Fernando Secco

The binding of Mg(II) and Ni(II) to synthetic polynucleotides

Equilibria and kinetics of the interactions of Mg2+ and Ni2+ with poly(U), poly(C) and poly(I) have been investigated at 25 degrees C, an ionic strength of 0.1 M, and pH 7.0 or 6.0. Analogous studies involving poly(A) were reported earlier. All binding equilibria were studied by means of the (usually small) absorbance changes in the ultraviolet range. This technique yields apparent binding constants which are fairly large for the interaction of Ni2+ with poly(A) (K = 0.9 X 10(4) M-1) and poly(I) (K approximately equal to 2 X 10(4) M-1) but considerably lower for the corresponding Mg2+ systems, Mg2+-poly(A) (K = 2 X 10(3) M-1) and Mg2+-poly(I) (K = 280 M-1). Each of the two pyrimidine nucleotides binds both metal ions with about the same strength (K approximately equal to 65 M-1 for poly(U) and K near 600 M-1 for poly(C]. In the case of poly(C) the spectral changes deviate from those expected for a simple binding equilibrium. In addition, the binding of Ni2+ to the four polynucleotides was measured by using murexide as an indicator of the concentration of free Ni2+. The results obtained by this technique agree or are at least consistent with those derived from the ultraviolet spectra. Complications are encountered in the binding studies involving poly(I), particularly at higher metal ion concentrations, obviously due to the formation of aggregated poly(I) species. Kinetic studies of the binding processes were carried out by the temperature-jump relaxation technique. Measurable relaxation effects of time constants greater than 5 microseconds were observed only in the systems Ni2+-poly(A) and Ni2+-poly(I). Such not-too-fast reaction effects are expected for processes which include inner-sphere substitution steps at Mg2+ or Ni2+. The relaxation process in Ni2+-poly(I) is characterized by (at least) four time constants. Obviously, the complicated kinetics again include reactions of aggregated poly(I). The absence of detectable relaxation effects in all other systems (except Mg2+-poly(I), the kinetics of which was not investigated) indicates that inner-sphere coordination of the metal ions to specific sites of the polynucleotides (site binding) does not occur to a significant extent. Rather, the metal ions are bound in these systems mainly by electrostatic forces, forming a mobile cloud. The differences in binding strength which are nevertheless observed are attributed to differences in the conformation of the polynucleotides which result in different charge densities.

Solvent effects on the thermodynamics and kinetics of coralyne self-aggregation.

The role of solvent effects on the thermodynamics and kinetics of the coralyne self-aggregation process has been investigated in ethanol-water mixtures of different compositions. The changes in the UV/visible spectra of coralyne and FAB/LSIMS mass spectrometry agreed well with the formation of a dimer species. 1D and 2D 1H experiments have allowed one to look into the features of the self-aggregation process and to determine the equilibrium constant and the deltaH0 and deltaS0 values for the aggregate formation in 0-50% ethanol-water mixtures. The kinetics of self-aggregation has been investigated by the T-jump chemical relaxation method, and the results have been interpreted in terms of dimer formation. The dependence of the relative viscosity of coralyne solutions on the dye concentration was studied in different ethanol-water mixtures. Finally, it was found that coralyne behaves as a solvatochromic indicator which is preferentially solvated according to the sequence ethanol > ethanol-water > water. All of the results concur in elucidating the relevant role of the hydrophobic interaction process of coralyne stack formation.

Role of the third strand in the binding of proflavine and pt-proflavine to poly(rA).2poly(rU): a thermodynamic and kinetic study.

The interactions of triple strands of poly(rA).2poly(rU) with proflavine (PR) and the proflavine cis-platinum derivative [{PtCl (tmen)} 2{NC 13H 7(NCH 2CH 2) 2}] (+) (PRPt) are examined at pH 7.0, T = 25 degrees C, and 0.2 M ionic strength by spectrophotometry, spectrofluorometry, circular dichroism, viscosimetry, stopped-flow, and T-jump relaxation techniques. The melting experiments demonstrate that both drugs tend to destabilize the triplex structure, although the PRPt effect is more relevant. By contrast, both drugs tend to slightly stabilize the duplex structure. The viscosity and circular dichroism measurements show that, at a low dye-to-polymer ratio ( C D/ C P), the binding is intercalative, whereas at high C D/ C P values, the external binding dominates. The binding kinetics and equilibria have been investigated over the C D/ C P region, where intercalation is operative. Both drugs bind to the RNA triplex according to the excluded site model. With PR, two kinetic effects have been observed, whereas with PRPt, only one has been observed. The results are interpreted according to the reaction schemes D + S right arrow over left arrow DS I, with PRPt, and D + S right arrow over left arrow DS I right arrow over left arrow DS II, with PR. The electrostatic contribution to the formation activation energy for DS I is similar (40%) for both systems. The results suggest that DS I is a partially intercalated species. Absence of the second step with PRPt is put down to groove interaction of the Pt-containing moiety, which prevents the PR residue from further penetration through the base pairs to form the fully intercalated complex, DS II. Comparison with the binding of the same drugs to the duplex reveals that the occupation of the major groove in poly(rA).2poly(rU) by the third strand plays a critical role in the kinetic behavior.

Solvent Effects on the Kinetics of the Interaction of 1-Pyrenecarboxaldehyde with Calf Thymus DNA

The kinetics of the interaction of a fluorescent probe, 1-pyrenecarboxaldehyde, with calf thymus DNA has been studied in different water/alcohol mixtures (ethanol, 2-propanol, and ter-butanol) at 25 degrees C, by using the stopped flow technique. The kinetic curves are biexponential and reveal the presence of two processes whose rates differ by about 1 order of magnitude on the time scale. The dependence of the reciprocal fast relaxation time on the DNA concentration is linear, whereas the concentration dependence of the reciprocal slow relaxation time tends to a plateau at high DNA concentrations. The simplest mechanism consistent with the kinetic results involves a simple two-step series mechanism reaction scheme. The first step corresponds to the formation of a precursor complex, (DNA/Py)(I), while the second one corresponds to full intercalation of the pyrene dye between the DNA base pairs. The values of the rate constants of both steps decrease as water activity decreases. The results have been discussed in terms of solvation of the species and changes in the viscosity of the solution.

Kinetic study of double-helix formation and double-helix dissociation of polyadenylic acid

The changes in the secondary structure of polyadenylic acid [poly(A)], caused by variations of pH at constant temperature, have been investigated. The position of the equilibrium 2 single-strands ⇌ double-strand is shifted towards the left-hand side by an increase in the ionic strength of the solution, and for constant values of the latter, by the addition of small amounts of divalent transition metal ions.The kinetics of the conformational changes have been investigated by T-Jump, stopped-flow and classical spectrophotometric methods. With the latter two techniques the reactions were initiated by changes in solution pH. The process of double-helix dissociation was found to involve at least three first-order steps. The process of double-helix formation begins with a second-order step leading, via a labile steady-state intermediate, to a multitude of mismatched double-strands. These, in turn, evolve to the final, completely formed double-helical form according to a series of first-order steps. The value of the second-order rate constant (105 dm3 mol–1 s–1) is similar to those found for the double-helix formation reactions of oligonucleotides. Increases in pH from 4 to 5.9 were used to study the conversion of a metastable double-stranded form of poly(A) into a form that is to be considered the most stable under the experimental conditions. This rearrangement process occurs through a series of three first-order steps, like the (much faster) process of double-helix dissociation.

Kinetics and equilibria of the interaction of 8-hydroxyquinoline with gallium(III) in water and sodium dodecyl sulfate solution.

The kinetics and equilibria of binding of gallium(III) to the 8-hydroxyquinoline (HQ) have been investigated in water and in the presence of sodium dodecyl sulfate (SDS) micelles. Moreover, the pKA1 and pKA2 of HQ and first hydrolysis constant of Ga3+ ion have been measured in water and SDS solution. The analysis of the kinetic and thermodynamic behavior reveals that the reactive form of Ga(III) is GaOH2+ in both cases. Although in water the only bound form of Ga(III) appears to be the deprotonated complex GaL, evidence for stabilization of the protonated form, GaHL on the micelle surface and stabilization of Ga3+ with respect to GaOH2+ is provided by the kinetic behavior in SDS. The addition of SDS at concentrations around the critical micellar concentration, results in a large enhancement of the rate of complex formation. The large catalytic effect produced by the SDS micellar solution provides a promising basis for the extraction of gallium from water using the HQ/SDS system. A procedure for gallium(III) extraction and recovery based on ligand modified-micellar enhanced ultrafiltration method, using the HQ/SDS system, is described.

Thermodynamics of coupled reactions by the analysis of chemical relaxation signals: the nickel(II)–murexide system

A temperature jump study of the NiII–murexide system, has been performed in buffered aqueous solution. The aim of the work is to provide a simple method for the evaluation of thermodynamic parameters of the involved proton transfer and complex formation steps by an analysis of the amplitudes of the relaxation signals. This method is based on a description of the system in terms of three ‘normal modes of reaction’ and it is demonstrated here that the slowest of them coincides with the concept of ‘apparent reaction’ introduced by Schwarzenbach in his theory of complexometric titrations. In analogy with classical titrations the experiments have been carried out by changing the metal ion concentration at constant ligand concentration and pH. In this way the amplitude of each relaxation curve represents a point of a ‘dynamic titration’ and a procedure is formulated whereby the analysis of the data provides simultaneously the equilibrium constant and the enthalpy of the slow normal reaction from simple linear plots.The equilibrium constants have been evaluated also by spectrophotometry and from the relaxation times. The results show that for the investigated system the accuracy of the dynamic method is comparable with that of conventional techniques. The circumstances where the dynamic titration becomes more informative than classical titration are discussed.

Route to Metallacrowns: The Mechanism of Formation of a Dinuclear Iron(III)-Salicylhydroxamate Complex

The equilibria and the kinetics of the binding of Iron(III) to salicylhydroxamic (SHA) and benzohydroxamic (BHA) acids have been investigated in aqueous solution (I = 1 M (HClO(4)/NaClO(4)), T = 298 K) using spectrophotometric and stopped-flow methods. Whereas Iron(III) forms a 1:1 complex (ML) with BHA, it forms both ML and M(2)L complexes with SHA. The presence of M(2)L in aqueous medium is corroborated by FTIR measurements. The reactive form of Iron(III) is the hydrolyzed species FeOH(2+), which binds to the O,O site in ML and to the O,O and O(P),N (P = phenolate) sites in M(2)L, inducing full deprotonation of the latter. The reaction pathway is discussed in terms of a multistep mechanistic scheme in which the metal-ligand interaction is coupled to hydrolysis and self-aggregation steps of Iron(III). The observation and characterization of M(2)L as a stable species is important because it contains the -Fe-O-N-Fe- sequence, which constitutes the repetitive motif of the SHA-based metallacrown ring and provides the rationale for 12-MC-4 metallacrowns. In the framework of this study, the kinetics of the Iron(III) dimerization and trimerization have also been investigated using the stopped-flow method to perform dilution jumps. The reaction scheme put forward involves two parallel steps (FeOH(2+) + FeOH(2+) and Fe(3+) + FeOH(2+)) that lead to formation of the Fe(2)(OH)(2)(4+) dimer and a slower step (FeOH(2+) + Fe(2)(OH)(2)(4+)) to form the trimer species. The kinetics of the last step have been investigated here for the first time, and the results deduced indicate that, of the two possible trimer structures reported in the literature, Fe(3)(OH)(3)(6+) and Fe(3)(OH)(4)(5+), the latter prevails by far.

Mechanism of complex formation: kinetics and equilibria of the gallium(III)–5-nitrosalicylate ion system

The equilibria between gallium(III) and the 5-nitrosalycylate ion, HA–, have been investigated by spectrophotometry in acid solution (pH 1–2.5). Two complexes are identified, their formation constants being K1=[GaA+][H+]/[Ga3+][HA–]= 49 ± 2 and K1′=[Ga(HA)2+]/[Ga3+][HA–]= 500 ± 55 dm3 mol–1 at 25 °C and I= 0.1 mol dm–3. The kinetics of the formation and decomposition of the complexes have been investigated by the temperature-jump technique. The complex [GaA]+ is formed in two pairs of ‘proton-ambiguous’ paths. Attributing the observed rate exclusively to the two reaction involving HA– on the one hand, and Ga3+ and [Ga(OH)]2+ on the other, one obtains upper limits for the rate constants, namely (4.1 ± 0.2)× 102 and (6.4 ± 0.7)× 103 dm3 mol–1 s–1, respectively. A fifth reaction, involving [Ga(OH)]2+ and HA–, makes a small contribution to the observed rate. The deprotonation of [Ga(HA)]2+ is rapid in comparison with its formation from Ga3+ and HA–. Comparison of the results with the previous data provides evidence for an associative mechanism.

Aggregation Features and Fluorescence of Hoechst 33258

The functionality of the bisbenzimide Hoechst 33258 in solution has been largely exploited in the quantification of DNA. Understanding of its behavior is essential to promote its widespread application and learning of biological processes. A detailed study of the dimerization process of the fluorescent blue dye Hoechst 33258 is carried out by isothermal titration calorimetry, absorbance, fluorescence, differential scanning calorimetry and T-jump kinetic measurements. The dimer/monomer ratio depends on the dye concentration and the ionic strength. The dimerization constant determined under physiological conditions (pH = 7.0; I = 0.10 M), KD = 3 × 10(4) M(-1), conveys that only micromolar concentrations of the dye can ensure reasonably high amounts of the monomer species in solution. For instance, for 10 μM dye content, the dimer prevails for I > 0.08 M, whereas the monomer is observed at low ionic strength, a key issue to be elucidated as long as the dimer species is more fluorescent than the monomer and the fluorescence intensity strongly relies on the ionic strength and the dye concentration.