Differential pulse voltammetric studies of ethidium bromide binding to DNA

The interaction of ethidium bromide (EtBr) with calf thymus DNA is investigated electrochemically with the use of differential pulse voltammetry (DPV) at two different ionic strengths of a solution (0.154 M and 0.02 M [Na+], pH 7.0). It is revealed that EtBr binds with DNA in more than one way. The appropriate values of constants (K) and number site sizes (n) of EtBr binding to DNA are determined. The values of binding constants are equal to 1.9 x 10(6) and 5.6 x 10(5) M(-1), and number site sizes to 9 and 3.6 for strong interactions at ionic strengths of solutions 0.02 and 0.154 M Na+ at 28 degrees C, respectively. For a weaker interaction, these parameters are equal to 7 x 10(4) and 8 x 10(4) M(-1) and 1.5 and 1 at the mentioned ionic strengths of solutions, respectively. Thus, EtBr interacts with DNA in more than one way--intercalative and electrostatic at low ionic strength, and semi-intercalative and electrostatic at a higher strength of the solution. These results are in good accordance with the ones obtained by spectroscopic (absorption and fluorimetric) methods.     

Adsorption behaviour of lactoferrin in oil-in-water emulsions as influenced by interactions with beta-lactoglobulin

Oil-in-water emulsions (pH 7.0 or pH 3.0) containing 30 wt% soya oil and various concentrations of lactoferrin were made in a two-stage valve homogenizer. The average droplet size (d32), the surface protein coverage (mg/m2) and composition, and the zeta-potential of the emulsions were determined. The value of d32 decreased with increasing lactoferrin concentration up to 1%, and then was almost independent of lactoferrin concentration beyond 1% at both pH 7.0 and pH 3.0. The surface protein coverage of the emulsions made at pH 7.0 increased almost linearly with increasing lactoferrin concentration from 0.3 to 3%, but increased only slightly in emulsions made at pH 3.0 at lactoferrin concentrations >1%. The surface protein coverage of the emulsions made at pH 3.0 was lower than that of the emulsions made at pH 7.0 at a given protein concentration. The emulsion droplets had a strong positive charge at both pH 7.0 and pH 3.0, indicating that stable cationic emulsion droplets could be formed by lactoferrin alone. When emulsions were formed with a mixture of lactoferrin and beta-lactoglobulin (beta-lg) (1:1 by weight), the charge of the emulsion droplets was neutralized at pH 7.0 suggesting the formation of electrostatic complexes between the two proteins. The composition of the droplet surface layer showed that both proteins were adsorbed, presumably as complexes, from the aqueous phase at pH 7.0 in equal proportions, whereas competitive adsorption occurred between lactoferrin and beta-lg at pH 3.0. At this pH, beta-lg was adsorbed in preference to lactoferrin at low protein concentrations (1%), whereas lactoferrin appeared to be adsorbed in preference to beta-lg at high protein concentrations.     

Electrochemical Study on the Interaction Betwwen Neutral Red and DNA

A voltammetric study of the interaction of neutral Red(NR) with DNA at a gold electrode in a phosphate buffer solution is described. After adding DNA in an NR solution, the reduction peak current of NR decreases. The binding mechahisms of NR to DNA in different pH ranges are different. The reduction peak potential of NR in a pH 7.0 phosphate buffer solution in the presence of DNA shifts positively, indicating that the binding of NR to DNA is intercalation action, but at pH=6.0 the reduction peak potential of NR shifts negatively, indicating that the binding of NR to DNA is electrostatic action. The formed complexes are DNA-NR when [NR]/[DNA]<0.18 and DNA-3NR when [NR]/[DNA]>0.35, respectively.     

Electrochemical Study on the Interaction Between Neutral Red and DNA

IntroductionDeoxyribonucleic acid( DNA) is the most im-portant germ plasma of most organisms.It playsan importantrole in the process ofstoring,copyingand transmitting germ messages.There have beenmany papers studying on the interaction betweensmall molecules and DNA since the1 960′s.Nowthe researches have become a field of common in-terest[1] .Those researches have contributed to theunderstanding of the way of the interaction be-tween DNA and protein.What is more,those re-searches are he…     

多柔比星稀土金属离子配合物与DNA相互作用的研究

用紫外分光光度法和荧光光谱法研究了多柔比星( Adriamycin,ADM)稀土金属离子配合物(ADM-M)与DNA的相互作用.结果发现,在pH=7.0时,ADM与Eu3+、yb3+能形成稳定配合物,该配合物可使DNA的最大吸收产生明显的减色效应及红移,并能够竞争置换溴化乙锭(EB)与DNA的结合点.KI猝灭试验发现D...     

Contributions of hydroxyethyl groups to the DNA binding affinities of anthracene probes

Contributions of hydroxyethyl functions to the DNA binding affinities of substituted anthracenes are evaluated by calorimetry and spectroscopy. Isothermal titration calorimetry indicated that binding of the ligands to calf thymus DNA (5 mM Tris buffer, 50 mM NaCl, pH 7.2, 25 degrees C) is exothermic. The binding constants increased from 1.5 x 10(4) to 1.7 x 10(6) M(-1) as a function of increase in the number of hydroxyethyl functions (0-4). DNA binding was accompanied by red-shifted absorption (approximately 630 cm(-1)), strong hypochromism (>65%), positive induced-circular dichroism bands, and negative linear dichroism signals. DNA binding, in general, increased the helix stabilities to a significant extent (DeltaT(m) approximately 7 degrees C, DeltaDeltaH approximately 3 kcal/mol, DeltaDeltaS approximately 6-20 cal/K.mol). The binding constants showed a strong correlation with the number of hydroxyethyl groups present on the anthracene ring system. Analysis of the binding data using the hydrophobicity parameter (Log P) showed a poor correlation between the binding affinity and hydrophobicity. This observation was also supported by a comparison of the affinities of probes carrying N-ethyl (Kb = 0.8 x 10(5) M(-1)) versus N-hydroxyethyl side chains (Kb = 5.5 x 10(5) M(-1)). These are the very first examples of a strong quantitative correlation between the DNA binding affinity of a probe and the number of hydroxyethyl groups present on the probe. These quantitative findings are useful in the rational design of new ligands for high-affinity binding to DNA.     

A novel bis-phenanthridine triamine with pH controlled binding to nucleotides and nucleic acids

The new bis-phenanthridine triamine is characterised by three pK(a) values: 3.65; 6.0 and >7.5. A significant difference in the protonation state of at pH = 5 (four positive charges) and at pH = 7 (less than two positive charges) accounts for the strong dependence of -nucleotide binding constants on nucleotide charge under acidic conditions, whereas at neutral pH all -nucleotide complexes are of comparable stability. All experimental data point at intercalation as the dominant binding mode of to polynucleotides. However, there is no indication of bis-intercalation of the two phenanthridine subunits in binding to double stranded polynucleotides, the respective complexes being most likely mono-intercalative. Thermal stabilisation of calf thymus DNA (ct-DNA) and poly A-poly U duplexes upon addition of is significantly higher at pH = 5 than at neutral conditions. This is not the case with poly dA-poly dT, indicating that the specific secondary structure of the latter, most likely the shape of the minor groove, plays a key role in complex stability. At pH = 5 acts as a fluorimetric probe for poly G (emission quenching) as opposed to other ss-polynucleotides (emission increase), while at neutral conditions this specificity is lost. One order of magnitude higher cytotoxicity of compared to its "monomer" can be accounted for by cooperative action of two phenanthridinium units and the charged triamine linker. The results presented here are of interest to the development of e.g. sequence-selective cytostatic drugs, and in particular for the possibility to control the drug activity properties over binding to DNA and/or RNA by variation of the pH of its surrounding.     

A simple helical macrocyclic polyazapyridinophane as a stereoselective receptor of biologically important dicarboxylates under physiological conditions

The interaction of a synthetic enantiopure azamacrocyclic receptor (L) with biologically important chiral dicarboxylates (A, 1-7) has been studied by means of potentiometric titrations in 0.15 M NaCl aqueous solution in a wide pH range. This macrocycle forms strong complexes of the type [HnLA](n-2) (with n = 0-5). As a general trend, the binding is much tighter at basic or neutral pH than in acidic medium. Interestingly, nonprotected excitatory amino acids (Asp and Glu) are strongly bound even at acidic pH. Regarding selectivity, the receptor showed stereoselective binding toward those substrates bearing an H-bonding donor at Calpha, being S-selective in most of the cases, except for glutamic acid. Thus, L displayed an excellent enantioselectivity for (S)-malate dianion (KS/KR = 11.50 at pH 10.0 and KS/KR = 6.86 at pH 7.0) and exhibited moderate enantiopreference for (S,S)-tartrate (KSS/KRR = 3.01 at pH 10 and KSS/KRR = 1.70 at pH 7.0). For this last anion, a very good diastereopreference was also observed (KSS/KRS = 8.46 at pH 10 and KSS/KRS = 4.99 at pH 7.0). On the contrary, L is smoothly R-selective toward (R)-Glu (KR/KS = 3.22 at pH 10 and KR/KS = 2.05 at pH 7.0) due to its longer and more flexible molecular structure. The stereoselectivity of the corresponding complexes decreased when decreasing pH values. For the hydroxy derivatives, mass spectrometry also reflected the trends observed by potentiometry and confirmed the receptor:dicarboxylate 1:1 stoichiometry of the supramolecular complexes. Additional experimental techniques were used to study the most stereoselective example. Solution studies by NMR suggested a good geometrical complementarity between the malate dianion and the receptor, which showed a predominant helical conformation in solution. Besides, self-diffusion rates (PGSE) of the diastereomeric complexes with malate also agree with binding data. Circular dichroism was also used in this case at different pH values, showing a very good correlation between the helical content of the receptor and the stereoselectivity of the molecular recognition process.     

Comparative studies on interactions of bovine serum albumin with cationic gemini and single-chain surfactants

The interactions of bovine serum albumin (BSA) with cationic gemini surfactants alkanediyl-alpha,omega-bis(dodecyldimethylammonium bromide) [C12H25(CH3)2N(CH2)(S)N(CH3)2C12H25]Br2 (designated as C12C(S)C12Br2, S = 3, 6, and 12) and single-chain surfactant dodecyltrimethylammonium bromide (DTAB) have been studied with isothermal titration microcalorimetry, turbidity, fluorescence spectroscopy, and circular dichroism at pH 7.0. Comparing with DTAB, C12C(S)C12Br2 have much stronger binding ability with BSA to induce the denaturation of BSA at very low molar ratio of C12C(S)C12Br2/BSA, and C12C(S)C12Br2 have a much stronger tendency to form insoluble complexes with BSA. The binding of C12C(S)C12Br2 to BSA generates larger endothermic peaks. The first endothermic peak is much stronger than that of the second endothermic peak. The double charges and strong hydrophobicity of the gemini surfactants are the main reasons for these observations. In addition, the spectra results show that the binding of DTAB to BSA only promotes BSA unfolding and aggregation, whereas the secondary structure of BSA is possibly stabilized by a small amount of C12C(S)C12Br2 , even if the small amount of binding C12C(S)C12Br2 could induce the loss of the tertiary structure of BSA. This result may be related to the double tails of gemini surfactants, which may generate the hydrophobic linkages between the nonpolar residues of BSA.     

Enantioseparation of phenothiazines in cyclodextrin-modified capillary zone electrophoresis: reversal of migration order

Enantioseparations of phenothiazines with gamma-cyclodextrin (gamma-CD) as a chiral selector were investigated using citrate and phosphate buffer electrolytes at pH 3.0. Reversal of the enantiomer migration order of promethazine, ethopropazine, and trimeprazine was observed by varying gamma-CD concentration in the range of 5-9 mM, 2.5-4.5 mM and 1.5-2.8 mM, respectively, using 100 mM citrate buffer at pH 3.0. As in the case of beta-CD, the (+)-enantiomers of phenothiazines possess greater binding strength to gamma-CD than the (-)-enantiomers. The evaluation of the binding constants and limiting mobility of the complexes formed between the enantiomers of phenothiazines and gamma-CD reveals that the binding strength of phenothiazines to gamma-CD and the differences in the binding constants and limiting mobility of the complexes are responsible for the enantiomer migration reversal. Both the binding constants and limiting mobility of the complexes between the (+)-enantiomers of phenothiazine and gamma-CD are greater than those of the corresponding (-)-enantiomers in a citrate buffer, while the binding constants of the complexes primarily determined the migration order of the enantiomers in a phosphate buffer. Compared with the results obtained using a phosphate buffer, we may conclude that citrate buffer which involves competitive complexation with chiral selector plays a significant role in the enantiomer migration reversal.     

A pyrazine-based fluorescence-enhancing ligand with a high selectivity for thymine in AP site-containing DNA duplexes

A fluorescent pyrazine derivative, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), is presented as a promising light-up ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 7.0 (I = 0.11 M, at 5 degrees C), DCPC can bind to thymine selectively over other nucleobases opposite an abasic site in DNA duplexes (5'-GTGTG CGTTG ANA TGGAC GCAGA-3'/3'-CACAC GCAAC TXT ACCTG CGTCT-5', X = abasic site, N = target nucleotide) with a dissociation constant of 2.6 microM. The binding of DCPC is accompanied by a significant enhancement of its fluorescence (lambda(max), 412 nm), and the response is highly selective to thymine base. These binding and sensing properties allow a clear detection of thymine-related mutations present in polymerase chain reaction (PCR) amplification products.     

Recognition of flexible peptides in water by transition metal complexes

This paper describes the design, synthesis, and evaluation of transition metal complexes capable of recognizing flexible histidine-containing peptides in aqueous medium (25 mM HEPES buffer, pH = 7.0, 25 degrees C). When the pattern of metal ions on a complex matches with the pattern of histidine moieties on the peptide, strong interaction (K = 1.2 x 10(6) M-1) can be achieved. The complex was highly selective (> 200:1) in discriminating similar flexible peptides differing only by one glycine unit.     

Influence of temperature and interactions with ligands on dissociation of dsDNA and ligand-dsDNA complexes of various types of binding. An electrochemical study

Several medicinally important compounds that bind to dsDNA strands via intercalation (C-1311, C-1305, EtBr), major groove binding (Hoechst 33258) and covalent binding (cis-Pt) were examined. The obtained results suggest that both the transfer of conformation B to C and the denaturation process, for the ligand-dsDNA complexes, except for covalently bound cis-Pt, took place at higher temperatures compared to the unbound helix. Furthermore, much lower currents of electrooxidation of guanine at 100 °C, compared to the currents obtained at this temperature for dsDNA in the absence of ligands, suggest that the binding of ligands affects the way the dsDNA denaturates at increased temperatures and leads to formation of different forms of DNA single strands. The voltammetric results were compared with the data of two spectroscopic techniques: UV-Vis and CD.     

两种有机磺酸配合物的合成、表征及与DNA键合性质

合成了2个新型有机磺酸配合物,[Cd(phen)₂(ans)₂]·H₂O (1)和[Pb(phen)₂(ans)₂]·H₂O (2) (phen=1,10-邻菲咯啉,ans=4-氨基-1-萘磺酸根),通过元素分析、红外光谱等对配合物进了表征,用X-射线单晶衍射方法测定了配合物的单晶结构。应用紫外-可见吸收光谱、荧光光谱及粘度测定方法研究了配合物与ctDNA的作用,发现2个配合物均以插入和氢键两种模式与ctDNA发生作用。     

Thermodynamic, kinetic and pH studies on the reactions of NCS-, N3-, and CH3CO2- with Fusarium galactose oxidase

Thermodynamic and kinetic studies on the X- = NCS-, N3-, and CH3CO2- replacement of H2O/OH- at the CuII exogenous site of the tyrosyl-radical-containing enzyme galactose oxidase (GOaseox) from Fusarium (NRR 2903), have been studied by methods involving UV-vis spectrophotometry (25 degrees C), pH range 5.5-8.7, I = 0.100 M (NaCl). In the case of N3- and CH3CO2- previous X-ray structures have confirmed coordination at the exogenous H2O/OH- site. From the effect of pH on the UV-vis spectrum of GOaseox under buffer-free conditions, acid dissociation constants of 5.7 (pK1a; coordinated H2O) and 7.0 (pK2a; H+Tyr-495) have been determined. At pH 7.0 formation constants K(25 degrees C)/M-1 are NCS- (480), N3- (1.98 x 10(4)), and CH3CO2- (104), and from the variations in K with pH the same two pKa values are seen to apply. No pK1a is observed when X- is coordinated. From equilibration stopped-flow studies rate constants at pH 7.0 for the formation reaction kf(25 degrees C)/M-1 s-1 are NCS- (1.13 x 10(4)) and N3- (5.2 x 10(5)). Both K and kf decrease with increasing pH, consistent with the electrostatic effect of replacing H2O by OH-. In the case of the GOaseox Tyr495Phe variant pK1a is again 5.7, but no pK2a is observed, confirming the latter as acid dissociation of protonated Tyr-495. At pH 7.0, K for the reaction of four-coordinate GOaseox Tyr495Phe with NCS- (1.02 x 10(5) M-1) is more favorable than the value for GOaseox. Effects of H+Tyr-495 deprotonation on K are smaller than those for the H2O/OH- change. The pK1a for GOasesemi is very similar (5.6) to that for GOaseox (both at CuII), but pK2a is 8.0. At pH 7.0 values of K for GOasesemi are NCS- (270 M-1), N3- (4.9 x 10(3)), and CH3CO2- (107).     

Tripropyltin(iv) complexes with some selected bioligands in 50% v/v dioxane/water mixture

The interaction of the tri-n-propyltin(IV) (TPT) with some selected bioligands having a variety of model functional groups were investigated using potentiometric technique at 25 degrees C and 0.1 M ionic strength in 50% v/v dioxane/water mixture. TPT is hydrolyzed to give [(C3H7)3SnOH], [(C3H7)3Sn(OH)2]-, and [((C3H7)3Sn)2OH]+. Amino acids and DNA constituents form 1:1 and 1:2 complexes. Peptides form 1:1 complexes and the corresponding deprotonated amide species. The protonated complexes are formed with amino acids, peptides and some DNA constituents. The hydrolysis constants and the stepwise formation constants of the complexes formed in solution were calculated using the nonlinear least-square program MINIQUAD-75. The participation of different ligand functional groups in binding to organotin is discussed. The speciation diagrams of the various complex species were evaluated as a function of pH.     

Complex Formation Reactions of Dimethyltin(IV) with Some Zwitterionic Buffers

Equilibrium studies in aqueous solution are reported for dimethyltin(IV) complexes of zwitterionic buffers, such as bicine and tricine (L). Stoichiometry and stability constants for the complexes formed were determined at 25°C and ionic strength 0.1M NaNO₃. The results showed the best fit of the titration curves were obtained for complexes MLH, ML, ML₂, MLH_-1, and MLH_-2 with the hydrolysis products of the dimethyltin(IV) cation. The bonding sites of the dimethyltin(IV) complexes with bicine and tricine at different pH were characterized in the solid state by elemental analyses, FTIR, and TG analysis. The molecular formula of the complexes synthesized at pH=3.0 is [(CH₃)₂Sn(L)(H₂O)]Cl while in neutral and alkaline media the hydrolytic species are formed.     

Reactions of platinum(II) complexes with sulfur- and histidine-containing peptides: a model for selective platination of peptides and proteins

The reactions between two monofunctional platinum complexes [Pt(Me₄dien)Cl]⁺ (Me₄dien = 1,1,7,7-tetramethyl-diethylenetriamine) and [Pt(Et₄dien)Cl]⁺ (Et₄dien = 1,1,7,7-tetraethyldiethylenetriamine) and the peptides, N-acetylated L-methionyl-L-histidine (MeCO–Met–His) and glutathione (GSH), have been investigated by ¹H-n.m.r. spectroscopy and u.v.–vis. spectrophotometry. The reactions of the platinum(II) complexes with MeCO–Met–His were carried out at room temperature and at pH 3.0 and 7.0, whereas with GSH the reactions were studied only at pH 3.0. No binding of these two platinum complexes to the sulfur atom of methionine or to nitrogen atoms of histidine residue of MeCO–Met–His was observed during the first 24 h. When the reaction was followed further, after 24 h very slow binding of [Pt(Me₄dien)Cl]⁺ to the N3 nitrogen atom of imidazole was observed. Both platinum complexes react with the sulfur atom of the cysteine residue in GSH. Kinetic data show that GSH reacts twice as fast with [Pt(Me₄dien)Cl]⁺ than with [Pt(Et₄dien)Cl]⁺. Our findings indicate that sterically crowded platinum(II) complexes are only capable of reacting with the sulfhydryl group of the cysteine residue. This influences the design of new platinum(II) complexes for selective covalent modification of peptides and proteins.     

Interaction of anticancer herbal drug berberine with DNA immobilized on the glassy carbon electrode

The interaction of anticancer herbal drug berberine with double-strand DNA (dsDNA) and single-strand DNA (ssDNA) in solution, dsDNA immobilized on the glassy carbon electrode prepared by Langmuir-Blodgett technique, were investigated by electrochemical techniques (cyclic voltammetry, differential pulse voltammetry) and UV spectroscopy. The presence of DNA results in a decrease of the currents and a negative shift of the electrode potentials from the DPV curves of berberine, indicating the dominance of electrostatic interactions. The spectroscopy data confirmed that the predominant interaction between berberine and DNA is electrostatic. The binding of berberine with DNA, when analyzed in terms of the cooperative Hill model, yields the binding constant K(a)=2.2(+/-0.2)x10(4) M(-1), corresponding to the dissociation equilibrium constant K(d)=4.6(+/-0.3)x10(-5) M, which in the range of the applied concentrations of DNA (bp) and berberine, and a Hill coefficient m=1.82(+/-0.08) in Britton-Robinson buffer solution (0.05 M, pH 5.72) at T=298 K (25 degrees C). Apparently, at least two molecules of berberine have to bind as a couple to cause, e.g., the "elementary event" of current change. The results are suggestive for further fruitful applications of this anticancer herbal drug and DNA-modified electrodes.     

Effect of time on the interfacial and foaming properties of beta-lactoglobulin/acacia gum electrostatic complexes and coacervates at pH 4.2

The electrostatic complexation between beta-lactoglobulin and acacia gum was investigated at pH 4.2 and 25 degrees C. The binding isotherm revealed a spontaneous exothermic reaction, leading to a DeltaHobs = -2108 kJ mol(-1) and a saturation protein to polysaccharide weight mixing ratio of 2:1. Soluble electrostatic complexes formed in these conditions were characterized by a hydrodynamic diameter of 119 +/- 0.6 nm and a polydispersity index of 0.097. The effect of time on the interfacial and foaming properties of these soluble complexes was investigated at a concentration of 0.1 wt % at two different times after mixing (4 min, referred as t approximately 0 h and t = 24 h). At t approximately 0 h, the mixture is mainly made of aggregating soluble electrostatic complexes, whereas after 24 h these complexes have already insolubilize to form liquid coacervates. The surface elasticity, viscosity and phase angle obtained at low frequency (0.01 Hz) using oscillating bubble tensiometry revealed higher fluidity and less rigidity in the film formed at t approximately 0 h. This observation was confirmed by diminishing bubble experiments coupled with microscopy of the thin film. It was thicker, more homogeneous and contained more water at t approximately 0 h as compared to t = 24 h (thinner film, less water). This led to very different gas permeability's of Kt approximately 0 h = 0.021 cm s(-1) and Kt=24 h) = 0.449 cm s(-1), respectively. Aqueous foams produced with the beta-lactoglobulin/acacia gum electrostatic complexes or coacervates exhibited very different stability. The former (t approximately 0 h) had a stable volume, combining low drainage rate and mainly air bubble disproportionation as the destabilization mechanism. By contrast, using coacervates aged for 24 h, the foam was significantly less stable, combining fast liquid drainage and air bubble destabilization though fast gas diffusion followed by film rupture and bubble coalescence. The strong effect of time on the air/water interfacial properties of the beta-lactoglobulin/acacia gum electrostatic complexes can be understood by their reorganization at the interface to form a coacervate phase that is more fluid/viscous at t approximately 0 h vs rigid/elastic at t = 24 h.