Pressure effects on thermal preference behaviour in gammarid amphipods from 600–1000m in Lake Baikal

Temperature preference behaviour of gammarid crustaceans from depths between 600–2000 m in Lake Baikal was studied in a system which provided a stable temperature gradient at pressures ranging from 50–150 atm. At the pressure of their habitat, these animals show well-defined modal distributions of sojourn temperatures around mean values from 3.0–5.5°C, av. 3.9 ± 0.3°C; mean modal T_p is estimated at 3.5°C. Year-round habitat temperatures are 3.0–3.6°C. The effect of changing pressure upon sojourn temperatures was explored over the range 50–150 atm. The slope of the mean sojourn temperature/pressure curves was 2.1°C/100 atm, significantly greater than 0. Mean nodal temperature estimates indicate that the corresponding slope in the range of 50–100 atm is 3°C/100 atm, and in the range of 100–150 atm, is likely to exceed 5°C/100 atm.     

Effects of gaseous anaesthetics and inert gases on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine was studied under high pressures of helium (340 atm), nitrogen (340 atm), nitrous oxide (43 atm), cyclopropane (4.4 atm) and $\text{n-}\text{propane}$ (8.2 atm), using a turbidimetric technique. Helium and nitrogen increased the transition temperature by 0.021 and 0.006°C/atm, respectively, compared with 0.024°C/atm for hydrostatic pressure. Nitrous oxide reduced the transition by 0.58°C/atm. The hydrocarbon gases spread the transition width and lowered the transition temperature with increasing effect at higher doses. Comparisons with other membrane probes are made and the concentration of gases in the bilayer which lower the transition temperature by 1°C are estimated, in mol%: He, 10.2; N₂, 13.2; N₂O, 9.04; $\text{n-}\text{C}{}_3\text{H}{}_8$, 6.3 and cyclopropane, 12.8.     

The effects of aqueous neutral-salt solutions on the melting temperatures of deoxyribonucleic acids

The helical stability of a variety of DNA samples, ranging in base composition from 0 to 72 mole-% GC, has been studied by heat denaturation at neutral pH in increasing concentrations of LiCl, NaCl, KCl, CsCl, Li₂SO₄, and K₂SO₄. The variation of melting temperature with average base composition, dT_m/dX_GC, was found to decrease drastically in the concentrated salt media, e.g., from 41°C in 0.006M LiCl to 29°C in 3.2M LiCl, and from 39°C in 0.003M Li₂SO₄ to 18°C in 1.6M Li₂SO₄. At the same time, the thermal transition is much more cooperative in the concentrated salt solutions than at low ionic strength. Indeed, at limiting salt concentrations, the transition breadth seems to reach a minimum value irrespective of the compositional heterogeneity of the DNA samples. Attempts to correlate the observed decrease of dT_m/dX_GC with predicted changes in the enthalpy of melting, deduced from a simple theoretical treatment, experimental data on the binding of counterions and water to DNA, and experimental data on thermal denaturation, were unsuccessful. However, the strongly reduced composition dependence of the melting temperature can be understood in terms of a destabilizing effect of the concentrated salt media on GC-base pairs. It is suggested, though not proven, that the destabilization involves the displacement of water molecules from the DNA helix.     

Helix-coil dynamics of a Z-helix hairpin

The helix–coil transition of a Z-helix hairpin formed from d(C-G)₅T₄(C-G)₅ has been characterized by equilibrium melting and temperature jump experiments in 5M NaClO₄ and 10 mM Na₂HPO₄, pH 7.0. The melting curve can be represented by a simple all-or-none transition with a midpoint at 81.6 ± 0.4°C and an enthalpy change of 287 ± 15 kJ/mole. The temperature jump relaxation can be described by single exponentials at a reasonable accuracy. Amplitudes measured as a function of temperature provide equilibrium parameters consistent with those derived from equilibrium melting curves. The rate constants of Z-helix formation are found in the range from 1800 s^?1 at 70°C to 800 s^?1 at 90°C and are associated with an activation enthalpy of ?(50 ± 10) kJ/mole, whereas the rate constants of helix dissociation are found in the range from 200 s^?1 at 70°C to 4500 s^?1 at 90°C with an activation enthalpy +235 kJ/mole. These parameters are consistent with a requirement of 3–4 base pairs for helix nucleation. Apparently nucleation occurs in the Z-helix conformation, because a separate slow step corresponding to a B to Z transition has not been observed. In summary, the dynamics of the Z-helix–coil transition is very similar to that of previously investigated right-handed double helices.     

A dilatometric investigation of the effects of general anaesthetics,alcohols and hydrostatic pressure on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The effect of general anaesthetics, alcohols and hydrostatic pressure on the thermal transition in dipalmitoyl phosphatidylcholine multilayer liposomes has been measured using dilatometry. The volume increasse at the transition (ΔV_t) is 0.0350 ± 0.0003 ml/g. the transition temperature (T_t) 41.84 ± 0.09°C and the width of the transition 1.025 ± 0.18°C. ΔH calculated by the Clapeyron-Clausius equation is 8.4 kcal/mol. The n-alcohols C₃C₅ reduced the transition temperature without affecting the transition width which was however, increased by n-hexanol. Trichloroethylene, the fluorescent probe N-phenyl-1-naphthyl-amine, and methoxyflurane all increased the transition width (reduced the cooperativity of the transition) with a simultaneous depression of T_t. Methoxyflurane caused a two-stage transition expansion. Diethyl ether's effect has similarities with both the C₃ and C₆ alcohols. Generally ΔV_t was unaffected by the agents.Pressure increased T_t by 0.0238°C/atm linearly over the range 1–300 atm in both treated and untreated liposomes, and therefore cannot be said to antagonize anaesthetics. In both treated and untreated liposomes ΔV_t and the width of the transition were unaffected by pressure. Pressure thus reverses the effects of anaesthetics on T_t but not their spread of the transition width.     

Puccinellia festucaeformis (Host) Parl.: Germinazione e crescita iniziale in funzione della salinità del substrato

Abstract

Puccinellia festucaeformis (Host) Parl.: germination and early growth on different salt substrates. Germination behaviour of Puccinellia festucaeformis seeds and early growth of seedlings at different experimental conditions was analysed. The following growth substrates were utilized: NaCl, KCl, KNO₃, MgCl₂, MgSO₄, Na₂SO₄, NaNO₃, CaCl₂ at the decreasing concentrations of 0.50, 0.25, 0.12, 0.06M. Caryopses were allowed to imbibe and grow at alternating temperatures (10°-20°C or 20°-30°C) in the dark for 3 days. Seedling were grown for 15 days, at controlled light and temperature conditions, in the same nutrient substrates as those used for the germination experiments.

The germination experiments showed a high tolerance to salts up to 0.25M solution and for the whole range of MgSO₄ concentrations. High growth temperatures increased the depressive effects of salt concentrations. Seedling growth was highly reduced when salt concentration was higher than 0.12M. High salt tolerance - maximum shoot and root growth - was showed by seedling allowed to grow on 0.50M MgSO₄.

Germination and growth condition of Puccinellia festucaeformis is discussed in relation to the ecological features of this species and to its possible importance as bioindicator of MgSO₄ rich natural substrates.     

Pressure-induced elevation of phase transition temperature in dipalmitoylphosphatidylcholine bilayers: An electron spin resonance measurement of the enthalpy of phase transition

The application of 136 atm of helium pressure to an aqueous dispersion of dipalmitoylphosphatidylcholine increased the temperature of the primary phase transition at 40.4 ± 0.2 °C by 3.0 °C. The lower temperature pretransition at 30.5 ± 0.5 °C, thought to be due to phosphate headgroup reorganization, was increased by 1.7 °C. Addition of 4% dipalmitoylphosphatidic acid to the dipalmitoylphosphatidylcholine affected the phase transition in the head group region more than in the hydrocarbon chain region. The pressure and temperature data obtained, taken together with the literature value for the bilayer volume expansion during solid-fluid phase transition, and inserted into the Clausius-Clapeyron equation yield a ΔH value of 8.8 kcal/mole for this phase transition. This value is within experimental error of the ΔH value obtained from differential scanning calorimetry and serves to support the validity of the data and the experimental technique. Phase transition was observed by electron spin resonance measurement of the exclusion of the small spin label Tempo (2,2,6,6-tetramethylpiperidine-N-oxyl) from the solid domains of the bilayer. This result offers a possible explanation for the direct antagonism by high pressure of the effects of the inhalation anesthetics.     

Helix-coil transitions in nucleoprotein: Computer-generated curves for independent melting of double-stranded regions with fixed ends

Helix-coil transitions in nucleoprotein at low salt concentrations are known to be characterized by two phases of the process: independent melting of uncomplexed “naked” regions without rearrangement of proteins, followed, at higher temperatures, by melting of complexed DNA. Blocking at the ends of these regions increases their thermal stability and three is a shift of 10–20°C in t_m of the melting profiles. In this study the basic assumption is that the loop entropy effect is mainly responsible for such stabilization. Calculations are made using conventional h-c transition theory for a system of independently melted segments with fixed ends. Segments are either homosize or have randomly distributed lengths. Calculated melting curves are used to obtain t_m, and transition width-dependence on segment length (or average length when randomly distributed) and on the nucleation parameter σ. Base-pair heterogeneity is taken into account by averaging over different base-pair distributions in the individual segments, using Gaussian distribution around the overall (G+C)-content. It is shown that this causes only an additional widening of the transition but no additional t_m shift. Comparison is made with similar systems in the literature. The main conclusion drawn is that the treatment proposed may be useful for analysis of the lower temperature melting phase in nucleoprotein at low counterion concentrations. It may be used as an independent method to reveal the features of nucleoprotein structure.     

Halophilic Properties of Metal Binding Protein Characterized by High Histidine Content from Chromohalobacter salexigens DSM3043

Periplasmic metal binding protein characterized by high histidine content was cloned from moderate halophile, Chromohalobacter salexigens. The protein, termed histidine-rich metal binding protein (HP), was expressed in and purified from E. coli as a native form. HP bound to Ni- and Cu-loaded chelate columns with high affinity, and Co- and Zn-columns with moderate affinity. Although the secondary structure was not grossly altered by the addition of 0.2–2.0 M NaCl, the thermal transition pattern was considerably shifted to higher temperature with increasing salt concentration: melting temperature was raised by ~20 °C at 2.0 M NaCl over the melting temperature at 0.2 M NaCl. HP showed reversible refolding from thermal melting in 0.2–1.15 M NaCl, while it formed irreversible aggregates upon thermal melting at 2 M NaCl. Addition of 0.01–0.1 mM NiSO₄ stabilized HP against thermal melting with high reversibility, while addition above 0.5 mM resulted in irreversible melting due to aggregation.     

NMR study on molecular mobility of DNA molecules in solution

The molecular mobility of calf thymus DNA molecules in solution has been discussed in terms of correlation time τ calculated from measurements of longitudinal T₁ and transverse T₂ magnetic relaxation times. The influence of DNA concentration and ionic strength of the solution upon freedom of movement of DNA molecules was studied for native and denatured DNA and also during thermal helix-coil transition. The dependence of τ values on temperature was carried out by comparing the values of correlation times τ_tat given temperature with the correlation time τ₂₀ at 20°C. The molecular rotation of DNA at 20°C and at higher ionic strength at 0.15 and 1.0.M NaCl is described by τ values of the order of 1.0–1.2 × 10^?8 and was reduced slightly with increase of temperature below the helix-coil transition. The molecular rotation of DNA in 0.02MNaCl was lower at 20°C as compared to DNA in solvents with higher NaCl concentrations and increases rapidly with increase of temperature in the range 20–60°C. The values of correlation time are characterized by fast increase at temperatures above the spectrophotometrically determined beginning of melting curve. The beginning of this increase is observed at about 65, 80, and 85°C for DNA in 0.02, 0.15, and 1.0MNaCl, respectively. Values of correlation time for denatured DNA are in all cases about 1.1–1.4 times that for native DNA. The obtained results are discussed in terms of conformation of DNA molecules in solution as well as in terms of water dipole binding in DNA hydration shells.     

An Alternative Ionic Conductivity Mechanism for Plastic Crystalline Salt–Lithium Salt Electrolyte Mixtures

The phase behavior and ionic conductivity of tetraethylammonium bis(trifluoromethane‐sulfonyl)imide (Et₄NTFSI) salt mixtures with LiTFSI have been examined. In addition, the phase behavior and crystal structure of neat LiTFSI is also reported. Two (1‐x) Et₄NTFSI‐(x) LiTFSI (x = 0.50 and 0.67, where x is the mol fraction) mixed‐salt crystalline phases form. Large variations in ionic conductivity are observed; these are attributed to solid‐solid phase transitions of the neat Et₄NTFSI salt creating disordered plastic crystalline phases and the formation of a low‐melting eutectic composition between the neat Et₄NTFSI salt and the 1/1 Et₄NTFSI/LiTFSI (x = 0.50) phase. Although Et₄NTFSI and LiTFSI melt at 102 and 234 °C, respectively, the two salts form a eutectic system with a melting temperature of 32 °C. Based upon the findings reported, a new conductivity mechanism is proposed for plastic crystalline salt‐lithium salt electrolytes which is not ascribed to solid‐state diffusion/conduction.     

Hemoglobin senses body temperature

When aspirating human red blood cells (RBCs) into 1.3 μm pipettes (ΔP = −2.3 kPa), a transition from blocking the pipette below a critical temperature T _c = 36.3 ± 0.3°C to passing it above the T _c occurred (micropipette passage transition). With a 1.1 μm pipette no passage was seen which enabled RBC volume measurements also above T _c. With increasing temperature RBCs lost volume significantly faster below than above a T _c = 36.4 ± 0.7 (volume transition). Colloid osmotic pressure (COP) measurements of RBCs in autologous plasma (25°C ≤ T ≤ 39.5°C) showed a T _c at 37.1 ± 0.2°C above which the COP rapidly decreased (COP transition). In NMR T₁-relaxation time measurements, the T₁ of RBCs in autologous plasma changed from a linear (r = 0.99) increment below T _c = 37 ± 1°C at a rate of 0.023 s/K into zero slope above T _c (RBC T₁ transition). In conclusion: An amorphous hemoglobin–water gel formed in the spherical trail, the residual partial sphere of the aspirated RBC. At T _c, a sudden fluidization of the gel occurs. All changes mentioned above happen at a distinct T _c close to body temperature. The T _c is moved +0.8°C to higher temperatures when a D₂O buffer is used. We suggest a mechanism similar to a “glass transition” or a “colloidal phase transition”. At T _c, the stabilizing Hb bound water molecules reach a threshold number enabling a partial Hb unfolding. Thus, Hb senses body temperature which must be inscribed in the primary structure of hemoglobin and possibly other proteins. This article is dedicated to Ludwig Artmann who died on July 21, 2001 on a beautiful summer day during which we performed experiments far away. Ludwig Artmann was a man who encouraged us to be strong and to study hard no matter what were the costs.     

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.     

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.     

Effects of elevated pCO2 on the metabolism of a temperate rhodolith Lithothamnion corallioides grown under different temperatures

Coralline algae are considered among the most sensitive species to near future ocean acidification. We tested the effects of elevated pCO₂ on the metabolism of the free‐living coralline alga Lithothamnion corallioides (“maerl”) and the interactions with changes in temperature. Specimens were collected in North Brittany (France) and grown for 3 months at pCO₂ of 380 (ambient pCO₂), 550, 750, and 1000 μatm (elevated pCO₂) and at successive temperatures of 10°C (ambient temperature in winter), 16°C (ambient temperature in summer), and 19°C (ambient temperature in summer +3°C). At each temperature, gross primary production, respiration (oxygen flux), and calcification (alkalinity flux) rates were assessed in the light and dark. Pigments were determined by HPLC. Chl a, carotene, and zeaxanthin were the three major pigments found in L. corallioides thalli. Elevated pCO₂ did not affect pigment content while temperature slightly decreased zeaxanthin and carotene content at 10°C. Gross production was not affected by temperature but was significantly affected by pCO₂ with an increase between 380 and 550 μatm. Light, dark, and diel (24 h) calcification rates strongly decreased with increasing pCO₂ regardless of the temperature. Although elevated pCO₂ only slightly affected gross production in L. corallioides, diel net calcification was reduced by up to 80% under the 1,000 μatm treatment. Our findings suggested that near future levels of CO₂ will have profound consequences for carbon and carbonate budgets in rhodolith beds and for the sustainability of these habitats.     

On the possibility of true phase transition in heterogeneous DNA during thermal denaturation under conditions close to equal stability of A+T and G+C pairs

The DNA helix–coil transition has been studied in the presence of high concentrations of manganese ions (about 10^?3M), which corresponds to the conditions close to equal stability of the A+T and G+C pairs, at the ionic strengths of 10^?1, 10^?2, and 1.6 × 10^?3M Na⁺. With the Mn²⁺ ion effect, the transition range is significantly reduced to not more than 0.2°C at 1.2 × 10^?3M Mn²⁺ and 1.6 × 10^?3M Na⁺. The melting curves display a sharp kink at the end of the helix–coil transition, which is interpreted as an indication of the second-order phase transition. It is shown that the melting curves obtained can be approximated by a simple analytical expression 1 – θ = exp[–a(t_c - t)], where θ is the DNA helix fraction, t_c is the phase transition temperature, and a is an empirical parameter characterizing the breadth of the melting range and responsible for the magnitude of a jump of the helicity derivative with respect to the temperature at the phase transition point.     

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.     

A Raman and calorimetric investigation of 3′, 5′ GpG

The Raman spectra of guanylyl (3′-5′) guanosine (GpG) in solution in H₂O and D₂O at pH 3–7 have been recorded at various temperatures between 0 and 80°C. The results are consistent with the existence in the lower temperature range of stable aggregates formed by the stacking of GpG tetramers. The aggregates melt cooperatively near 60°C, which results in important changes in the spectra. Among these, a large increase in intensity of some of the bands assigned to the guanine residues shows that unstacking of the bases occurs at the melting. Also apparent in the spectra are changes in the intensity and frequency of band attributable to molecular groups involved in intermolecular hydrogen bonding between adjacent molecules in the complex. The melting temperature of GpG decreases by approximately 15°C upon lowering the concentration from 5 × 10^?2 to 5 × 10^?4M, as shown by Raman, calorimetric, CD, and uv measurements. The experimentally determined ΔH and ΔS for the melting transition are 9 Kcal/mol and 28 e.u./mol, respectively. The aggregation of GpG in 1.5 × 10^?3M solutions was found to be very slow. The half-time of the process, which roughly follows first-order kinetics, is approximately 3 min at 10°C and 21 min at 35°C. The negative energy of activation associated with this reaction (?143 Kcal) indicated that the process involves intermediates whose concentrations decrease the temperatures raised, thus slowing down the overall process. The rate of disaggregation of GpG upon dilution to very low concentration is also extremely slow, indicating that the GpG aggregates, once formed, are very stable.     

Mutagen–nucleic acid complexes at the polynucleotide duplex level in solution: Intercalation of proflavine into poly(dA-dT) and the melting transition of the complex

The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.     

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.