalent) changes in the CD of this oligomer.     

23Na and flame photometric studies of the NMR visibility of sodium in rat muscle.

23Na nuclear magnetic resonance spectroscopy (NMR) is increasingly being used to study Na+ gradients and fluxes in biological tissues. However, the quantitative aspects of 23Na NMR applied to living systems remain controversial. This paper compares sodium concentrations determined by 23Na NMR in intact rat hindlimb (n = 8) and excised rat gastrocnemius muscle (n = 4) with those obtained by flame photometric methods. In both types of samples, 90% of the sodium measured by flame photometry was found to be NMR-visible. This is much higher than previously reported values. The NMR measurements for intact hindlimb correlated linearly with the flame photometric measurements, implying that one pool of sodium, predominantly extracellular, is 100% visible. From measurements on excised muscle, in which extracellular space is more clearly defined, the NMR visibility of intracellular Na+ was calculated to be 70%, assuming an extracellular space of 12% of the total tissue water volume and an extracellular NMR visibility of 100%. 23Na transverse relaxation measurements were carried out using a Hahn spin echo on both intact hindlimb (n = 1) and excised muscle (n = 2) samples. These showed relaxation curves that could each be described adequately using two relaxation times. The rapidly relaxing component showed a T2 value of 3-4 ms and the slowly relaxing component a T2 of 21-37 ms. A spin lattice relaxation (T1) measurement on intact hindlimb yielded a value of 51 ms. These relatively long relaxation times show that the quadrupolar relaxation effect of Na+ complexing to large macromolecules or being otherwise motionally restricted is relatively weak. This is consistent with the high NMR visibilities reported here.     

Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione.

S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.     

Competitive interactions of Co(NH3)6(3+) and Na+ with helical B-DNA probed by 59Co and 23Na NMR

59Co NMR is demonstrated to provide a useful probe of the interactions of Co(NH3)6(3+) with helical B-DNA. The association of Co(NH3)6(3+) with B-DNA produces relatively modest effects on the relaxation rate and chemical shift of 59Co, which indicate that the octahedral coordination shell remains intact and that no significant number of long-lived "outer-sphere" complexes are formed at specific sites on the DNA surface. Under conditions where essentially all of the cobalt complex is associated with DNA, the chemical shift of 59Co appears to depend on its binding density. This effect could be due to magnetic heterogeneity in the environments of Co(NH3)6(3+) adjacent to DNA. The local exchange reaction between Co(NH3)6(3+) and Na+ in the vicinity of DNA has been investigated by measuring 59Co chemical shifts and 23Na line widths concurrently. The number of sodium ions displaced by the association of one Co(NH3)6(3+) with DNA cannot be uniquely determined, but the data indicate that this number remains constant over at least the initial stage of a titration of NaDNA with NaCl. 59Co chemical shifts have been analyzed to construct binding isotherms for the association of cobalt hexaammine with DNA over a range of salt (NaCl) concentrations. The magnitudes of the resulting binding constants and their salt dependence are similar to those previously reported for the association of structurally diverse trivalent ligands, such as spermidine and trilysine, with helical nucleic acids. Therefore, these association equilibria appear to be governed primarily by electrostatic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy

The effects of 48 h fasting, administration of ethanol or 2,4-dinitrophenol, on the phosphorus-containing metabolites in liver in vivo have been determined utilizing 31P nuclear magnetic resonance spectroscopy. These measurements were combined with determinations of metabolite concentrations in livers which were freeze-clamped immediately after the NMR measurements were completed. Administration of sub-lethal amounts of dinitrophenol dramatically decreased ATP and increased Pi concentrations in liver in vivo as indicated by a 2.7-fold increase in the NMR-derived [Pi]/[ATP] ratio. Ethanol administration to fed animals increased the NMR-derived [Pi]/[ATP] ratio 27%; in contrast, the same amount of ethanol administered to fasted animals decreased the NMR-derived [Pi]/[ATP] ratio 30%. The NMR visible Pi and ADP represent about 50% and 15% of the total Pi and ADP, respectively. The phosphorylation potentials calculated from the NMR visible Pi and ADP were an order of magnitude higher than those obtained from metabolite concentrations in freeze-clamped tissue. There was no apparent correlation between the phosphorylation potentials derived from either the NMR spectral analyses or from metabolite concentrations and the hepatic [NAD+]/[NADH] ratio. The chemical shift of Pi indicated that ethanol administration elicited a decrease in pH of 0.1 unit in liver in vivo. Hepatic free [Mg2+] was increased 21% in fasted animals, but was unaffected by ethanol administration.     

The interaction of polyamines with DNA: a 23Na NMR study.

The interaction between a variety of polyamines, both naturally occurring and synthetic, and calf thymus DNA has been studied using 23Na NMR. The relaxation behaviour of 23Na reflects the extent of interaction of Na+ with DNA phosphate groups and therefore the extent of charge neutralisation of DNA phosphate groups (P) by polyamine amino and imino groups (N) in solutions of DNa, polyamine and Na+. The studies reveal that whereas spermine and spermidine are capable of expelling nearly all of the Na+ ions from DNA at N/P approximately 1, diamines such as putrescine and homologues of spermine and spermidine are capable of neutralising only roughly 50% of DNA phosphates. The results provide a challenge to current models of DNA-polyamine interactions.     

Interaction of an N-methylated polyamine analogue, hexamethonium(2+), with NaDNA: quantitative 14N and 23Na NMR relaxation rate studies of the cation-exchange process

The interactions of the divalent hexamethonium (Hex2+) cation with double-helical calf thymus DNA are investigated by means of 14N NMR and, indirectly, by means of 23Na NMR. During a titration of NaDNA with HexBr2, the displacement of Na+ from DNA by Hex2+ is monitored by concurrent measurements of the Lorentzian 14N signals and the bi-Lorentzian 23Na signals. The variations in the quadrupolar relaxation rates of 14N and 23Na are analyzed according to a simple two-state model for the competition between Hex2+ and Na+ associated with DNA. From this analysis parameters characterizing the exchange process are evaluated, and the following conclusions are drawn: (1) The association of one Hex2+ displaces 1.7-2.0 sodium ions from the vicinity of the DNA. (2) Cation accumulation near DNA neutralizes approximately half of the phosphate charge at all points in the titration. (3) The exchange coefficient characterizing the displacement of Na+ by Hex2+ is of the same order of magnitude as the exchange coefficients determined by NMR for other divalent cations such as Mg2+ and putrescine. These findings imply that the interaction of Hex2+ with DNA is primarily electrostatic in character. The transverse and longitudinal relaxation rates observed for 14N are analyzed under the assumption that the quadrupolar relaxation processes of 14N in Hex2+ associated with DNA can be characterized by a single-exponential correlation function with correlation time tau NB. The resulting value of tau NB, 7.8 +/- 0.8 ns, is 3 orders of magnitude greater than that estimated for Hex2+ in the absence of DNA and is only 3-4 times greater than correlation times reported for 23Na and other quadrupolar cations near DNA. These comparisons indicate that the observed enhancements in the relaxation rates of 14N are due mainly to slowing of the motions that modulate its quadrupolar interactions in Hex2+ near DNA. The magnitudes of tau NB and of the quadrupolar coupling constant of Hex2+ associated with DNA are consistent with the conclusion that this association is primarily electrostatic.     

Bacterial DNA supercoiling and [ATP]/[ADP]. Changes associated with a transition to anaerobic growth.

Shifting Escherichia coli from aerobic to anaerobic growth caused changes in the ratio of [ATP]/[ADP] and in negative supercoiling of chromosomal and plasmid DNA. Shortly after lowering oxygen tension, both [ATP]/[ADP] and supercoiling transiently decreased. Under conditions of exponential anaerobic growth, both were higher than under aerobic conditions. These correlations may reflect an effect of [ATP]/[ADP] on DNA gyrase, since in vitro [ATP]/[ADP] influences the level of plasmid supercoiling attained when gyrase is either introducing or removing supercoils. When the supercoiling activity of gyrase was perturbed by a mutation in gyrB, a shift to anaerobic conditions resulted in plasmid supercoil relaxation similar to that seen with wild-type. However, the low level of supercoiling in the mutant persisted during a time when supercoiling in wild-type recovered and then exceeded aerobic levels. Thus, changes in oxygen tension can alter DNA supercoiling through an effect on gyrase, and correlations exist between changes in supercoiling and changes in the intracellular ratio of [ATP]/[ADP].     

Spectroscopic investigations of the structure of DNA complexes with Mn2+ in UV and IR regions

Based on the data of UV and IR spectroscopy, electronic and vibrational circular dichroism, the interaction of manganese ions with DNA was investigated. It was shown that the binding of ions to DNA proceeds in three stages depending on the manganese-to-DNA phosphates molar ratio [Mn]/[P]. At the first stage ([Mn]/[P] < or = 1), the interaction of manganese ions with DNA phosphates occurs, causing a partial screening of their negative charge and the stabilization of the double helix. At the second stage (1 < [Mn]/[P] < 6), the ions interact with both the phosphates and the nitrogen bases of DNA. At this stage, it is possible for the manganese ion to coordinate simultaneously to the oxygen atom of the phosphate and the neighbouring base of DNA. At a higher [Mn]/[P] ratio, the destabilization of the double helix begins, and partial breakage of the hydrogen bonds between the nitrogen bases occurs.     

Matrix-dependent modulation of anisotropic effects on NMR spectra from 7Li+ and 23Na+ encapsulated in cryptands

⁷Li and ²³Na NMR spectra of the respective cations in gelatin and ι-carrageenan gels containing cryptand-[2.1.1] (for Li⁺) or cryptand-[2.2.2] (for Na⁺) displayed two transitions: the one at higher frequency corresponded to the cation surrounded by gel, the other to cation inside its appropriately sized cryptand. While binding to cryptands yielded much broader lines and shorter T ₁ relaxation times, anisotropic splitting in first order ⁷Li or ²³Na NMR spectra was not detected. Stretching the gels resulted in increasing the anisotropic electric field gradient tensor; thus, the NMR transitions of the cation in the gel were split (removal of degeneracy) to display its characteristic 3:4:3 triplet for spin = 3/2 nuclei. The transitions of the cryptand-bound cations (Li⁺-cryptand-[2.1.1] and Na⁺-cryptand-[2.2.2]) showed different extents of interaction with the electric field gradient tensor depending on the composition of the gel matrix. The NMR signal for ⁷Li⁺-cryptand-[2.1.1] in stretched gelatin gel showed a five-fold increased splitting as compared to the ⁷Li⁺ signal in the reference gel. In stretched ι-carrageenan gels, no anisotropic splitting from the cryptand-bound Li⁺ was recorded. Steady-state irradiation envelopes or z-spectra showed evidence of Li⁺ exchange between isotropic (cryptand) and anisotropic (gel) sites only at higher temperatures (55 °C). For Na⁺ bound to the cryptand-[2.2.2], anisotropic splitting (three-fold smaller compared with the ²³Na signal in the reference gel) was only recorded in stretched ι-carrageenan gels, whereas gelatin gels showed only anisotropic splitting for the ²³Na signal in the reference gel.     

The dimerization of ferrihaems. I. The effect of buffer ions and specific cations on deuteroferrihaem dimerization.

The dimerization of dueteroferrihaem in aqueous solution has been investigated using a parameter, named the dimerization index (Robs). This is defined as the ratio of extinction coefficients at wavelengths corresponding to Soret band maxima for the monomeric and dimeric species, respectively. For solutions containing mainly monomeric species, Robs greater than 2, whereas for solutions containing mainly dimeric species Robs less than 1. A computer programme has been applied to determine values of the dimerization constant, K, defined as: K = [dimer] [H+]/[monomer]2. Phosphate buffer anions and Tris . HCl buffer enhanced dimerization. Monovalent and divalent cations also increased dimerization, but in a specific manner. The magnitudes of their effects increased in the order K+ less than Na+ less than Li+ less than Sr2+ less than Mg2+ approximately or equal to Ca2+. Values of K were determined for several concentrations of Na+ and Sr2+. These data are interpreted in terms of a stabilization of the ferrihaem dimer by the formation of ion triplets with the added cation 'sandwiched' between carboxyl residues of the adjacent ferrihaem monomeric units. General guidelines are recommended for the choice of conditions which minimize dimerization.     

Relative affinities of divalent polyamines and of their N-methylated analogues for helical DNA determined by 23Na NMR

Interactions of divalent polyamines with double-helical DNA in aqueous solution are investigated by monitoring the decrease in 23Na NMR relaxation rates as NaDNA is titrated with H3N(+)-(CH2)m-+NH3, where m = 3, 4, 5, or 6. Analogous measurements are made for the same homologous series of methylated polyamines (methonium ions). The dependence of the 23Na relaxation rates on the amount of added divalent cation (M2+) is analyzed quantitatively in terms of a two-state model. The sodium ions are assumed to be in rapid exchange between a "bound" state, where they are close enough to DNA so that it affects their relaxation rate, and a "free" state in bulk solution, where their relaxation rate is the same as in solutions containing no DNA. The distribution of Na+ and M2+ between these states is described quantitatively in terms of an ion-exchange parameter: DM = (pMB)(1-pNaB)n/(pNaB)n(1-pMB), where pNaB and pMB are the fractions of Na+ and M2+ that are close enough to DNA to be considered bound (by the NMR criterion), and n is the number of sodium ions displaced from DNA by the binding of one M2+ ion. For each of the polyamines and methonium ions investigated here, equations derived from this two-state model yield acceptable fittings of the titration curves if roNa, the number of sodium ions bound per DNA phosphate when no competing cations are present, is assigned a value between 0.6 and 1.00. Within this range, changing the value assigned to roNa does change the best-fitted values of DM determined for these polyamines (DH) and for the methonium ions (DMe) but does not alter the following conclusions about the trends in these parameters. (1) For polyamines and methonium ions of the same m, DH exceeds DMe by factors that are significantly larger for m = 3 and 4 than for m = 5 and 6. (2) DH for m = 3 and 4 is larger than DH for m = 5 and 6. (3) DMe for m = 3 and 4 is smaller than DMe for m = 5 and 6.     

DNA structures from phosphate chemical shifts

For B-DNA, the strong linear correlation observed by nuclear magnetic resonance (NMR) between the ³¹P chemical shifts (δP) and three recurrent internucleotide distances demonstrates the tight coupling between phosphate motions and helicoidal parameters. It allows to translate δP into distance restraints directly exploitable in structural refinement. It even provides a new method for refining DNA oligomers with restraints exclusively inferred from δP. Combined with molecular dynamics in explicit solvent, these restraints lead to a structural and dynamical view of the DNA as detailed as that obtained with conventional and more extensive restraints. Tests with the Jun-Fos oligomer show that this δP-based strategy can provide a simple and straightforward method to capture DNA properties in solution, from routine NMR experiments on unlabeled samples.     

Reinvestigation of the copper(II)-carcinine equilibrium system: "two-dimensional" EPR simulation and NMR relaxation studies for determining the formation constants and coordination modes

The equilibria and solution structure of complexes formed between copper(II) and carcinine (beta-alanyl-histamine) at 2< or = pH< or =11.2 have been studied by EPR and NMR relaxation methods. Beside the species that have already been described in the literature from pH-potentiometric measurements, several new complexes have been identified and/or structurally characterized. The singlet on the EPR spectrum detected in equimolar solutions at pH 7, indicates the formation of an oligomerized (CuL)n(2n+) complex, with [NH2,Nim] coordination. The oligomerization is probably associated with the low stability of the ten-membered macrochelate ring, which would form in the mononuclear complex CuL2+. In presence of moderate excess of ligand the formation of four new bis-complexes (CuL2Hn(2+n), n=2,1 and 0/-1) was detected with [Nim][Nim], [NH2,Nim][Nim] and [NH2,N-,Nim][Nim] type co-ordination modes, respectively. At higher excess of ligand ([L]/[Cu2+]>10) and at pH approximately 7, the predominant species is CuL4H2(4+). The 1H and 13C relaxation measurements of carcinine solutions (0.6 M) in presence of 0 mM< or = [Cu2+](tot)< or = 5 mM at pH=6.8, allowed us to extract the carbon-to-metal distances, the electronic relaxation and tumbling correlation times, as well as the ligand exchange rate for the species CuL4H2(4+). According to these results, the metal ion is [4Nim] co-ordinated in the equatorial plane, while the neutral amino groups are unbounded. Since naturally occurring carcinine shows in vivo antioxidant property, the SOD-like activity of the copper(II)-carcinine system has also been investigated and the complex CuLH(-1) was found to be highly active.     

Characterization of chemical exchange between soluble and aggregated states of beta-amyloid by solution-state NMR upon variation of salt conditions

Alzheimer's disease (AD) is characterized by the accumulation of insoluble fibrillar aggregates of beta-amyloid peptides (Abeta), a 39-42 residue peptide, in the brain of AD patients. It is hypothesized that the disease causing form is not the fibrillar species but an oligomeric Abeta molecule, which is often referred to as the "critical oligomer" of Abeta. We show in this paper that Abeta(1-40) undergoes chemical exchange between a monomeric, soluble state and an oligomeric, aggregated state under physiological conditions. In circular dichroism spectroscopy, we observe for this intermediate an alpha-helical structure. The oligomer is assigned a molecular weight of >100 kDa by diffusion-ordered spectroscopy-solution-state NMR spectroscopy (NMR). We can show by saturation transfer difference NMR experiments that the oligomer is related to monomeric Abeta. This experiment also allows us to identify the chemical groups that are involved in interactions between mono- and oligomeric Abeta molecules. Variation of the anionic strength in the buffer induces a shift of equilibrium between mono- and oligomeric states and possibly allows for the stabilization of these intermediate structures.     

ATP-phosphocreatine metabolism catalyzed by creatine kinase. Comparison of saturation transfer (NMR) and isotope labeling technics

Unidirectional fluxes from ATP to phosphocreatine (PCr) catalyzed by MM-isoenzyme of creatine kinase (CK) were measured by using 31P-NMR saturation transfer technique and by means of radioactively labeled [gamma-32P]ATP. At 30-37 degrees C and pH 7.4 in a wide range of [PCr]/[creatine] ([PCr]/[Cr]) ratios (0.2 to 3.0) both of these methods gave similar results, thus showing that magnetization (saturation) transfer allows to determine fluxes close to real ones under "physiological" conditions. However, at [PCr]/[Cr] ratio higher than 5 ([ADP] less than 30 microM) or at decreased temperatures (7-15 degrees C, [PCr]/[Cr] approximately 1) fluxes determined by saturation transfer substantially exceeded those measured with the radioactive label. These data imply that under "physiological" conditions phosphoryl group transfer is actually rate-determining step of the CK reaction. On the contrary, at high [PCr]/[Cr] values or at low temperature the control step could be shifted from the phosphoryl group transfer or distributed among other steps of the reaction.