The binding of Mg++ ions to polyadenylate, polyuridylate, and their complexes

The binding of Mg ⁺⁺ to polyadenylate (poly A), Polyuridylate(poly U), and their complexes, poly (A + U) and poly (A + 2U), was studied by means of a technique in which the dye eriochrome black T is used to measure the concentration of free Mg^?. The apparent binding constant K_X = [MgN]/[Mg⁺⁺][N], N = site for Mg⁺⁺ binding (the phosphate group of the nucleotide), was found to decrease rapidly as the extent of binding increased and, at low extents of binding, as the concentration of Na^? increased in poly A, poly (A + U), and poly (A + 2U), and somewhat less so in poly U. K_x is generally in the range 10⁴ > K_X > 10². The cause of these dependences is apparently, primarily, the displacement of Na⁺ by Mg⁺⁺ in poly U and poly (A + U) on the basis of the similarity of extents of displacement measured in this work and those measured potentiometrically. was calculated and was found to approach zero as the concentration of Na⁺ increased. In poly U, poly (A + U), and poly (A + 2U) at low ΔH′ v.H. > 0, about + 2 kcal/“mole.” In poly A, also at low salt, ΔH′ v.H. ≈ ?4 kcal/“mol” for the initial binding of Mg⁺⁺, and increases to +2 kcal/“mol” at saturation. This enthalpic variation probably accounts for the anticooperativity in the binding of Mg⁺⁺ not ascribable to the displacement of Na⁺⁺.     

A calorimetric investigation of the binding of indole and phenylethane boronic acid to chymotrypsin

The heat of formation of the chymotrypsin-phenylethane boronic acid complex has been observed calorimetrically from pH 4 to 8 at 25 degrees C and is found to be pH-dependent, changing from near -6 kcal/mol at pH 4 to -13 kcal/mol at pH 8. The heat of formation of the chymotrypsin-indole complex is a nearly constant -6 kcal/mol over most of the same pH range. alpha-Chymotrypsin has been purified by pH gradient elution from an immobilized lima bean inhibitor column. Solutions of the enzyme up to 400 microM, prepared in this manner, have a zero heat of dilution from pH 5 to 8 in 0.1 M KCl, with or without added 0.05 M Tris, N-(tris[hydroxy-methyl]methyl-2-amino) ethanesulfonic acid, 4-morpholineethanesulfonic acid, or acetate buffers. Binding of phenylethane boronic acid causes a pH-dependent decrease in proton binding to chymotrypsin; the decrease in proton binding evoked by formation of the indole complex is much less, with a much smaller pH dependence. The calorimetric and proton-binding results are applied to a model for boronic acid binding (Hanai, K. (1976) J. Biochem. (Tokyo) 79, 107-116). We conclude that the thermodynamics of formation of the trigonal boronic acid complex are quite similar to those for the formation of the noncovalent complex formed by indole and related ligands. The trigonal-tetrahedral tautomerism in the boronic acid-chymotrypsin complex is characterized by thermodynamic changes similar to those accompanying the binding of virtual substrates to chymotrypsin.     

The mode of Mg++ binding to oligonucleotides. Inner sphere complexes as markers for recognition?

Large changes of UV absorbance and CD spectra as well as specific relaxation processes with time constrants around 50 mus are found for the association of Mg++ with A(pa)n. The Mg++ binding constants strongly increase with increasing n. The relaxation data demonstrate that a large fraction of Mg++ bound to short A(pA)n forms inner sphere complexes (ISC), with H2O molecules from the inner hydration sphere of Mg++ exchanged against some site (s) of the oligomer. This fraction decreases from about 85% for A(pA)4 to less than 10% for A(pA)17. A parallel decrease is observed in the relative change of CD spectrum upon Mg++ binding from 77.5% for A(pA)4 to 13.4% for (pA)17. The rate of ISC formation decreases with increasing n suggesting some (probably sterical) hindrance effect at high n. The data support the conclusion that Mg++ favours the formation of outer sphere complexes with linear polynucleotides and require a special chain folding for ISC. Measurements of Mg++ binding to C(pC)5, U(pU)5, I(pI)5 and d[A(pA)5] did not give evidence for the formation of ISC, indicating that both specific base and sugar residues are required for ISC. These results suggest the possibility that Mg++ISC ARE USED FOR SPECific recognition of nucleic acid sequences.     

A comparison of van't Hoff and calorimetric heats of binding to DNA using an ethidium selective electrode

A liquid membrane electrode selective for ethidium ion was used to measure free ethidium in mixtures with calf thymus DNA. Electrode response was unaffected by variation in ionic strength from 1 mm to 0.5 m, and was not degraded over the temperature range studied. DNA-ethidium binding isotherms obtained with the electrode at 17.4, 25.4, 30.1, and 40.6 °C were fitted to a single class of excluded sites model for $\text{}\text{?}$gn ranging from 0.01 to 0.16. van't Hoff analysis of these data yielded ΔH = ?8300 cal/mol ethidium bound (in 0.5 m KCl, 10 mm Tris buffer, pH 10, 1 mm EDTA). Direct calorimetric measurements of the heat of complex formation led to a value of ?7600 cal/mol at 25 °C in the same medium; the two results were not significantly different at the 95% confidence level. The agreement supports the validity of the ethidium selective electrode, and illustrates its utility in the study of ligand binding to nucleic acids and related materials.     

Proton magnetic resonance studies of the binding of oligopeptides containing tryptophan to polyribonucleotides poly A, poly U and poly C

The binding of oligopeptides Lys-Trp-Gly-Lys OtBu, Lys-Gly-Trp-Lys OtBu and Lys-Trp-Lys to Polyadenylic, Polycytidylic and Polyuridylic acid has been studied by Proton NMR at 90 MHz and 500 MHz at oligopeptide/Polynucleotide ratios ranging from 0.01 to 0.20 at 275-365 K. Downfield shift of 0.01-0.2 ppm at 296 K of the H2, H8 and H1' resonances of Poly A due to binding with oligopeptides is accompanied by a marked narrowing of resonance lines of Poly A. The ring protons of tryptophan shift upfield by 0.3-0.6 ppm at 296 K on binding to Poly A. Changes in chemical shift of both adenine and tryptophan protons on binding are much smaller at 355 K than that at 275 K. These observations are ascribed to intercalation of the tryptophan ring in the adenine bases resulting in partial destacking of adenine bases in Poly A. Using the magnetic anisotropy ring current shifts, an overlap geometry of tryptophan ring in the adenine has been proposed. Addition of oligopeptides to Poly C and Poly U, on the other hand, suggests that tryptophan ring does not stack in Poly U and Poly C.     

Mg2+ ion effect on conformational equilibrium of poly A . 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg²⁺ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na⁺) and high (0.1 M Na⁺) ionic strengths. Four complete phase diagrams were obtained for Mg²⁺–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10⁻⁵–10⁻²) M Mg²⁺. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg_t²⁺]_cr=(4.5±1.0)×10⁻⁵ M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na⁺ contents in the solution. Such a value is observed for Ni²⁺ ions too. The phase diagram of the (A2U+Mg²⁺) complex with 0.01 M Na⁺ has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg²⁺ range. In (AU+Mg²⁺) phase diagram at 0.01 M Na⁺ the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na⁺. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.     

Effect of Mg++ and polyamines on proflavine binding to T2 DNA

Proflavine binding may be used as a probe of the environment and interactions of DNA. In this paper we report the effects of the divalent cations Mg⁺⁺ and putrescine and the trivalent cation spermidine on the proflavine–Na DNA binding equilibrium. Difference spectroscopy at 430 nm was used to determine apparent proflavine–DNA binding constants K at several concentrations of each cation for temperatures between 15 and 43°C, and at a constant total ionic strength of 0.1M. Mg⁺⁺, putrescine, and spermidine all have greater effects on K than expected on the basis of ionic strength alone in the order spermidine > Mg⁺⁺ ? putrescine. van't Hoff analysis of K(T) enabled calculation of ΔH° and ΔS°, which are affected differently by each cation. These differences are discussed qualitatively in terms of such concepts as release of condensed counterions, localized or unlocalized condensation, hydration, and restriction of molecular and internal rotation.     

Polarographic investigation of binding of Cu++ and Cd++ by DNA

The equilibrium binding constants of Cd⁺⁺ and Cu⁺⁺ to native and denatured calf thymus DNA were determined polarographically. The binding constants are an exponential function of the potential at the binding site and as such they vary with ionic strength and with the charge on the DNA molecule. The correlation between the fraction of sites occupied by heavy metal ions and between the thermal stability of DNA in solution is discussed.     

On the binding of actinomycin and of daunomycin to DNA: a calorimetric and spectroscopic investigation

The “strong” binding of two antibiotics, actinomycin D and daunomycin, to native DNA (calf-thymus) in dilute aqueous solution has been studied by means of calorimetric and spectroscopic measurements. In essence our results show: (1) Daunomycin interaction with DNA is an exothermic process, all features of which depend in a discontinuous way on the fraction of DNA binding sites engaged by the drug. Fluorescence data indicate that such a discontinuous trend should be independent of the GC content of DNA. (2) Actinomycin binding to DNA is, on the contrary, characterized by a positive enthalpy. For such binding, no discontinuity appears discernible with increasing the molar ratio of drug to DNA (phosphorous) on the basis of calorimetric and fluorescence data. (3) Both antibiotics can be bound simultaneously to DNA: our results would suggest that their binding sites on the biopolymer are independent.Discussion is focussed on the possible information derivable from our data on whether or not intercalation may indeed be the main process through which each antibiotic considered “strongly” interacts with DNA.     

A calorimetric study of the binding of lisinopril, enalaprilat and captopril to angiotensin-converting enzyme

The angiotensin I-converting enzyme (ACE; EC.3.4.15.1) is a dipeptidyl carboxypeptidase that plays a central role in blood pressure regulation. The somatic form of the enzyme is composed of two highly similar domains, usually referred to as N and C domains, each containing one active site. Nevertheless, a 1:1 stoichiometry for the binding of lisinopril, captopril or enalaprilat to somatic pig lung ACE is shown by isothermal titration calorimetry (ITC) and enzymatic assays. The binding of the three inhibitors at neutral pH is very tight and the enthalpy changes are positive, indicating that the binding is entropically driven. The origin of this thermodynamic signature is discussed under the new structural information available.     

Interaction of metallic cations with DNA VI. Specific binding of Mg++ and Mn++

The binding of Mg²⁺ and Mn²⁺ by DNA by a divalent cation specific electrode and by ultracentrifugation. Both techniques give similar results for the stoichiometry of the reaction. An oscillating densiemete allowed us to detect small changes of volume accompanying the binding. The reaction was also followed by circular dichroism measurements. Interpretation of the results is only possible if one assumes an electrostate site-binding of Mg²⁺ to phosphate group, and a chelation Mn²⁺ between the phosphate group and the N₇ of the guanine. Physical modifications accompanying these two types of binding are discused and compared to the role of these cations in some biological systems involving DNA.     

Cooperative binding of a platinum metallointercalation reagent to poly(A).poly(U).

The cationic complex (2-hydroxyethanethiolato)(2,2',2'-terpyridine)platinum(II), [(terpy)Pt(HET)]+, binds cooperatively to poly(A).poly(U) by intercalation. The melting temperature of poly(A).poly(U) in low-salt buffer is increased by 6 degrees C in the presence of [(terpy)Pt(HET)]+, indicating stabilization of the duplex structure by the bound platinum reagent. Viscosity measurements provide evidence for comparable lengthening of the polynucleotide in the presence of [(terpy)Pt(HET)]+ and the intercalating dye, ethidium bromide. Scatchard plots of the binding of [(terpy)Pt(HET)]+ to poly(A).poly(U) and poly(I).poly(C), determined through ultracentrifugation pelleting methods, show large positive curvature, reflecting the strong cooperativity associated with the platinum complex-RNA interaction. The characteristics of the binding isotherms are interpreted in terms of a model where cooperative pair units of [(terpy)Pt(HET)]+ intercalate into the double-stranded polymer. At saturation, two platinum molecules are bound for every three base pairs. This stoichiometry may be compared with the nearest-neighbor-exclusion binding observed previously in the interaction of [(terpy)Pt(HET)]+ and the ethidium cation with DNA, in which one intercalator occupies every other interbase-pair site at saturation. The striking differences observed in the interaction of [(terpy)Pt(HET)]+ with DNA and RNA suggest that drug recognition is sensitive to the constraints imposed by nucleic acid secondary structure.     

A calorimetric and equilibrium investigation of the hydrolysis of lactose

The thermodynamics of the hydrolysis of lactose to glucose and galactose have been investigated using both high pressure liquid chromatography and heat-conduction microcalorimetry. The reaction was carried out over the temperature range 282-316 K and in 0.1 M sodium acetate buffer at a pH of 5.65 using the enzyme beta-galactosidase to catalyze the reaction. For the process lactose(aq) + H2O(liq) = glucose(aq) + galactose(aq), delta G0 = -8.72 +/- 0.20 kJ.mol-1, K0 = 34 +/- 3, delta H0 = 0.44 +/- 0.11 kJ.mol-1, delta S0 = 30.7 +/- 0.8 J.mol-1.K-1, and delta Cop = 9 +/- 20 J.mol-1.K-1 at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. Thermochemical cycle calculations using enthalpies of combustion and solution, entropies, solubilities, activity coefficients, and apparent molar heat capacities have also been performed. These calculations indicate large discrepancies which are attributable primarily to errors in literature data on the enthalpies of combustion and/or third law entropies of the crystalline forms of the substrates.     

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase.

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.