Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Abstract

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α₂M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α₂M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α₂M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α₂M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α₂M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α₂M as compared to the native α₂M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α₂M and makes it immunogenic. The presence of neo-antigenic determinants on modified α₂M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease.

Communicated by Ramaswamy H. Sarma     

Preferential recognition of peroxynitrite modified human DNA by circulating autoantibodies in cancer patients

Peroxynitrite (ONOO⁻) has been vastly implicated in mutagenesis and cancer development. Present study probes the antigenicity of peroxynitrite damaged DNA (ONOO⁻-DNA) in cancer patients. Purified human placental DNA was damaged by the synergistic action of sodium nitroprusside (SNP) and Pyrogallol for 3 h at 37 °C. Binding characteristics of cancer autoantibodies as well as experimentally induced anti-peroxynitrite-DNA (anti-ONOO⁻-DNA) antibodies were assessed by ELISA and band shift assay. DNA modifications produced single strand breaks, decreased melting temperature (T_m), hyperchromicity in UV spectrum and decreased fluorescence intensity. The ONOO⁻-DNA induced high titre antibodies in experimental animals. Cancer autoantibodies exhibited enhanced binding with the modified DNA as compared to the native form. Lymphocyte DNA from cancer patients showed appreciable recognition of anti-ONOO⁻-DNA IgG as compared to the DNA from healthy subjects. The peroxynitrite modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients.     

Polyacrylamide gel as a medium for DNA dissociation and reassociation

Several properties of thermal denaturation and renaturation of DNA in polyacrylamide gels were investigated: (1) Following electrophoresis the DNA band was scanned and shown to increase in absorbance with increasing temperature. The increase was proportioned to DNA concentration across the peak. (2) The dependence of theT _m on salt concentration over a hundred fold range was similar to that found for DNA in free solution. (3) Denaturation of several DNA samples ranging in G+C content from 26 to 71% was compared in gels and free solution. The relationship betweenT _m and % G+C was virtually identical for both sets of DNAs. (4) The kinetics of DNA renaturation in the gel was followed. Reassociation of bacteriophageT ₄ DNA was 2nd order and proceeded more rapidly in polyacrylamide gels than in free solution.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

Chitosan-Soyprotein Interaction as Determined by Thermal Unfolding Experiments

Chitosan interaction with soybean β-conglycinin β₃ was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T _m) was 63.4 °C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T _m value was elevated by 7.7 °C, whereas the T _m elevation upon addition of chitosan hexamer (GlcN)₆ was 2.2 °C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T _m elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T _m elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with β-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.     

Sex- and age-related changes in the biophysical properties of cuticular lipids of the housefly,Musca domestica

We examined the biophysical properties of cuticular lipids isolated from the housefly, Musca domestica. Melting temperatures (T_m) of surface lipids isolated from female houseflies decreased from 39.3 °C to 35.3 °C as the females attained sexual maturity and produced sex pheromone, whereas those prepared from males did not change with age. Lipids melted over a 10–25 °C temperature range, and their physical properties were a complex function of the properties of the component lipids. The T_m of total cuticular lipids was slightly below that of cuticular hydrocarbons (HC), the predominant lipid fraction. Hydrocarbons were further fractionated into saturated, unsaturated, and methyl-branched components. The order of decreasing T_m was total alkanes > total HCs > methyl-branched alkanes > alkenes. For 1-day-old flies, measured T_ms of hydrocarbons were 1.3–5.5 °C lower than T_ms calculated from a weighted average of T_ms for saturated and unsaturated components. For 4-day-old flies, calculated T_ms underestimated T_m by 11–14 °C. © 1995 Wiley-Liss, Inc.     

Influence of DPPE surface undulations on melting temperature determination: UV/Vis spectroscopic and MD study

One of the most distinguished quantities that describes lipid main phase transition, i.e. the transition from the gel (L_β(′)) to the fluid (L_α) phase, is its melting temperature (T_m). Because melting is accompanied by a large change in enthalpy the, L_β(′) → L_α transition can be monitored by various calorimetric, structural and spectroscopic techniques and T_m should be the same regardless of the metric monitored or the technique employed. However, in the case of DPPE multilamellar aggregates there is a small but systematic deviation of T_m values determined by DSC and FTIR spectroscopy. The aim of this paper is to explain this discrepancy by combined UV/Vis spectroscopic and MD computational approach. Multivariate analysis performed on temperature-dependent UV/Vis spectra of DPPE suspensions demonstrated that at 55 ± 1 °C certain phenomenon causes a small but detectable change in suspension turbidity, whereas a dominant change in the latter is registered at 63.2 ± 0.4 °C that coincides with T_m value determined from DSC curve. If this effect should be ignored, the overall data give T_m value the same as FTIR spectra data (61.0 ± 0.4 °C). As the classical MD simulations suggest that about 10° below T_m certain undulations appear at the surface of DPPE bilayers, we concluded that certain discontinuities in curvature fluctuations arise at reported temperature which are to some extent coupled with lipid melting. Ultimately, such events and the associated changes in curvature affect T_m value measured by different techniques.     

MOLECULAR EVOLUTIONARY DIVERGENCE AMONG NORTH AMERICAN CAVE CRICKETS. II. DNA-DNA HYBRIDIZATION

Single-copy DNA divergence among 23 populations of cave crickets belonging to two genera (Euhadenoecus and Hadenoecus) has been determined by DNA-DNA hybridization employing the TEACL method. These same populations have been studied for allozyme variation (Caccone and Sbordoni, 1987). In addition, a European relative (Dolichopoda laetitiae) has been included as an outgroup for rooting the phylogeny. One of the most remarkable findings is the large degree of DNA divergence among these species and populations. A ΔT_m of up to 5°C has been found between populations of the same species; even further divergence is indicated by a lowered normalized percentage of reassociation. A phylogeny was constructed and tested for synchrony of rates, i.e., a molecular clock. Statistically, we could not reject the clock hypothesis. Attempts to calibrate the clock led to the conclusion that these insects are among the fastest evolving (with respect to single-copy DNA) groups yet studied—at least as fast as Drosophila and sea urchins—where a ΔT_m of 1°C indicates 0.5 to 1.5 MY since the last common ancestor. In general, the phylogeny derived from the DNA data agrees with that derived from isozymes. Nei's D and ΔT_m are correlated; in this group a D of 0.1 corresponds to a ΔT_m of about 1.5°C. This indicates that, relative to total single-copy DNA, the protein-coding regions of the genome are slowly evolving.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

DNA damage and photosynthesis in Antarctic and Arctic Sanionia uncinata (Hedw.) Loeske under ambient and enhanced levels of UV-B radiation

The response of the bipolar moss Sanionia uncinata (Hedw.) Loeske to ambient and enhanced UV‐B radiation was investigated at an Antarctic (Léonie Island, 67°35′ S, 68°20′ W) and an Arctic (Ny‐Alesund, 78°55′ N, 11°56′ E) site, which differed in ambient UV‐B radiation (UV‐BR: 280–320 nm) levels. The UV‐BR effects on DNA damage and photosynthesis were investigated in two types of outdoor experiments. First of all, sections of turf of S. uncinata were collected in an Arctic and Antarctic field site and exposed outdoors to ambient and enhanced UV‐BR for 2 d using UV‐B Mini‐lamps. During these experiments, chlorophyll a fluorescence, chlorophyll concentration and cyclobutyl pyrimidine dimer (CPD) formation were measured. Secondly, at the Antarctic site, a long‐term filter experiment was conducted to study the effect of ambient UV‐BR on growth and biomass production. Additionally, sections of moss turf collected at both the Antarctic and the Arctic site were exposed to UV‐BR in a growth chamber to study induction and repair of CPDs under controlled conditions. At the Antarctic site, a summer midday maximum of 2·1 W m^?2 of UV‐BR did not significantly affect effective quantum yield (ΔF/F_m′) and the ratio of variable to maximal fluorescence (F_v/F_m). The same was found for samples of S. uncinata exposed at the Arctic site, where summer midday maxima of UV‐BR were about 50% lower than at the Antarctic site. Exposure to natural UV‐BR in summer did not increase CPD values significantly at both sites. Although the photosynthetic activity remained largely unaffected by UV‐B enhancement, DNA damage clearly increased as a result of UV‐B enhancement at both sites. However, DNA damage induced during the day by UV‐B enhancement was repaired overnight at both sites. Results from the long‐term filter experiment at the Antarctic site indicated that branching of S. uncinata was reduced by reduction of ambient summer levels of UV‐BR, whereas biomass production was not affected. Exposure of specimens collected from both sites to UV‐BR in a growth chamber indicated that Antarctic and Arctic S. uncinata did not differ in UV‐BR‐induced DNA damage. It was concluded that S. uncinata from both the Antarctic and the Arctic site is well adapted to ambient levels of UV‐BR.     

Intermediates in the strand-separation transition of T7 DNA.

Sedimentation velocity runs as a function of temperature in the region of the alkaline helix-coil transition have enabled us to demonstrate the existence of stable two-stranded intermediates in the strand-separation process for T7 DNA. The strand-separation transition under these conditions has an intrinsic breadth of ～1°C, and within this temperature range (T_m + 2°C < T < T_m + 3°C) the intermediate forms are progressively converted (as a function of temperature) to single-stranded DNA. Parallel characterizations of the strand-separation transition by viscosity and absorbance–renaturation studies in the alkaline solvent are entirely consistent with the sedimentation experiments. Comparison of the experimental mean sedimentation coefficient of the intermediate forms with theoretical predictions for branched structures suggests that in these molecules the two strands are connected at a single point, not centrally located with respect to the ends of the molecule.     

EVOLUTIONARY IMPLICATIONS OF DNA DIVERGENCE IN THE DROSOPHILA OBSCURA GROUP

Using DNA–DNA hybridization, we have determined the degree of single-copy DNA (scDNA) divergence among eight species of the Drosophila obscura group. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinis subgroup are more closely related to American members of the obscura subgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔT_m of 6.5°C can form hybrids whereas species separated by a ΔT_m of 2.5°C cannot. As with other groups of Drosophila, the obscura group has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscura and the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔT_m of 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscura group may be evolving considerably slower than in the melanogaster subgroup.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

Oligonucleotides containing a peptide internucleotide linkage. A novel class of modified oligonucleotides

Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where X = H, (S)-CH₃, and (R)-CH₃. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared to that of native ones (ΔT _m∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (ΔT _m∼+2°C per modification).     

Liposomes destabilize tRNA during heat stress

Biomembranes play an important role in cellular response to heat stress. In this study, we focus on the interaction between liposomes and tRNA. Upon heat treatment we determined circular dichroism spectra of tRNA in presence of liposomes prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol (Ch). To compare thermal stability, midpoint temperature (T_m) of tRNA was calculated from normalized θ₂₀₈. Addition of POPC/Ch liposomes decreased the T_m value of tRNA from 48°C to 38°C. We conclude that POPC/Ch liposomes interact with tRNA and destabilize its conformation under heat stress.     

Thermal denaturation of Na- and Li-DNA in salt-free solutions

Thermal denaturation of Na- and Li-DNA from chicken erythrocytes was studied by means of scanning microcalorimetry in salt-free solutions at DNA concentrations (C_p) from 4.5 · 10^?2 to 1 · 10^?3 moles of nucleotides/liter (M). Linear dependencies of DNA melting temperature (T_m) vs lgC_p were obtained: ((1)) ((2)) for Na- and Li-DNA, respectively. Microcalorimetry data were compared with the results of spectrophotometric studies at 260 nm of DNA thermal denaturation in Me-DNA + MeCl solutions at C_p ? (6–8) · 10^?5 M and C_s = 0–40 mM (Me is Na or Li, C_s is salt concentration). It was found that Eqs. (1) and (2) are valid in DNA salt-free solutions over the C_p range 6 · 10^?5?4.5 · 10^?2M. Protonation of DNA bases due to the absorption of CO₂ from air in Na-DNA + NaCl solutions affects DNA melting parameters at C_s < 4 mM. Linear dependence of T_m on lga₊ is found in Na-DNA + NaCl at C_s > 0.4 mMin the absence of contact of solutions with CO₂ from air (a₊ is cation activity). A dependence of [dT_m/dlga₊] on Li⁺ activity was observed in Li-DNA + LiCl solutions at C_s < 10 mM: [dT_m/dlga₊] increases from 17°–18° at C_s > 10 mM to 28°–30° at C_s ? 0.2–0.4 mM. Spectrophotometric measurements at 282 nm show that this effect was caused by protonation of bases in fragments of denatured DNA in neutral solutions. The Poisson–Boltzmann (PB) equation was solved for salt-free DNA at the melting point. The linear dependence of T_m vs lgC_p was interpreted in terms of Manning's condensation theory. PB and Manning's theories fit the experimental data if charge density parameter (ξ) of denatured DNA is in the range 1.8–2.1 (assuming for native DNA ξ = 4.2). Specificity of Li ions in interactions with DNA is discussed. © 1994 John Wiley & Sons, Inc.     

Melting point depression of DNA by tetraaklylammonium bromides

Varied concentrations of tetramethyl- (TMAB), tetraethyl- (TEAB), tetrapropyl- (TRAB), tetrabutyl- (TBAB), tetrapentylammonium bromide (TPAB) and ammonium bromide are monitored for effects on DNA sedimentation, viscosity and melting temperature. No changes are observed at 25–30°C for the hydrodynamic properties of DNA when treated with the quaternary ammonium bromide. At higher temperatures, 30–100°C, the tetraalkyl-ammonium bromides reduce the temperature required for conversion of the DNA double-strand helix to the coil (T_m). A form of Debye-Huckel equation ($\text{T}{}_{\mathrm{m}}\text{=a}\text{mu;}\text{2}\text{+ b}$) accurately describes the results. Ionic strength of the quaternary ammonium compounds is represented by μ/2. The potency of tetraalkylammonium bromide in reducing T_m (“a” in the T_m equation) is correlated with the hydrophobicity of the molecule (number of carbon atoms), r = 0.999 and F = 526 (r represents the correlation coefficient and F represents the test value for significance of the equation evaluated by least squares analysis). The T_m of rat liver chromatin is also reduced in a similar fashion by TPAB. The T_m of poly(dG-dC) is not altered by 0.1 M TBAB whereas the T_m of poly(dA-dT) is greatly reduced by the compound.It is thought that the tetraalkylammonium bromide preferentially binds to the coil of DNA during heating and that a preferentially hydrophobic interaction may occur with the adenine-thymine bases.     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.