Conformation and molecular and ionic transformations of polycytidylic acid immobilized in multilayer Langmuir and polyelectrolyte films

Multilayer films of complexes of polycytidylic acid with dioctadecyldimetylammonium were obtained by the Langmuir-Blodgett method (LB films), and complexes of poly(C) with polycations (poly-L-lysisne, polyethyleneimine, polyallylamine) were obtained by the method of alternate adsorption (polyionic assembly) from solutions of oppositely charged polyelectrolytes on the solid carrier (SA films). It was shown that poly(C) exists in SA films in a single-stranded state irrespective of whether in the starting solution it occurred in the single-stranded nonprotonated or double-stranded protonated conformation. Conversely, in the LB film poly(C) preferred to be in a double protonated conformation. UV-spectra of water-insoluble LB and SA films at different pH values of surrounding water medium were investigated. Proton titration curves of poly(C) immobilized in LB films were obtained. The analysis of the shape of titration curves showed that the molecular-ionic transformation of poly(C) in LB films is accompanied by both the conformational transition of the polynucleotide and the molecular rearrangement in the whole film. Poly(C) was found to transform from the double- to single-stranded state and vice versa in the "deprotonation-protonation" cycle of LB film due to cooperative release/binding of hydrogen ions by cytosine bases. In contrast, poly(C) "protonation-deprotonation" in SA films occurred without conformational transitions of the polynucleotide. As opposed to poly(C) in solution a rather big hysteresis of forward and back titration curves was found for both types of multilayer films, indicating molecular rearrangements in films. The reason for the structural transformations of poly(C) upon fabrication of LB or SA films and the mechanism of molecular ionic transformations of poly(C) in films are discussed in terms of a simple model of ion exchange. An assumption about the nature of structural transformations of LB and SA films during their protonation-deprotonation is put forward.     

Ionic strength dependence of the hysteresis in the polyriboadenylate-polyribouridylate system.

The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.     

Swelling of brome mosaic virus as studied by intensity fluctuation spectroscopy.

The swelling of brome mosaic virus induced by pH and temperature has been investigated with intensity fluctuation spectroscopy. A special light-scattering cell was designed which permits titrations within the cell in which Stokes radii, pH and temperature are measured simultaneously. Freshly prepared viruses in the presence of EDTA at 20 °C show a partially irreversible swelling in a first titration cycle (pH 5.8 to 7.2 to 5.8): the viruses do not recontract completely to the original compact form. All further titration cycles lead to a closed hysteresis with respect to the Stokes radii. These are stable in the swelling branch (obtained when adding base) of the titration cycle, but metastable in the contraction branch (i.e. when adding acid). In the latter, the Stokes radii relax to the values observed in the swelling branch within a few hours. If MgCl₂ is present, fresh viruses have slightly bigger radii than with EDTA, and the initial titration cycle is closed. It exhibits a similarly pronounced hysteresis with a metastable contraction branch as in the abscence of divalent cations. However, further titrations do not exhibit significant hysteresis. An irreversible swelling of the viruses can be induced in the absence of Mg²⁺ by raising the temperature from 5 ° to 35 °C at a constant pH value. In the presence of MgCl₂, Stokes radii are not affected by temperature.     

Hysteresis and conformational changes in ribosomal ribonucleic acid

Both rat liver and Escherichia coli rRNA in 0.1m-sodium chloride were titrated with acid or alkali over the range pH3-7 at approx. 0 degrees C. rRNA did not bind acid reversibly and hysteresis was observed, i.e. the plot of acid bound to rRNA against pH had the form of a loop showing that the amount of acid bound at a particular pH depended on the direction of the titration. Although the boundary curves were reproducibly followed on titration from pH7 to 3 and from pH3 to 7, points within the loop were ;scanned', e.g. by titration from pH7 to a point in the range pH3-4 followed by titration with alkali to pH7. It is inferred that the ;lag' in the release of certain bound protons is at least 1 pH unit, that at least about 9-15% of the titratable groups (adenine and cytosine residues) that are involved in this process and that the free energy dissipated in completing a cycle is approx. 4.2kJ/mol (1kcal/mol) of nucleotide involved in hysteresis. The interpretation of the ;scanning' curves was illustrated by means of a cycle of possible changes in the conformation of a hypothetical nucleotide sequence that allows formation of poly(A).poly(AalphaH(+))-like regions in acidic solutions. It is also inferred that the extent of ;hysteresis' might depend on the primary nucleotide sequence of rRNA as well as on secondary structure.     

Protonated polynucleotides structures - 23. The acid-base hysteresis of poly(dG).poly(dC).

The large hysteresis observed during the acid-base titration of poly(dG). poly (dC) was studied by CD and potentiometric scanning curves. Intermediate scanning loops as well as the equilibrium and metastable branches of the hysteresis loop have been determined. The potentiometric titrations showed, however, that the various complexes were not discrete entities, but were linked in "polycomplexes" as had been already suggested. This prevented a thermodynamic study of the system. The acid-base titration was further investigated as a function of ionic strength and temperature. The pK's showed considerably lower ionic strength dependence than observed for polyribonucleotide complexes. The thermal transitions permitted to establish the relative stabilities of the various complexes between pH 2.5 and pH 12.0.     

Temperature dependence of the binding of endotoxins to the polycationic peptides polymyxin B and its nonapeptide

The interaction between endotoxins-free lipid A and various lipopolysaccharide (LPS) chemotypes with different sugar chain lengths-and the polycationic peptides polymyxin B and polymyxin nonapeptide has been investigated by isothermal titration calorimetry between 20 and 50 degrees C. The results show a strong dependence of the titration curves on the phase state of the endotoxins. In the gel phase (<30 degrees C for LPS and <45 degrees C for lipid A), an endothermic reaction is observed, for which the driving force is an entropically driven endotoxin-polymyxin interaction, due to disruption of the ordered water structure and cation assembly in the lipid A backbone and adjacent molecules. In the liquid crystalline phase (>35 degrees C for LPS and >47 degrees C for lipid A) an exothermic reaction takes place, which is mainly due to the strong electrostatic interaction of the polymyxins with the negative charges of the endotoxins, i.e., the entropic change DeltaS is much lower than in the gel phase. For endotoxins with short sugar chains (lipid A, LPS Re, LPS Rc) the stoichiometry of the polymyxin binding corresponds to pure charge neutralization; for the compounds with longer sugar chains (LPS Ra, LPS S-form) this is no longer valid. This can be related to the lower susceptibility of the corresponding bacterial strains to antibiotics.     

Stability of a Lac repressor mediated "looped complex"

The quantitation of the stability of a protein-mediated "looped complex" of the Lac repressor and DNA containing two protein-binding sites whose centers of symmetry are separated by 11 helical turns (114 bp) was accomplished by footprint and gel mobility-shift titration techniques. Lac repressor binding to this DNA was only moderately cooperative; a cooperative free energy of -1.0 kcal/mol was calculated in a model-independent fashion from the individual-site loading energies obtained from the footprint titration studies. In order to partition the cooperative binding energy into components representing the dimer-tetramer association of Lac repressor and the cyclization probability of the intervening DNA, advantage was taken of the presence of experimental measures that were in proportion to the concentration of the looped complex present in solution. One measure was the DNase I hypersensitivity observed in footprint titrations in bands located between the two binding sites. The second measure resulted from the electrophoretic resolution in the gel mobility-shift titrations of the band representing the doubly liganded "tandem complex" from the band representing the singly liganded complexes, including the looped complex. Analysis of the footprint and mobility-shift titration data utilizing this additional information showed that approximately 65% of the molecules present in solution are looped complexes at pH 7.0, 100 mM KCl, and 20 degrees C when the binding sites on the DNA are saturated with protein. Reconciliation of the observed low binding cooperativity and the high proportion of looped complexes could only be obtained when the titration data were analyzed by a model in which Lac repressor tetramers dissociate into dimers in solution. The proportion of looped complexes present in solution is highly dependent on the dimer-tetramer association constant, delta Gtet. This result is consistent with the determination by high-pressure fluorescence techniques that Lac repressor tetramers dissociate with an association free energy comparable to their DNA-binding free energies [Royer, C. A., Chakerian, A. E., & Matthews, K. S. (1990) Biochemistry 29, 4959-4966]. However, when the value of delta Gtet of -10.6 kcal/mol (at 20 degrees C) reported by Royer et al. (1990) is assumed, the titration data demand that tetramers bind DNA with much greater affinity than dimers: a result inconsistent with the destabilization of tetramers by the operator observed in the dimer-tetramer dissociation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids.

The reactions between purified anti-poly A. poly U and-poly I. poly C. antibodies (IgG and IgM), and synthetic and natural polynucleotides were visualized at the molecular level. This was achieved by the use of fine tungsten bidirectional shadowing of molecules adsorbed onto thin carbon films, combined with dark field electron microscopic observation. A progression was observed from monogamous multivalency (binding of a single multifunctional antigen molecule with several combining sites of the same antibody molecule simultaneously) (Crothers and Metzger, 1972, Immunochemistry, 9, 341-357), to aggregation. Different types of figures were observed, among which loops formed by the coiling of the antigen around a single IgM molecule were very frequently seen. The tendency of IgG antibodies to bind cooperatively to certain antigens was also noted. In contrast, cross-links were seldom encountered. The cross-reactivity of different polynucleotides was also assessed by a quantitative analysis. The length of antigen associated to an antibody molecule (either IgG or IgM) was also measured.     

Proton titration curve of yeast iso-1-ferricytochrome c. Electrostatic and conformational effects of point mutations.

The proton titration curves of yeast iso-1-ferricytochrome c and selected point mutants of this protein have been determined between pH 3 and 11 at 10 and 25 degrees C with a computer-controlled titration system. Initial titration of the wild-type protein to acidic pH followed by subsequent titrations to alkaline and then acidic pH demonstrates hysteresis, with one more group (28.7) titrating between pH 11 and 3 than originally titrated (27.7) between pH 3 and 11. Initial titration to alkaline pH, however, resulted in observation of the same number of groups in both directions of titration (28.7 vs 28.6). At 10 degrees C, 7.5 fewer groups were found to titrate over the same range of pH. Titration curves obtained for six cytochrome c mutants modified at Arg-38, Phe-82, Tyr-48, and Tyr-67 were analyzed by subtraction of the corresponding titration curve for the wild-type protein to produce difference titration curves. In most cases, the effects of these mutations as revealed in the difference titration curves could be accounted for as either the result of introduction of an additional group titrating within this pH range, the result of a change in the pK of a titrating residue, and/or the result of a change in the pK for either the first acidic or the first alkaline protein conformational transition. In addition to demonstration of the electrostatic consequences of the mutations in cytochrome c studied here, this study establishes the general usefulness of precise proton titration curve analysis in the characterization of variant proteins produced through recombinant genetic techniques.     

Incorporation of styrene enhances recognition of ribonuclease A by molecularly imprinted polymers

Ribonuclease A (RNase A) is an RNA-cleaving enzyme characterized by its high conformational stability and strong catalytic activity. This enzyme is ubiquitous in living organisms and is difficult to inactivate. In polymerase chain reaction (PCR) RNase activity is removed by adding inhibitors. Molecularly imprinted polymers (MIPs) with high selectivity, high stability, low cost and facile synthesis could prove useful in extraction of target molecules, such as RNase A, from reaction mixtures. In this investigation, MIPs were synthesized from the monomers styrene and polyethyleneglycol 400 dimethacrylate (PEG400DMA) in several different ratios. Styrene as a functional monomer gave MIPs with a higher affinity for RNase A than other functional monomers tested, according to both enzyme-linked immnuosorbent assay (ELISA) and isothermal titration calorimetry (ITC). The optimum volume ratio of styrene/PEG400DMA was 20/100 at 25 degrees C, and this ratio maximized the rebinding efficiency of RNase A to MIPs. Isothermal titration calorimetry was also used, and could be useful to design the composition of molecularly imprinted polymers for various target molecules.     

Metastable secondary structures in ribosomal RNA: a new method for analyzing the titration behavior of rRNA

The pH titration behavior of E. coli rRNA in the acid range has been analyzed by combining spectrophotometric and potentiometric titration data. The “simplest” model for the system, which considers as possible reactions the protonation of adenine (A), cytosine (C), and guanine (G) residues along with the opening of A·U and G·C base pairs, does not adequately account for the titration properties. It is postulated that extra reactions may occur in addition to those in the “simplest” model, and a new analytical method was developed to deal with this situation. Our approach yields the ultraviolet spectral changes which accompany the extra reactions, from which the nature of these reactions can in principle be deduced. The calculations also give, at each pH, the extents of the extra reactions as well as the extents of those reactions which comprise the “simplest” model. We infer that in acidic RNA solutions of 0.1M ionic strength there occur at least two extra reactions, each of which involves G residues. We propose that in the pH range 6.0 ≥ pH ≥ 3.8 triple-stranded helical sequences, presumably protonated G·C·G, are formed. These regions are replaced at lower pH by acid-stable structures involving G·G and A·A base pairs. In solutions of lower ionic strength (I = 0.01M) no triple strands are formed, but G·G and A·A regions seem to develop even at pH values as high as 6.0. At I = 0.1M, an acid–base titration cycle between pH 7 and 2.8 is not reversible; rRNA shows true hysteresis behavior. We conclude that in ribosomal RNA's, which are generally G-rich, guanine residues may participate in hitherto unpredicted conformations, some of which may be metastable while others are equilibrium structures.     

The properties of brain galactocerebroside monolayers

Using a Langmuir film balance we have compared the properties of films of the brain galactocerebrosides at 37 degrees C. There are two types of cerebroside in brain, those with an alpha-hydroxy substituent on the acyl chain (HFA) and those without (NFA). At equivalent pressures the areas of both cerebroside films are significantly less than the areas of films of the brain glycerolipids, the choline and ethanolamine phosphatides. The isotherm of NFA galactocerebrosides has two discontinuities, one at low and one at high film pressure, while the isotherm of HFA galactocerebrosides is a smooth curve at all film pressures. Below the high-pressure transition the area of the NFA film is significantly larger than the area of the HFA film. When compressed beyond the high-pressure transition there is a marked hysteresis between compression and expansion isotherms of the NFA galactocerebrosides. The pressures of both films continue to rise steeply when they are compressed into areas which are too small for them to exist as simple monolayers. We conclude that under compression cerebroside films form bilayer structures; that bilayer formation starts at low pressure and occurs progressively as the HFA cerebroside monolayer is compressed, but occurs more abruptly in the NFA cerebroside monolayer at the high-pressure-transition region of the isotherm. A study of pure cerebrosides with a single defined acyl chain shows that there is a correlation between the relative volumes of the hydrophobic and hydrophilic parts of the molecule and the ease of bilayer formation. The larger the relative volume of the hydrophilic group the more readily the cerebroside forms a bilayer film. Other brain lipids added to cerebroside monolayers have sharply differing effects on their areas. The areas of films containing cholesterol are less than the areas calculated by adding the areas of the pure components multiplied by their mole fractions. On the other hand, the area of phosphatidylcholine-containing films is much larger than calculated.     

Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior

Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center. Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1. Reproducible hysteresis is demonstrated in the redox titrations. In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV. However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2. A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated. In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1). The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.     

Surface modification of chitosan films. Effects of hydrophobicity on protein adsorption

The surface of chitosan films was modified using acid chloride and acid anhydrides. Chemical composition at the film surface was analyzed by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). ATR-FTIR data verified that the substitution took place at the amino groups of chitosan, thus forming amide linkages, and the modification proceeded to the depth at least 1 microm. Choices of molecules substituted at the amino groups of the glucosamine units did affect the hydrophobicity of the film surface, as indicated by air-water contact angle analysis. The surface became more hydrophobic than that of non-modified film when a stearoyl group (C(17)H(35)CO-) was attached to the films. The reaction of chitosan films with succinic anhydride or phthalic anhydride, however, produced more hydrophilic films. Selected modified films were subjected to protein adsorption study. The amount of protein adsorbed, determined by bicinchoninic acid (BCA) assay, related to the types of attached molecules. The improved surface hydrophobicity affected by the stearoyl groups promoted protein adsorption. In contrast, selective adsorption behavior was observed in the case of the chitosan films modified with anhydride derivatives. Lysozyme adsorption was enhanced by H-bonding and charge attraction with the hydrophilic surface. While the amount of albumin adsorbed was decreased possibly due to negative charges that gave rise to repulsion between the modified surface and albumin. This study has demonstrated that it is conceivable to fine-tune surface properties which influence its response to bio-macromolecules by heterogeneous chemical modification.     

Letter: Hysteresis of proton binding to tobacco mosaic virus protein associated with metastable polymerization.

Proton binding to tobacco mosaic virus protein at 20 °C has been found to exhibit a reproducible hysteresis which results from the metastability of high molecular weight helical, virus-like rods. In a titration from pH 4 or 5 to 7, the time for depolymerization of such rods, as measured by ultracentrifugation, decreases from days to minutes over a range of about a tenth of a pH unit, near pH 6·6 at 20 °C. Relative to the extent of proton binding in the depolymerized state at 4 °C, the magnitude of the hysteresis near pH 6·2 corresponds to more than 50% of the protons bound per subunit in the equilibrium polymerized state.     

Surface shear viscometry as a probe of protein-protein interactions in mixed milk protein films adsorbed at the oil-water interface

Time-dependent surface viscosities are reported for films adsorbed from binary mixtures of the proteins alpha-lactalbumin, beta-lactoglobulin and beta-casein. The measurements were made at a planar interface between n-tetradecane and various protein solutions (10(-3) wt% of each protein, pH 7, 25 degrees C) using a Couette-type torsion-wire surface viscometer operating at very low shear-rate. Differences in behaviour between simultaneous and sequential exposure of the pairs of proteins to the interface were investigated. Some experiments were performed with chemically modified beta-lactoglobulin samples whose disulphide bonds had been cleaved and blocked. Displacement of one protein by another (e.g. alpha-lactalbumin by beta-casein) is indicated by a sudden drop in surface viscosity immediately after addition of the second protein. In systems containing beta-lactoglobulin, the long-time surface viscosity is very sensitive to the adsorption time of beta-lactoglobulin prior to addition of the second protein. Blocking the disulphide bonds in beta-lactoglobulin leads to a much faster approach to a steady-state surface viscosity. This is interpreted in terms of a much more rapid unfolding of the disordered molecules of modified beta-lactoglobulin at the oil-water interface. We conclude that surface viscosity experiments give useful and sensitive information about competitive adsorption and cooperative interactions in mixed protein films.     

Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.     

Nanocomposite films based on xylan-rich hemicelluloses and cellulose nanofibers with enhanced mechanical properties

Interest in xylan-rich hemicelluloses (XH) film is growing, and efforts have been made to prepare XH films with improved mechanical properties. This work described an effective approach to produce nanocomposite films with enhanced mechanical properties by incorporation of cellulose nanofibers (CNFs) into XH. Aqueous dispersions of XH (64-75 wt %), sorbitol (16-25 wt %), and CNF (0-20 wt %) were cast at a temperature of 23 °C and 50% relative humidity. The surface morphology of the films was revealed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The thermal properties and crystal structure of the films were evaluated by thermal analysis (TG) and X-ray diffraction (XRD). The surface of XH films with and without CNF was composed primarily of nanonodules, and CNFs were embedded in the XH matrix. Freeze-dried XH powder was amorphous, whereas the films with and without CNF showed a distinct peak at around 2θ = 18°, which suggested that XH molecules aggregated or reordered in the casting solution or during water evaporation. Furthermore, the nanocomposite films had improved thermal stability. XH film with 25 wt % plasticizer (sorbitol, based on dry XH weight) showed poor mechanical properties, whereas incorporation of CNF (5-20 wt %, based on the total dry mixture) into the film resulted in enhanced mechanical properties due to the high aspect ratio and mechanical strength of CNF and strong interactions between CNF and XH matrix. This effective method makes it possible to produce hemicellulose-based biomaterials of high quality.     

Protonated polynucleotides structures - 22.CD study of the acid-base titration of poly(dG).poly(dC)

The acid-base titration (pH 8 --> pH 2.5 --> pH 8) of eleven mixing curve samples of the poly(dG) plus poly(dC) system has been performed in 0.15 M NaCl. Upon protonation, poly(dG).poly(dC) gives rise to an acid complex, in various amounts according to the origin of the sample. We have established that the hysteresis of the acid-base titration is due to the non-reversible formation of an acid complex, and the liberation of the homopolymers at the end of the acid titration and during the base titration: the homopolymer mixtures remain stable up to pH 7. A 1G:1C stoichiometry appears to be the most probable for the acid complex, a 1G:2C stoichiometry, as found in poly(C(+)).poly(I).poly(C) or poly(C(+)).poly(G).poly(C), cannot be rejected. In the course of this study, evidence has been found that the structural consequences of protonation could be similar for both double stranded poly(dG).poly(dC) and G-C rich DNA's: 1) protonation starts near pH 6, dissociation of the acid complex of poly(dG).poly(dC) and of protonated DNA take place at pH 3; 2) the CD spectrum computed for the acid polymer complex displays a positive peak at 255 nm as found in the acid spectra of DNA's; 3) double stranded poly(dG).poly(dC) embedded in triple-stranded poly(dG).poly(dG).poly(dC) should be in the A-form and appears to be prevented from the proton induced conformational change. The neutral triple stranded poly(dG).poly(dG).poly(dC) appears therefore responsible, although indirectly, for the complexity and variability of the acid titration of poly(dG).poly(dC) samples.     

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein "freezing" to the surface. In essence: the antifreeze proteins are "melted off" the ice at the bulk melting point and "freeze" to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.