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.     

Elasticity of native and cross-linked polyelectrolyte multilayer films

Mechanical properties of polyelectrolyte multilayer films were studied by nanoindentation using the atomic force microscope (AFM). Force-distance measurements using colloidal probe tips were systematically obtained for supported films of poly(L-lysine) and hyaluronan that are suited to bio-application. Both native and covalently cross-linked films were studied as a function of increasing layer number, which increases film thickness. The effective Young's modulus perpendicular to the film, Eperpendicular, was determined to be a function of film thickness, cross-linking, and sample age. Thick PEM films exhibited a lower Eperpendicular than thinner PEM, whereas the Young's modulus of cross-linked films was more than 10-fold larger than native films. Moduli range from approximately 20 kPa for native films up to approximately 800 kPa for cross-linked ones. Young's moduli increased slightly with sample age, plateauing after approximately 4 weeks. Spreading of smooth muscle cells on these substrates with pre-attached collagen proved to be highly dependent on film rigidity with stiffer films giving greater cell spreading.     

Volume changes accompanying the thermal denaturation of deoxyribonucleic acid. I. Denaturation at neutral pH

Dilatometric measurements were made to determine the change in apparent specific volume φ of DNA resulting from thermal denaturation in neutral solution, φ increased continuously with temperature in the range 10–85°C. No deviations from a monotonically rising curve were observed in the φ versus temperature profile in the region of the melting temperature. The results are interpreted in terms of a partial loss of the preferentially bound DNA hydration shell. The nature of the well known buoyant density difference between native and denatured DNA was investigated by evaluating the densities in a series of cesium salt gradients at constant temperature. Extrapolation of the results to zero water activity indicates that the partial specific volumes of anhydrous native and denatured DNA are equal. The density difference at nonzero water activities is attributed to decreased hydration in the denatured state. The absence of a related change in φ accompanying the denaturation in the dilatometric experiments suggests that the probable volume change associated with loss of bound water during denaturation is accompanied by other compensatory volume effects. The possible nature of these volume effects is discussed.     

Thermostability of DNA and its connection with the glassing process

Using differential scanning calorimetry, the thermal denaturation of calf thymus DNA with different content of water (from 12 to 92%) was investigated. Dependences of melting temperature and enthalpy on the biopolymer hydration degree were established. Within the range of water concentrations from 92 to 50% the values of thermodynamic parameters of denaturation were obtained being in good agreement with the published data. Besides, a calorimetric manifestation of renaturation process at different cooling conditions after denaturation was studied. Special attention was paid to thermal properties of denatured and native DNA in the samples containing only the bound water. The temperature dependence of heat capacity in the denatured samples, which have completely lost their renaturation ability due to the proper thermal treatment, demonstrated a characteristic jump of thermal capacity. The value of this jump has been determined to be equal to 1.0 cal/g. degree C, related to dry weight, and almost not dependent on humidity. Temperature position of the jump (Tg) depends on the content of water which serves as a plasticizer. It is shown that the observed anomaly demonstrates all the properties characteristic of vitrification process in synthetic polymers and proteins. General similarity of thermal properties of the samples of native DNA, containing only the bound water, with those of denatured DNA also indicates a transition from the glassy into the rabber-like state. A possibility of existence of both native and denatured DNA in the glassy state at room temperature for the samples with low humidity (about 25%) has been demonstrated experimentally. It can be suggested that the formation of glassy state at dehydration of native DNA ensures its thermostability and the ability of restoration of its functional properties at a subsequent dehydration.     

SULFHYDRYL GROUPS IN FILMS OF EGG ALBUMIN

1. The same number of SH groups reduces ferricyanide in surface films of egg albumin as in albumin denatured by urea, guanidine hydrochloride, Duponol, or heat, provided the ferricyanide reacts with films while they still are at the surface and with the denatured proteins while the denaturing agent (urea, heat, etc.) is present. 2. The SH groups of a suspension of egg albumin made by clumping together many surface films react with ferricyanide in the same sluggish and incomplete manner as do the groups in egg albumin denatured by concentrated urea when the urea is diluted or in albumin denatured by heat when the solution is allowed to cool off. 3. The known change in configuration of the egg albumin molecule when it forms part of a surface film explains why SH groups in the film react with ferricyanide whereas those in native egg albumin do not. In the native egg albumin molecule groups in the interior are inaccessible to certain reagents. A film is so thin that there are no inaccessible groups. 4. Because of the marked resemblance in the properties of egg albumin in surface films and of egg albumin after denaturation by the recognized denaturing agents, it may be supposed that the same fundamental change takes place in denaturation as in film formation—indeed, that film formation is one of the numerous examples of denaturation. This would mean that in general the SH groups of denatured egg albumin reduce ferricyanide and react with certain other reagents because they are no longer inaccessible to these reagents.     

Low-angle light-scattering studies on alkali- and heat-denatured DNA

The molecular weight of native DNA is shown to decrease by at least a factor of two on denaturation by heat or alkali. This result is obtained only if low-angle (<30°) light-scattering measurements are used. High-angle measurements (>30°) do not reveal a decrease in molecular weight on denaturation due to the incorrect value for native DNA. The dn/dc value for both native and denatured DNA is 0.166 ml/g ± 0.003 ml/g. Methods are described for the clarification of native and denatured DNA solutions for light scattering by the use of membrane filters.     

Endonucleases for UV-irradiated and depurinated DNA in barley chloroplasts.

Lysates of barley chloroplasts release more radioactivity into acid soluble form from UV-irradiated and alkylated-depurinated E. coli [3H] DNA than from intact DNA. By means of affinity chromatography on depurinated DNA-cellulose and/or UV irradiated DNA-cellulose and by electrophoresis in polyacrylamide gels, four activities on depurinated DNA were separated. One of these contained activity against heavily UV-irradiated /270 J.m-2/ native DNA. In addition, two other nucleases specific towards UV-DNA were separated. One of them was active on native and heat denatured DNA irradiated with 10 J . m-2 UV, whereas the other was predominantly active on native UV-irradiated DNA.     

Thermal transition of DNA measured by NMR spin-echo technique

The thermal helix–coil transition of DNA can be studied by means of the spin-echo technique. The longitudinal spin–lattice relaxation time T1 and the transvense spin–spin relaxation time T2 of the DNA sample show a similar phase transition as observed spec-trophotometrically with increasing and decreasing temperatures. Four slopes on the T1 and T2 temperature relationship curves were found and interpreted as functions of nonrelational hydration of the DNA molecule. The T1 and T2 values differ depending on the native or denatured state of the DNA molecule. The importance of the dynamic equilibrium between water molecules in the hydration lattice and steps in the denaturation of the DNA molecule are discussed. This phenomenon may be directly related to the nonrotational hydration.     

Electron microscopic identification of supercoiled regions in complex DNA structures

When intracellular lambda replicative intermediates (theta structures) are intercalated with psoralen and then irradiated with long wavelength ultraviolet light (u.v.), interstrand crosslinks are produced. After purification and denaturation of these theta structures, a global difference in denaturation can be observed by electron microscopy; parental sections are essentially native whereas daughter segments are highly denatured. This difference can be explained if parental sections are covalently continuous (and therefore able to supercoil) and daughter segments are not. Due to the higher thermal stability of supercoiled DNA, parental DNA will remain native while daughter sections will denature. Because these structures are crosslinked, the thermal treatment does not lead to dissociation of the highly denatured daughter strands. Experiments with simple negatively supercoiled plasmid circles support the above conclusions. When circles are crosslinked with psoralen-u.v. and then denatured, they remain native because of the higher thermal stability of covalently closed structures. If the circles are linearized before heating but after the psoralen-u.v. treatment, the thermal stability effect is eliminated and the molecules become highly denatured. In this case, however, the crosslinking density is found to be higher than in samples linearized before psoralen-u.v. treatment. This, therefore, shows that crosslinking density also reflects the superhelical state of the molecule at the time of psoralen-u.v. treatment. Two different properties can be used to discriminate between supercoiled and covalently discontinuous domains in complex DNA structures. First, supercoiled regions remain native while covalently discontinuous segments denature following a thermal treatment. This effect requires that covalent continuity exists up to and during the heating treatment. Second, because negative superhelicity enhances psoralen intercalation, crosslinking density is higher in these regions. Even if supercoiled domains are destroyed after the psoralen-u.v. treatment, the imprint of superhelicity is retained and can be recognized as a higher than normal crosslinking density.     

Polarographic reducibility of denatured DNA

The de polarographic behavior of native and denatured DNA at pH 7.0 was studied. Whereas native DNA was polarographically inactive under the given conditions, denatured DNA yielded a reduction polarographic step at the potential of about ?1.4 V. Native DNA produced a single desorption wave on ac polarograms, while denatured DNA yielded, in addition to this wave, another more negative wave approximately corresponding, as to its potential, to the dc polarographic step of denatured DNA. The behavior of apurinic acid was similar to that of denatured DNA. The course of DNA denaturation at elevated temperature was studied by means of the two above techniques and changes at temperatures below the melting temperature observed. This finding is in agreement with earlier results obtained by oscillopolarographic and the pulse-polarographic method.     

Effect of magnesium ions on heat denaturation of DNA

The dependence of animal DNA denaturation on magnesium ion concentration has been studied in the range (10(-6)--10(-1) M with sodium ion content of 10(-3) and 10(-2) M. Special attention has been given to the effect of multivalent metallic impurities bound to DNA. An increase of DNA thermal stability has been shown to occur in the magnesium concentration rage of 10(-6)--10(-4) M. At concentrations exceeding 10(-3) M the T M begins to decrease. The dependence of the DNA melting range on magnesium ion concentration has a maximum at approximately 10(-5) M Mg2+. At low magnesium and sodium ion concentrations a strong asymmetry of the melting curves has been observed. This effect can be described in terms of the melting theory for DNA complexed with small molecules and is explained by magnesium ion redistribution from the denatured portions of DNA to native ones. The method for calculation of melting curves in the DNA-ligand system has been proposed. Studies of thermal denaturation parameters have been shown to be an effective method for the estimation of binding constants of ligands to native and denatured DNA.     

Hydration-dehydration of adsorbed protein films studied by AFM and QCM-D

The hydration-dehydration process of an adsorbed human serum albumin film has been studied using atomic force microscopy (AFM) and a quartz crystal microbalance (QCM). All measurements were performed with identically prepared protein films deposited on highly hydrophilic substrates. Both techniques are shown to be suitable for following in situ the kinetics of protein hydration, and for providing quantitative values of the adsorbed adlayer mass. The results obtained by the two methods have been compared and combined to study changes of physical properties of the films in terms of viscosity, shear, Young's modulus, density and film thickness. These properties were found to be reversible during hydration-dehydration cycles.     

Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins

Calorimetric measurements of absolute heat capacity have been performed for hydrated (11)S-globulin (0 < C(H(2)O) < 25%) and for lysozyme in a concentrated solution, both in the native and denatured states. The denaturation process is observed in hydrated and completely anhydrous proteins; it is accompanied by the appearance of heat capacity increment (Delta(N)(D)C(p)), as is the case for protein solutions. It has been shown that, depending on the temperature and water content, the hydrated denatured proteins can be in a highly elastic or glassy states. Glass transition is also observed in hydrated native proteins. It is found that the denaturation increment Delta(N)(D)C(p) in native protein, like the increment DeltaC(p) in denatured protein in glass transition at low water contents, is due to additional degrees of freedom of thermal motion in the protein globule. In contrast to the conventional notion, comparison of absolute C(p) values for hydrated denatured proteins with the C(p) values for denatured proteins in solution has indicated a dominant contribution of the globule thermal motion to the denaturation increment of protein heat capacity in solutions. The concentration dependence of denaturing heat absorption (temperature at its maximum, T(D), and thermal effect, DeltaQ(D)) and that of glass transition temperature, T(g), for (11)S-globulin have been studied in a wide range of water contents. General polymeric and specific protein features of these dependencies are discussed.     

Complexing and denaturation of DNA by methylmercuric hydroxide. II. Ultracentrifugation studies

When increasing concentrations of methylmercuric hydroxide are added to a Cs₂SO₄ solution of native DNA, the buoyant density of DNA is unaltered until a critical concentration is reached above which there is a cooperative transition to denatured DNA which now binds so much CH₃HgOH that it becomes very dense and nonbuoyant. As increasing concentrations of methylmercuric hydroxide are added to a Cs₂So₄ solution of denatured DNA, the buoyant density gradually increases, indicating a gradual increase in the amount of methylmercury cation bound. The denatured DNA methylmercury complex becomes nonbuoyant at the same concentration of methylmercuric hydroxide as does the native DNA. These results support our previous interpretation that CH₃HgOH reacts with the imino NH bonds of thymine and guanine in nucleic acids. The reaction occurs more or less independently at the different binding sites for denatured DNA, but it occurs cooperatively with simultaneous denaturation for native DNA. The nature of the transition of denatured DNA to the nonbuoyant state is not known, but it is probably due to an abrupt decrease in the degree of hydration of the DNA when its density and hydrophobic character are sufficiently increased by the binding of the methylmercury cation. Direct measurements of the amount of methylmercury bound by DNA, as observed by preparative ultracentrifugation, confirm approximately the buoyant density results as to the amount of methylmercury bound. The possibility of using methylmercuric hydroxide as a reagent for the separation of complementary strands, depending on then thymine of their thymine plus guanine content, is discussed.     

Study of novel rosin-based biomaterials for pharmaceutical coating

The film forming and coating properties of Glycerol ester of maleic rosin (GMR) and Pentaerythritol ester of maleic rosin (PMR) were investigated. The 2 rosin-based biomaterials were initially characterized in terms of their physicochemical properties, molecular weight (Mw), and glass transition temperature (Tg). Films were produced by solvent evaporation technique on a mercury substrate. Dibutyl sebacate plasticized and nonplasticized films were characterized by mechanical (tensile zzzz strength, percentage elongation, and Young's modulus), water vapor transmission (WVT), and moisture absorption parameters. Plasticization was found to increase film elongation and decrease the Young's modulus, making the films more flexible and thereby reducing the brittleness. Poor rates of WVT and percentage moisture absorption were demonstrated by various film formulations. Diclofenac sodium-layered pellets coated with GMR and PMR film formulations showed sustained drug release for up to 10 hours. The release rate was influenced by the extent of plasticization and coating level. The results obtained in the study demonstrate the utility of novel rosin-based biomaterials for pharmaceutical coating and sustained-release drug delivery systems.     

Zinc ion-DNA polymer interactions

The adjacent GN7-M-GN7 cross-linking and adjacent G-M-G sandwich-complex models for DNA metal ion binding were evaluated both with native DNAs differing in GC content as well as with the synthetic polymers poly [(dGdC)]2, poly[(dAdT)]2, and poly[(dAdC)(dGdT)]. The effect of Zn2+ was studied in depth, and limited studies were also performed with Co2+ and Mg2+. The results were compared to the extensive information available on Cu2+ binding to native DNAs and poly[(dAdT)]2. At high ratios of metal/base (R), Zn2+ caused all native DNAs to denature with the same melting temperature Tm, approximately 61 degrees C. A similar pattern was reported previously for Cu2+, but the typical Tm was approximately 35 degrees C. The extent of renaturation on cooling DNAs denatured in the presence of Zn2+ increased with GC content, as reported previously for Cu2+. These results, together with previously reported similarities, strongly indicate that the DNA binding characteristics of the two cations are similar. By comparison of the Tm values and hyperchromicity changes monitored at 260 and 282 nm, it is clear that, during thermal denaturation in the presence of Zn2+, both AT and GC regions were denatured, even at high R. The Tm vs R profile for the native DNAs was typical. The rise at low R and subsequent decrease at high R were inversely and directly related, respectively, to GC content. Except for poly[(dAdT)]2, where Tm increased with R, the other synthetic polymers exhibited the increase/decrease pattern. Poly[(dAdC)(dGdT)] gave a Tm value at high R of 54 degrees C. In the absence of Zn2+, this polymer exhibited little hypochromicity on cooling of denatured polymer. However, in the presence of Zn2+, nearly complete hypochromicity was observed, although the midpoint of the cooling curve was lower than the Tm value by approximately 15 degrees C at R = 10. These characteristics were similar to those with native DNAs, although viscosity and CD studies suggested that the "renatured" polymer was not identical to the unheated polymer. Furthermore, addition of Zn2+ after denaturation nearly completely reversed the absorption increase. This finding contrasts with those for native DNAs, where the Zn2+ must be present during denaturation in order to reverse the absorption increase nearly completely on cooling. With some caveats, poly[(dAdC)(dGdT)] appears to be a good model for native DNAs since its properties, including CD and uv changes on addition of Zn2+ to premelted and melted polymer, parallel those of the native polymers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Southern transfer of native DNA using nylon membranes

Transfer of native or denatured DNA from gels or filter manifolds was compared using nylon or nitrocellulose membranes. The results were comparable when denatured DNA was used, but only nylon membranes were able to retain native DNA. Although retention of the native DNA was less efficient the bound DNA could be rapidly denatured in situ, avoiding the need to soak gels in alkaline denaturation solution and neutralizing buffer.     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)