Description of the binding of chitosan to DNA at different ionic strengths in terms of the theories of ion condensation and adsorption

The binding of chitosan molecules to DNA in solutions of different ionic strength has been studied. The data were analyzed in terms of the model of ion condensation and the thermodynamic theory of the binding of protracted ligands to DNA. Combining these approaches made it possible to estimate the sterical and energetic characteristics of chitosan-DNA binding and establish the dependence of the chitosan-DNA binding constant on the ionic strength of solution.     

Influence of ionic strength on Hoechst 33258 binding with DNA

The binding of Hoechst 33258 with DNA at various ionic strengths of solution and different ligand concentrations has been investigated. Existence of more than one type of interactions of Hoechst 33258 with DNA has been revealed, which were very sensitive to the ionic strength. Hoechst 33258 doesn't show specificity to AT sequences of DNA at low ionic strength. High affinity binding mode becomes obvious at high ionic strength. The values of binding constants and binding site sizes for revealed strong and weak interactions have been determined.     

The effect of temperature on the interaction of Yersinia pseudotuberculosis lipopolysaccharide with chitosan

The mechanism of binding of lipopolysaccharide (LPS) from Yersinia pseudotuberculosis to low-molecular-weight chitosan was investigated using sedimentation analysis, centrifugation in glycerol and percoll density gradients, and isopicnic centrifugation in cesium chloride. The LPS interaction with chitosan was shown to be a multistage process that depended on time and reaction temperature. A stable LPS-chitosan complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). This temperature caused the LPS dissociation and promoted its binding to chitosan. The LPS binding to chitosan results in further dissociation of the endotoxin and formation of the complex with a molecular weight that is tens of times less than the initial molecular weight of LPS. The obtained complex remained stable in solutions of high ionic strength.     

Phosphorylated protamines. II. Circular dichroism of complexes with DNA, dependency on ionic strength.

The influence of protamine phosphorylation upon the conformation of nucleoprotamine complexes was studied at different ionic strengths using circular dichroism. The sharp onset of CD spectral changes upon decreasing the NaC1 concentrationwas correlated with the beginning of complex formation and can be used to determine apparent binding affinities in terms of a critical ionic strength. It is show that phosphorylation strongly reduces the binding strength of protamines towards DNA. Directly mixed and reconstituted complexes reveal differences in their CD spectra, which decrease with increasing ionic strength. Spectra of complexes between threefold phosphorylated clupeine Z and DNA obtained by reconstitution or direct mixing at higher ionic strength resemble the phi-type spectra of DNA and are unique for the phosphorylated species. The implications of protamine phosphorylation for chromatin or DNA condensation havebeen discussed.     

Binding of ethidium bromide and quinacrine hydrochloride to nucleic acids and reconstituted nucleohistones

Studies of binding of ethidium bromide and quinacrine hydrochloride to native DNA at low ionic strength indicate that for both compounds the binding is selective, with about one binding site for about four nucleotides. Annealing of unfractionated histones to DNA by a salt-gradient dialysis method slightly decreases the binding of the dyes to DNA. Similar observations made with reconstituted preparations by using individual histone fractions reveal that the arginine-rich histones (histones H3 and H4) are most effective in decreasing the binding. The binding studies with ethidium bromide at high ionic strength and with denatured DNA show that strong dye binding to DNA is strongly dependent on the ionic strength and on the secondary structure of DNA. The histones are not effective in decreasing the dye binding under conditions of high ionic strength. The results are consistent with the observations [Oliver & Chalkley (1974) Biochemistry13, 5093-5098; Axel, Melchoir, Sollner-Web & Felsenfield (1974) Proc. Natl. Acad. Sci. U.S.A.71, 4101-4105] that histones form some kind of surface structures on DNA through non-specific interactions and [Kornberg & Thomas (1974) Science184, 865-868; Kornberg (1974) Science184, 868-871; D'Anna & Isenberg (1974) Biochemistry13, 4992-4997; Vandegrift, Serra, Marve & Wagner (1974) Biochemistry13, 5087-5092] that the tendency of arginine-rich histones to aggregate may be an important factor in determining the structure of chromatin.     

Biospecific fractionation of chitosan

We have previously reported that, although a fully de-N-acetylated chitosan does not bind to hen egg white lysozyme, chitosans with a low fraction of N-acetylated units (FA) bind biospecifically to lysozyme with an affinity strongly dependent upon pH and ionic strength and without concomitant cleavage of glycosidic linkages. In this study, we report on the fractionation of a low FA chitosan with low molecular weight by biospecific adsorption of the chitosan molecules containing N-acetyl groups to immobilized lysozyme. Hen egg white lysozyme was immobilized to CNBr-activated Sepharose 4B, and a chitosan with a fraction of N-acetylated units of 0.045 and a weight average degree of polymerization (DPw) of 22 was applied to the column at suitable conditions for biospecific binding (pH 5.7, 0.15 M NaCl). The chitosan could be separated into two fractions, one that was not adsorbed to the lysozyme-column and one that was adsorbed and could be eluted by decreasing the pH and the ionic strength (0.08 M acetic acid of pH 3.0). The fractions were analyzed and the fraction that was not adsorbed was found to be fully de-N-acetylated chitosan with a DPw of 18, whereas the fraction that was adsorbed was a chitosan with FA of 0.080 and DPw of 24. Experimental data were compared with data from theoretical calculations, which was used to calculate the fraction of chitosan molecules with and without acetyl groups, showing good correlation between experimental and theoretical results.     

Nonhistone chromosomal protein HMG 1 interactions with DNA. Fluorescence and thermal denaturation studies

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.     

The potential role of proteoglycans in cationic lipid-mediated gene delivery. Studies of the interaction of cationic lipid-DNA complexes with model glycosaminoglycans.

Recent evidence supports a role for proteoglycans in polycation-mediated gene delivery. Therefore, the interaction of glycosaminoglycans with cationic lipid-DNA complexes (CLDCs) has been characterized using a combination of biophysical approaches. At low ionic strength, CLDCs bind to heparin-derivatized Sepharose particles, with the ratio of cationic lipid to DNA controlling the binding. Incorporation of the helper lipids cholesterol or 1,2-dioleoyl-phosphatidylethanolamine increases the amount of bound CLDC. Heparin also induces the aggregation of CLDCs, with cholesterol reducing this effect. Isothermal titration calorimetry demonstrates an endothermic heat for the binding of heparin to CLDCs at low ionic strength, whereas circular dichroism studies suggest a heparin-stimulated release of DNA from CLDCs at a greater than 20-fold charge excess. Increasing the ionic strength to 0.11 reduces CLDC binding to heparin beads, and greatly enhances the release of DNA from CLDCs by heparin. The ability of the cell surface glycosaminoglycan heparan sulfate to release DNA from CLDCs is more sensitive than heparin to the incorporation of the cholesterol or 1,2-dioleoyl-phosphatidylethanolamine. Titration calorimetry reveals an exothermic heat for the interaction glycosaminoglycans with CLDCs at higher ionic strength. These results are consistent with the direct involvement of proteoglycans in transfection.     

Modulation of binding of DNA to the C-terminal domain of p53 by acetylation

The binding of nonspecific DNA to the C-terminal negative regulatory domain (CTD) of p53 modulates its activity. The CTD is a natively unfolded region, which is subject to acetylation and phosphorylation at several residues as part of control. To measure the effect of covalent modification on binding to DNA, we synthesized a series of fluorescein-labeled CTD peptides with single and multiple acetylations at lysine residues that we had identified by NMR as making contact with DNA, and developed an analytical ultracentrifugation method to study their binding to DNA. Binding depended on ionic strength, indicating an electrostatic contribution. Monoacetylation weakened DNA binding at physiological ionic strength 2- to 3-fold, diacetylations resulted in further 2- to 3-fold decrease in the affinity, and tri- and tetraacetylations rendered DNA binding undetectable. Phosphorylation at S392 did not affect DNA binding. NMR spectroscopy showed binding to DNA did not induce significant structure into CTD, apart possibly from local helix formation.     

Bindings of RNAse to denatured and native deoxyribonucleic acids

The binding of pancreatic ribonuclease-A by denatured DNA, native DNA, poly-dA, and poly-dT, has been studied by a gel filtration method. With denatured DNA at pH 7.5, ionic strength 0.053M, there is one binding site per 12 nucleotides and the equilibrium binding constant per site is 9.7 × 10⁴ l./mole. The binding constant increases by a factor of 8 as the pH is decreased from 8 to 7. The strength of the binding of denatured DNA increases with decreasing ionic strength. At pH 7.5, native DNA binds about ? as strongly as does denatured DNA. The binding affinity increases in the order poly-dA, denatured DNA, and poly-dT. These results support the view that the binding of denatured DNA involves both electrostatic interactions between the negatively charged polynucleotide and the positively charged protein, and an interaction of the protein with a pyrimidine residue of the denatured DNA, and thus that the binding is basically similar to that between RNAse and its substrate RNA.     

The Sorption of Ketotifen Fumarate by Chitosan

The purpose of this investigation was to determine the mechanism of interaction between ketotifen fumarate and chitosan at different pH values. The specific surface area of chitosan was determined using gas sorption analyzer. The sorption experiments were conducted at pH 7 and 10 using two different particle size ranges of chitosan. The solutions were prepared at constant ionic strength and buffer concentration, with only varying the pH. The rotating bottle method was used for measuring the sorption. The average specific surface areas for the two different particle size ranges of chitosan were found to be 4.56 and 0.74 m²/g. The Langmuir-like equation and a model independent equation were both applied to the sorption experimental data. The extent of ketotifen uptake at pH 7 for small and large particles of chitosan was found to be 1,073 and 2,204 mg/g respectively. While the extent of ketotifen uptake at pH 10 for small and large particles of chitosan was found to be 4 and 11 mg/g respectively. The aforementioned results indicated that sorption of ketotifen fumarate at pH 7 is extremely high compared to pH 10 and that the sorption increases by decreasing the specific surface area of chitosan. Based on the results obtained, the following conclusions were reached. Ketotifen might be absorbed into the bulk structure of chitosan in addition to being adsorbed on the surface and the ability of chitosan to swell at pH 7 has a significant role in increasing its uptake.     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.     

The iso-competition point for counterion competition binding to DNA: calculated multivalent versus monovalent cation binding equivalence.

In this paper we introduce an important parameter called the iso-competition point (ICP), to characterize the competition binding to DNA in a two-cation-species system. By imposing the condition of charge neutralization fraction equivalence theta1 = ZthetaZ upon the two simultaneous equations in Manning's counterion condensation theory, the ICPs can be calculated. Each ICP, which refers to a particular multivalent concentration where the charge fraction on DNA neutralized from monovalent cations equals that from the multivalent cations, corresponds to a specific ionic strength condition. At fixed ionic strength, the total DNA charge neutralization fractions thetaICP are equal, no matter whether the higher valence cation is divalent, trivalent, or tetravalent. The ionic strength effect on ICP can be expressed by a semiquantitative equation as ICPZa/ICPZb = (Ia/Ib)Z, where Ia, Ib refers to the instance of ionic strengths and Z indicates the valence. The ICP can be used to interpret and characterize the ionic strength, valence, and DNA length effects on the counterion competition binding in a two-species system. Data from our previous investigations involving binding of Mg2+, Ca2+, and Co(NH3)63+ to lambda-DNA-HindIII fragments ranging from 2.0 to 23.1 kbp was used to investigate the applicability of ICP to describe counterion binding. It will be shown that the ICP parameter presents a prospective picture of the counterion competition binding to polyelectrolyte DNA under a specific ion environment condition.     

Circular dichroism spectroscopy of a cationic porphyrin bound to DNA

Recently, the porphyrin photosensitizer meso-tetra(4-N-methyl-pyridyl)-porphine was identified as a DNA-reactive agent demonstrating both electrostatic and intercalative binding. A series of porphyrin derivatives were synthesized and studied to see if similar compounds manifested identical behavior. One derivative, meso-tetra(p-N-trimethylanilinium) porphine did not exhibit intercalation behavior but did show avid binding and novel circular dichroic features when bound to B-form DNA. At an ionic strength of 1.02, the binding constant was found to be on the order of 10⁴ and higher at lower ionic strength. The large binding constants and induced optical activity suggest that at large porphyrin/DNA ratios the final porphyrin · DNA complex may take the form of a suprahelical helix.     

DNA-binding activity of tightly-bound nonhistone chromosomal proteins in chicken liver chromatin.

We have isolated a nonhistone chromosomal protein fraction from chicken liver chromatin which possesses high affinity and preferential sequence DNA binding. Residually DNA-bound nonhistone chromosomal proteins after 2.0 M NaCl extraction of bulk chromatin are isolated. Bound proteins are released by dissociation of the complexes in 5.0 M urea/3.0 M NaCl. We have investigated the in vitro DNA-binding properties of this class. In contrast to other DNA-binding NHCP whose activities have been studied, direct DNA-binding activity is observed which is not abolished under conditions of high ionic strength (to 3.0 M NaCl). Strong preference in binding fractionated homologous DNA is observed, while binding of heterologous (E. Coli) DNA is negligible. The fractionation of homologous DNA permits the isolation of DNA for which this protein class displays strong binding preference, presumably through a concentration of binding sites. The composite data suggest sequence-specific interaction between this protein class and DNA, which is not abolished by high ionic strength.     

The interaction of multiply charged intercalating heterocycles with DNA

The interaction with DNA of two aromatic nitrogen heterocycles, 1 and 2 , which at pH 6 have two positive charges on their ring systems and two cationic side chains, have been determined. A third similar compound, 3 , with a single side chain and reduced ring charge, was analyzed as a control. Viscometric titrations with sonicated DNA indicated that all three compounds bind to DNA by intercalation. Spectrophotometric binding studies as a function of ionic strength indicated that both 1 and 2 bind to DNA as tetracations at pH 6. These are the first examples of intercalators with two charges directly on the intercalating ring system. Dissociation kinetics experiments as a function of ionic strength confirmed that 1 and 2 bind to DNA as tetracations. Compound 1 has a G · C base-pair binding preference, 2 seems to prefer binding to alternating pyrimidine–purine sequences regardless of the composition, and 3 has no significant binding specificity.     

Binding of cationic porphyrin to isolated DNA and nucleoprotein complex: quantitative analysis of binding forms under various experimental conditions

We studied the complex formation of tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with double stranded DNAs and T7 phage nucleoprotein complex. We analyzed the effect of base pair composition of DNA, the presence of capsid protein, and the composition of the microenvironment on the distribution of TMPyP between binding forms as determined by the decomposition of porphyrin absorption spectra. No difference was found in the amount of bound TMPyP between DNAs of various base compositions; however, the ratio of TMPyP binding forms depends on the AT/GC ratio. The presence of protein capsid opposes the binding of TMPyP to DNA. This behavior offers a possibility to investigate the protein capsid integrity due to the analysis of porphyrin binding. Increasing ionic strength of monovalent ions decreases the amount of bound porphyrin through the inhibition of intercalation, but does not influence the quantity of groove-binding forms when TMPyP interacts with isolated DNA. In the case of the nucleoprotein complex the groove-binding is also inhibited already at 140 mM ionic strength. The presence of 1 mM divalent cations (Mg(2+), Ca(2+), Cu(2+) and Ni(2+)) in a buffer solution of 70 mM ionic strength does not influence significantly the free to bound ration of TMPyP when it interacts with isolated DNA. The contribution of binding forms is remarkably different in Mg(2+)/Ca(2+) and Cu(2+)/Ni(2+) containing solutions. Transition metals significantly decrease the binding sites for intercalation in both DNA and nucleoprotein complex, but facilitate the groove-binding of TMPyP to isolated DNA.     

The comparative analysis of interaction between acridine orange and DNA-containing systems]

The interaction between acridine orange (AO) and diluted and concentrated solutions of DNA, DNP systems and chromatin suspension at the physiologic ionic strength was investigated. The effect of AO on DNP systems was also investigated. It was shown that highest possible number of AO molecules bound to DNA made up 70% of the total number of nucleotides. The model of AO binding to DNA is proposed and used for calculation of constants of stronger and weaker AO-binding capacities equal to 6-10(6) M-1 and 1,7-10(5) M-1, respectively. The AO-DNA binding constants in DNP-complex are five as low. The primary number of binding sites in chromatin suspension made up 10% of the corresponding sites in DNA and increased as AO was adsorbed. AO induced the supercontraction of oriented DNP systems at the physiologic ionic strength and the appearance of the low-temperature melting hump.     

The dependence of DNA repair induced by genetically hazardous exposures on the ionic strength of the medium containing the cells]

The dependence of UV-induced unscheduled DNA synthesis (UDS) in non-stimulated lymphocytes of human peripheral blood on the ionic strength (mu) of the culture medium has been shown. With the level of mu lower or higher than physiological (mu ph) the UDS significantly decreases. The effect of modification of mu due to changes in ionic strength is absent in the lymphocytes of patients with the classic form of xeroderma pigmentosum. This phenomenon may become useful for development of a new test revealing cells with genetically or physiologically changed system of UV-induced DNA repair. Mechanisms of investigated phenomenon, particularly their dependence on the chromatin structure, as well as the influence of ionic strength on binding the repair enzymes with DNA are discussed.     

Porphyrin intercalation and non-specific 'edge on' outside binding to natural DNA

A theoretical two-mode binding model for porphyrin binding to natural DNA is presented. One of the binding modes is assumed to be base sequence specific with binding sites n base-pairs long. The other binding mode has binding sites which consist of only one base-pair and can involve cooperativity. The model fits satisfactorily to data for H2TMPyP-4, Cu(II)TMPyP-3 and Cu(II)TMPyP-4 binding to calf thymus DNA in both a high (mu congruent to 1.0 M) and a low (mu congruent to 0.2 M) ionic strength buffer. The results show that the fraction of porphyrin bound in the non-specific mode reaches a maximum at certain input DNA to porphyrin concentrations ratios. The value of this maximum decreased, and its position shifted to higher DNA to porphyrin concentration ratios for binding in the high ionic strength buffer. The value of the cooperativity parameter obtained through the fitting process suggests that the non-specific binding is positively cooperative. The results are compared with the data analysed using other techniques.