Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

Thermodynamics of the interaction of sodium-n-dodecyl sulphate with Aspergillus niger catalase in high ionic strength aqueous solutions

The thermodynamic parameters for the interaction between sodium n-dodecyl sulphate (SDS) and Aspergillus niger catalase in aqueous solution at pH 3.2 and 6.4 have been measured by microcalorimetry and equilibrium dialysis over a range of ionic strength from 0.05 to 0.2 at 25 degrees C. Binding isotherms have been interpreted in terms of theoretical models (Hill equation and Wyman binding potential). The Gibbs energies of interaction become increasingly negative with increase in ionic strength and the entropies of interaction become increasingly positive. The ionic strength dependence of the Gibbs energies are much greater than predicted by the Debye-Hückel limiting law indicating a strongly ionic strength dependent hydrophobic contribution to the interactions.     

Hemoglobin-membrane interaction at physiological ionic strength and temperature

The spin-label electron paramagnetic resonance (EPR) technique has been used to study the interaction between human hemoglobin and erythrocyte membranes as a function of temperature and ionic strength. We show, for the first time, experimental evidence for the existence of the interaction at physiological pH, ionic strength and temperature. In addition to the pH dependence that we have previously reported, the interactions are also temperature and ionic strength dependent. Using a simple two-state equilibrium model to analyze the EPR data, we obtain an equilibrium dissociation constant of about 8.1 +/- 5.6 X 10(-5) M for hemoglobin-membrane systems in 5 mM phosphate with 150 mM NaCl at pH 7.4 and 37 degrees C.     

Acridine orange interaction with DNA: Effect of ionic strength

BackgroundThe study of acridine orange (AO) spectral characteristics and the quenching of its singlet and triplet excited states by TEMPO radical at its binding to DNA in the function of the DNA concentration and in the absence and presence of NaCl is reported.MethodsThe study was performed using steady-state and time resolved optical absorption and florescence, fluorescence correlation spectroscopy and resonant light scattering techniques.ResultsThe presence of different species in equilibrium: AO monomers and aggregates bound to DNA, has been demonstrated, their relative content depending on the DNA and the AO concentrations. At high DNA concentration the AO monomers are protected against the contact with other molecules, thus reducing the AO excited state quenching. The addition of NaCl reduces the AO binding constant to DNA, thus reducing the AO and DNA aggregation.ConclusionsThe interaction of AO with DNA is a complex process, including aggregation and disaggregation of both components. This modifies the AO excited state characteristics and AO accessibility to other molecules. The salt reduces the DNA effects on the AO excited state characteristics thus attenuating its effects on the AO efficacy in applications.General significanceThis study demonstrates that the interaction of photosensitizers with DNA, depending on their relative concentrations, can both decrease and increase the photosensitizer efficacy in applications. The salt is able to attenuate these effects.     

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.     

Diamine oxidase from horse kidney: ionic strength dependence of stability and activity

Diamine oxidase was prepared from horse kidney by a procedure involving heat denaturation at 50 degrees C, ammonium sulfate fractionation, chromatography on hydroxyapatite and on G-200 Sephadex columns. This procedure gave about 1000 fold purification over the crude kidney cortex homogenate. The enzyme preparations thus obtained are stable only at high ionic strength. The effect on enzyme activity of salt concentration and various stabilizing agents have been investigated. The horse kidney diamine oxidase is irreversibly inhibited by carbonyl reagents and shows substrate specificity quite similar to other animal diamine oxidases.     

Thermodynamics and dissociation constants of carboxylic acids at high ionic strength and temperature

Dissociation constants (pK_a) of oxalic, iminodiacetic, citric, nitrilotriacetic, ethylenediaminetetraacetic, trans-1,2 diaminocyclohexanetetraacetic acid and diethylenetriaminepentaacetic acid have been determined potentiometrically using a glass electrode at an ionic strength of 6.60 m (NaClO₄) and temperatures of 0-60 °C. The constants of iminodiacetic, nitrilotriacetic and diethylenetriaminepentaacetic acid were measured at 25 °C at ionic strengths from 0.30 to 6.60 m (NaClO₄). The thermodynamic parameters for the dissociation of these carboxylic acids were derived from the temperature dependence of the dissociation constants. The Specific Ion Interaction Theory (SIT) and the Parabolic model successfully described the ionic strength dependencies of the pK_a values. The variation of the pK_a values at high ionic strengths as a function of the type and concentration of supporting electrolyte is discussed and compared with literature data.     

The ionic strength dependence of the cooperativity factor for DNA melting

The melting temperature for the d(AT)24.d(AT)24 stretch, located inside the DNA helix and terminally, have been determined in a wide range of ionic strength values (0.01 - 1 M Na+). The cooperativity factor was calculated from the shifts in the melting temperature of the stretch due to its different boundary conditions. With the sodium concentration decreasing from 1 M to 0.01 M the cooperativity factor dropped by three orders of magnitude, its change being less marked at high than at low ionic strength.     

The dependence of the molecular dynamics of calmodulin upon pH and ionic strength

The mobilities of several fluorescent probes placed at different locations on calmodulin in the absence of Ca2+ have been found to depend upon the charge, ionic strength, and temperature. In general, the time decay of fluorescence anisotropy could be fitted with two rotational correlation times. The shorter of these reflects primarily the motion of the probe itself, while the longer corresponds to the motion of a major portion of the molecule. An increase in ionic strength or a decrease in net charge results in a decrease in the relative amplitude of the shorter correlation time, while an increase in temperature produces an increase in its amplitude. These results are consistent with, and suggest, that an increase in probe mobility accompanies an expansion of the calmodulin molecule under conditions of high electrostatic stress.     

Thermodynamic characterization of daunomycin-DNA interactions: microcalorimetric measurements of daunomycin-DNA binding enthalpies

We report the first direct determination of binding enthalpies for the complexation of monomeric daunomycin with a series of 10 polymeric DNA duplexes. These measurements were accomplished by using a recently developed stopped-flow microcalorimeter capable of detecting reaction heats on the microjoule level. This enhanced sensitivity allowed us to measure daunomycin-DNA binding enthalpies at monomeric drug concentrations (e.g., 10-20 microM), thereby precluding the need to correct for daunomycin self-association, as has been required in previous batch calorimetric studies [Remeta, D. P., Marky, L. A., & Breslauer, K. J. (1984) Abstracts of Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, 838a; Breslauer, K. J., Remeta, D. P., Chou, W. Y., Ferrante, R., Curry, J., Zaunczkowski, D., Snyder, J. G., & Marky, L. A. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8922-8926]. We correct the published daunomycin-DNA binding enthalpies measured by batch calorimetry at higher drug concentrations (e.g., 0.5-1.0 M) for the enthalpy contribution associated with the binding-induced disruption of drug aggregates. The requisite correction term was obtained from a van't Hoff analysis of temperature-dependent NMR measurements on daunomycin solutions. We find remarkable agreement between the net binding enthalpies derived from these corrected batch calorimetric data and the corresponding binding enthalpies measured directly by stopped-flow microcalorimetry. The enhanced sensitivity of the stopped-flow instrument also allowed us to evaluate the influence of drug binding density on the daunomycin-DNA binding enthalpies. This assessment was accomplished by conducting stopped-flow calorimetric measurements over a range of seven different drug-to-phosphate ratios (r). For most of the 10 DNA host duplexes studied, we find that the daunomycin binding enthalpies exhibit small but significant r dependencies. The sensitivity of the stopped-flow instrument also enabled us to detect significant dilution enthalpies for several of the drug-free DNA duplexes, a quantity generally assumed to be negligible in previous studies. We discuss the binding enthalpies, their dependence on binding density, and the duplex dilution enthalpies in terms of the influence of base composition, sequence, conformation/hydration, and binding cooperativity on the sign and the magnitudes of the daunomycin-DNA binding enthalpy data reported here.     

Deamidation of glutaminyl residues: dependence on pH, temperature, and ionic strength

We have shown the dependence of the deamidation half-times of the peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly upon pH, temperature, and ionic strength. Increase in temperature or ionic strength, variation of pH to pH′s higher or lower than pH 6, and the use of phosphate buffer rather than Tris buffer at high pH all decrease the half-time of dcamidation. Temperature increase of 20°C or pH change of 2 pH units decreases the half-time about fivefold, while increase of one ionic strength unit decreases the half-time about twofold. In pH 7.4, I = 0.2, 37.0°C phosphate buffer, the deamidation half-times are 663 ± 74 and 389 ± 56 days respectively for the two peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly.These experiments should serve as a warning to peptide and protein experimenters that even the more stable glutaminyl residues are unstable with respect to deamidation in certain solvent conditions. These experiments also provide, along with previously reported experiments on asparaginyl peptides (7), some quantitative data to help with the extrapolation of in vitro deamidation experiments to in vivo deamidation conditions.     

Effect of ionic strength on the interaction between aldolase and actin-containing filaments

The effect of ionic strength on the adsorption of aldolase to synthetic thin filaments derived from rabbit skeletal muscle has been investigated by partition equilibrium experiments, the results being interpreted in terms of the intrinsic association constant for the interaction of four sites on aldolase with two sites per filament repeat unit. At physiological ionic strength, values of 10,000 and 2000 m^?1 were obtained for this equilibrium constant in the absence and presence, respectively, of calcium ions. Comparison of binding curves obtained with synthetic thin filaments and myofibrils indicated a lesser extent of enzyme adsorption to the myofibrillar system, a difference attributed to the covert nature of many of the potential binding sites on the filaments in the assembly of the myofibril. On the basis of the quantitative information on the effect of ionic strength on the adsorption of aldolase, a case is made for the probable occurrence of the enzyme-filament interaction as a physiologically significant phenomenon in skeletal muscle.     

Erythrocyte agglutination by wheat germ agglutinin: ionic strength dependence of the contact seam topology

Abstract

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K_D of 138 ± 55 nM, a ΔH of ?9708 ± 1195 cal/mol and a ΔS of ?1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K_D of 3.2 ± 0.8 μM, a ΔH of ?2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.     

Electro-optical properties of carboxymethylcellulose. I. Concentration and ionic strength dependence

The experimental conditions for studying the electro-optical properties of a natural, modified polyelectrolyte, carboxymethylcellulose (DS 1.3; DP 180) were determined. The transient Kerr effect was found to be a function of CMC concentration, field strength, and ionic strength, I. If the concentration and I were low enough (c < 20 mg.l^?1), saturation was obtained for field strengths of approximately 15 kV.cm^?1. The optical anisotropy was shown to be independent of I; the electrical anisotropy decreased sharply when I increased. These results are discussed in connection with polarization theories of polyelectrolytes. The molecular dimensions of carboxymethylcellulose, calculated from the birefringence kinetics, suggest that the molecule is a rigid rod.     

Electric charge distribution in proteins and ionic strength dependence of reaction rate

The ionic strength dependence of the reaction rate between protein and dichloride anion radical has been investigated by flash photolysis of aqueous chloride-containing lysozyme, ribonuclease A, or insulin. The rate constant for the reaction of lysozyme or ribonuclease A with dichloride anion radicals decreases with increasing ionic strength, while it increases for insulin. The dependence was found to obey an equation derived from the theory of Debye and Hückel or the equation of Wherland and Gray for lysozyme within experimental errors. For ribonuclease A, however, it deviates largely from these equations. In the case of insulin a moderate deviation was observed. The different behavior in the ionic strength dependence is discussed in terms of the electric charge distribution in the protein molecules.     

Histone-histone interaction mediates chromatin unfolding at physiological ionic strength

High-resolution thermal denaturation data on chicken erythrocyte chromatin are reported over 4 orders of magnitude in NaCl concentration which includes the physiological region. A novel technique using critical-point polyacrylamide sols instead of ordinary solvents effectively stabilizes chromatin against precipitation at high salt concentrations. These sols are optically transparent from 260 to 320 nm and are thermally stable over the temperature ranges studied. At Na+ ion concentrations below 10 mM, the polyacrylamide slightly destabilizes chromatin at the nucleosome level, possibly through interactions of histones H1 and H5 with the carboxylic acid residues. At the same low salts, polyacrylamide stabilizes pure DNA against denaturation, presumably by mechanically stabilizing it against helix-distorting thermal fluctuations. In both cases, however, the polyacrylamide sols are entirely noninvasive at higher salts. Prominent low-temperature thermal transitions are observed in chromatin at and above 100 mM NaCl which evidently are associated with conformational changes in DNA. Our results are in accord with the idea that histone-histone interactions at physiological ionic strengths (approximately 100 mM Na+) may be comparable to histone-DNA interactions and hence may be sufficient to promote the destabilization of the DNA helix in chromatin under these conditions. The biological implications of this are discussed, and a possible model for the local decondensation of chromatin under physiological conditions is proposed.     

Electrostatic effects in hemoglobin: Bohr effect and ionic strength dependence of individual groups.

The electrostatic treatment applied in the preceding paper in this issue [Matthew, J. B., Hanania, G.I.H., & Gurd, F.R.N. (1979) Biochemistry (preceding paper in this issue)] to the titration behavior of individual groups in human deoxyhemoglobin and oxyhemoglobin was applied to the computation of the alkaline Bohr effect at various values of ionic strength. The enhanced proton binding of deoxyhemoglobin in the pH range of 6--9 was accounted for at ionic strength 0.01 M by the effects of the unique charge distributions of ionizable groups in the two quaternary states. At ionic strength 0.10 M the effects of 2--4 bound anions had to be considered in addition in the deoxyhemoglobin charge configuration. At the higher ionic strength 10 groups per tetramer contributed to the Bohr effect, whereas 28 groups were contributory at the lower ionic strength. The ionic strength dependence of individual groups in the two tetrameric structures as well as in the alpha-chain monomer was explained in terms of the electrostatic treatment. This examination showed that the differences in electrostatic behavior of deoxy- and oxyhemoglobin follow from particular dissymmetries in their configurations with respect to charge and static solvent accessibility.