The temperature dependence of water and counter ion order in soap-water mesophases. A deuterium and sodium NMR study

Deuterium magnetic resonance (DMR) spectra of the water and hydrocarbon chains in potassium and sodium palmitates and sodium magnetic resonance spectra of sodium in sodium palmitate demonstrate correlations between water, hydrocarbon chain and counter ion order. In the lamellar phase of potassium palmitate the order parameters inferred from DMR splittings of D₂O and the first few methylene chain segments initially increase and then decrease with increasing temperature. This is explained in terms of a model where the lipid-water structure at low temperatures imposes a direction for which all the order parameters are smaller than for the higher temperature structure for purely geometric reasons. As temperature increases the structuring effect of water decreases and there is an “apparent” increase in order until at even higher temperatures there is an intrinsic decrease in order parameter. In addition, for potassium palmitate the DMR splittings of D₂O and the first few methylene segments indicate a “phase transition” within the liquid crystalline phase.     

The state of water in biological systems as studied by proton and deuterium relaxation

Careful experiments on the measurement of the intensity of the deuterium NMR signal for ²H₂O in muscle and in its distillate were performed, and they showed that all ²H₂O in muscles is “NMR visible.”The spin-lattice relaxation time (T₁) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to −70°C. T₁ values of deuterons in ²H₂O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to −20°C. Calculations on T₁ for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T₁ values compared to pure water and the frequency dependence of T₁ are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.     

The effect of deuterium oxide on the stability of the collagen model peptides H‐(Pro‐Pro‐Gly)10‐OH,H‐(Gly‐Pro‐4(R)Hyp)9‐OH,and Type I collagen

The collagen triple helix has a larger accessible surface area per molecular mass than globular proteins, and therefore potentially more water interaction sites. The effect of deuterium oxide on the stability of collagen model peptides and Type I collagen molecules was analyzed by circular dichroism and differential scanning calorimetry. The transition temperatures (T_m) of the protonated peptide (Pro‐Pro‐Gly)₁₀ were 25.4 and 28.7°C in H₂O and D₂O, respectively. The increase of the T_m of (Pro‐Pro‐Gly)₁₀ measured calorimetrically at 1.0°C min^?1 in a low pH solution from the protonated to the deuterated solvent was 5.1°C. The increases of the T_m for (Gly‐Pro‐4(R)Hyp)₉ and pepsin‐extracted Type I collagen were measured as 4.2 and 2.2°C, respectively. These results indicated that the increase in the T_m in the presence of D₂O is comparable to that of globular proteins, and much less than reported previously for collagen model peptides [Gough and Bhatnagar, J Biomol Struct Dyn 1999, 17, 481–491]. These experimental results suggest that the interaction of water molecules with collagen is similar to the interaction of water with globular proteins, when the ratio of collagen to water is very small and collagen is monomerically dispersed in the solvent. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 93–101, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Deuteration and solvent composition effects in the helix–coil transition of poly-γ-beiizyl-L-glutamate

The effects of deuteration and of changes in solvent composition on the thermo dynamics of the helix–coil transition have been studied by calorimetric and optical measurements in the poly-γ-benzyl-L -glutamate–dichloroacetic acid–1,2-dichloro-ethanc system. For a given solvent composition, deuteration of the polypeptide and of the acid lowers the transition temperature T_c, while an increase in the volume fraction of acid in the solvent raises T_c. A rise in T_c is accompanied by a decrease in both the van't Hoff and the calorimetric heats of transition, but at different rates. The result is a temperature dependency in the Zimm-Bragg cooperation parameter σ. Possible causes of this result and its implications are discussed.     

Effects of deuterium on the thermal stability of the poly A-poly U helix

The effect of deuterium on the thermal stability of the polynecleotide double helix formed by the homopolymers polyadenylie acid (poly A)and polyuridylic acid (poly U)has been invertigated by measuring the best capacity as a function of temperature in an automatic scanning adiabatic calorimeter. Hydrogen-bounded and deuterium-bonded helical conformations of the polynecleotides used have been melted in H₂O and D₂O₂ respectively, as solvent. Within the limits of experimental error, there is no dfference in the measured enthallpy change accompanying the helix-random coil transition. The enthalpy change ΔH is 6.6 Kcal/MBP ub any case. The half-conversion temperatures T_m differ by two degrees. T_m for poly (AU) in H₂O is 45.8°C, T_m for poly (AU) in D₂is 47.7.°C.     

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.     

Ordering in aqueous polysaccharide solutions. II. Optical rotation and heat capacity of aqueous solutions of a triple-helical polysaccharide schizophyllan

Deuterium oxide solutions of schizophyllan, a triple-helical polysaccharide, undergoing an order-disorder transition centered at 17 degrees C, were studied by optical rotation (OR) and heat capacity (C(p)) to elucidate the molecular mechanism of the transition and water structure in the solution and frozen states. The ordered structure at low temperature consisted of the side chains and water in the vicinity forming an ordered hydrogen-bonded network surrounding the helix core and was disordered at higher temperature. In the solution state appeared clearly defined transition curves in both the OR and C(p) data. The results for three samples of different molecular weights were analyzed theoretically, treating this transition as a typical linear cooperative transition from the ordered to disordered states and explained quantitatively if the molecular weight polydispersity of the sample was considered. The excess heat capacity C(EX)(p) defined as the C(p) minus the contributions from schizophyllan and D(2)O was estimated. In the frozen state it increased with raising temperature above 150 K until the mixture melted. This was compared with the dielectric increment observed in this temperature range and ascribed to unfreezable water. From the heat capacity and dielectric data, unfreezable water is mobile but more ordered than free water. In the solution state, the excess heat capacity originates from the interactions of D(2)O molecules as bound water and structured water, and so forth. Thus the schizophyllan triple helix molds water into various structures of differing orders in solution and in the solid state.     

The interaction of mequitazine with phospholipid model membranes

The effect of increasing concentrations of mequitazine, a quinuclidinylmethyl-phenothiazine, on the phase transition temperature (T_c), the broadening of the transition peak, the enthalpy and entropy of transition of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholine (DPPC) liposomes was studied. Pest critical micelle concentrations of mequitazine (CMC = 5.23 X 10^?2M), caused broadening of the transition peak and lowering of the T_c of pure liposomes. The ratio of peak heights from the nuclear magnetic resonance (NMR) spectra of egg phosphatidycholine liposomes was used as a criterion for assessing the interaction of the drug with phospholipid membranes. Mequitazine interacts with both the polar head groups and hydrophobic membrane interior.     

Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor

Deuterium oxide solutions of a triple-helical polysaccharide schizophyllan, undergoing an order-disorder transition centered around 17 degrees C, were studied by the time-domain reflectometry (TDR) to obtain dielectric dispersions in the solution and frozen states. In the solution state, the dispersion below the transition temperature is resolved in three dispersions (relaxation times at 0 degrees C) ascribed to side chain glucose residue (1; 102 ns), structured water (s; 2.0 ns) and bulk water (h), respectively, from low to high frequencies. Bulk water is divided into slow water (h2; 0.04 ns) and free or pure water (h1; 0.02 ns). Above the transition temperature structured water almost disappears and is compensated by slow water. Structured water is similar to bound water for proteins but different from it because of this transition behavior. Another dispersion (l) seen at the lowest frequency is assigned to the rotation of side-chain glucose residue coupled with hydrated water. Parts of this dispersion and structured water are suggested to constitute bound water. In the frozen state were observed a major dispersion (h; 0.14 ns) and a minor one (m; 28 ns), which were ascribed to considerably mobile and less mobile waters. They are similar to but not exactly the same as that for unfreezable water in bovine serum albumin solutions argued by Miura et al. (Biopolymers, 1995, Vol. 36, p. 9). Water is molded into different structures by the triple helix.     

Nuclear Magnetic Resonance Evidence using D2O for Structured Water in Muscle and Brain

The electric quadrupole moment of the deuterium nucleus provides a nuclear magnetic resonance (NMR) probe of electric field gradients, and thereby of organization of tissue water. 8-17% of H₂O in rat muscle and brain was replaced by D₂O from 50% deuterated drinking water. The peak height of the steady-state NMR spectrum of D in muscle water was 74% lower than that of an equal concentration of D₂O in liquid water. Longitudinal NMR relaxation times (T₁) of D in water of muscle and brain averaged 0.092 and 0.131 sec, respectively, compared with 0.47 sec in D₂O in liquid water. Transverse NMR relaxation times (T₂) averaged 0.009 and 0.022 sec in D₂O of muscle and brain, respectively, compared with 0.45 sec in D₂O in liquid water. These differences cannot be explained by paramagnetic ions or by magnetic inhomogeneities, which leaves increased organization of tissue water as the only tenable hypothesis. Evidence was also obtained that 27% of muscle water and 13% of brain water exist as a separate fraction with T₂ of D₂O less than 2 × 10^-3 sec, which implies an even higher degree of structure. Each of the two fractions may consist of multiple subfractions of differing structure.     

Formation of a new 5'-guanylic acid helix in neutral solution

5′-GMP forms in neutral solution a regular, ordered structure, presumably helical. The ordered structure is different from that previously reported at pH 5 and is not associated with gel formation. The transition is more cooperative than that of the pH 5 helix, but has a lower T_m.     

Fluctuations, exchange processes, and water diffusion in aqueous protein systems: A study of bovine serum albumin by diverse NMR techniques

Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T₁ and T₂ of D₂O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (C_s ≈ 0.3) and the onset of protein tumbling (C₀ ≈ 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be C_c^w ≈ 0.43.     

During B-Z Transition There is No Large Scale Breakage of Watson-Crick Base Pairs A Direct Demonstration Using 500 MHz 1H NMR Spectroscopy

Abstract

Monitoring of the Watson-Crick GNH1 proton in poly(dG-dC)-poly(dG-dC) at 500 MHz in 90% H₂0:10% D₂o at 30° C as a function of NaCl concentration (1.5 to 3.6 M), demonstrates that the bases retain Watson-Crick pairing throughout the transition. This observation unequivocally demonstrates that during the B-Z transition there is no large scale and detectable base pair opening and that macroscopically the phenomenon can be described as a direct helix to helix transition. We present frame by frame, an energetically sound stereodynamical trajectory for this transfiguration from right-handed B-DNA to left-handed Z-DNA.     

Thermodynamics of the helix-coil transition in polypeptides in mixed organic solvents: the influence of inert solvent and side chain

The thermally induced coil–helix transition of poly(γ-benzyl-L -glutamate) (PBLG) and poly(γ-methyl-L -glutamate) (PMLG) in binary solvent mixtures was investigated by calorimetric and optical rotatory dispersion (ORD) measurements. Dichloroacetic acid was the common active solvent, and the inert solvent was one of the chlorinated hydrocarbons, such as chloroform, 1,3-dichloropropane, 1-chlorobutane, or 1-chlorooctane. The thermodynamic parameters characterizing the intramolecular polypeptide and polypeptide–solvent interactions were calculated using the Karasz and Gajnos theoretical model [(1973) J. Phys. Chem. 77 , 1139–1145]. It was found that the enthalpy (ΔH₁) and entropy (ΔS₁) of helix stabilization in the absence of the active solvent depend on the inert solvent, but only in the case of PBLG. This is explained by the additional helix stabilization achieved by the stacking of the benzyl groups. The stacking is more pronounced in less polar chlorinated hydrocarbons with longer aliphatic chains. The results obtained indicate that the maximum helix stability is reached in chlorinated hydrocarbons with 12 C atoms. In the case PMLG, with an aliphatic ester side group, ΔH₁ and ΔS₁ are independent of the inert solvent. The ORD measurements were used to determine the maximum fraction of helicity attained at constant solvent composition and the transition temperature, T_c, at the point where f_H = 0.5. It was found that, for the same solvent composition, T_c was higher than the temperature of the midpoint of the calorimetric peak. This is explained by the fact that the maximum fraction of helicity is less than unity. The finite transition width was taken into account by calculating the phase boundaries for different fractions of helicity using the value of σ estimated from the calorimetric and van't Hoff enthalpies in the usual manner.     

Theory of melting of the triple helix poly (A + 2U) for a 1 : 2 mixture of Poly A to Poly U

The Zimm-Bragg theory is extended to treat the melting of the triple helix poly (A + 2U) for a solution with a 1 : 2 mole ratio of poly A to poly U. Only the case for long chains is considered. For a given set of parameters the theory predicts the fraction of segments in the triple helix, double helix, and random coil states as a function of temperature. Four nucleation parameters are introduced to describe the two order–disorder transitions (poly (A + 2U) ? poly A + 2 poly U and poly (A + U) ? poly A + poly U) and the single order–order transition (poly (A + 2U) ? poly (A + U) + poly U). A relation between the nucleation parameters is obtained which reduces the number of independent parameters to three. A method for determining these parameters from experiment is presented. From the previously published data of Blake, Massoulié and Fresco⁸ for [Na⁺] = 0.04, we find σ_T = 6.0 × 10^?4, σ_D = 1.0 × 10^?3, and σσ* = 1.5 × 10^?3. σ_T and σ_D are the nucleation parameters for nucleating a triple helix and double helix, respectively, from a random coil region. σσ* is the nucleation parameter for nucleating a triple helix from a double helix and a single strand. Melting curves are generated from the theory and compared with the experimental melting curves.     

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cell. I. Metabolic inactivation impairs both CD and LD antigen signals

The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T_c) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T_c responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T_c precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T_c from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T_c. It is concluded that, in addition to the traditional view that these treatments damage the “LD” signal to helper T lymphocytes, metabolic inactivation also impairs recognition of “CD” determinants by precursor T_c.     

The effect of salts on the Nmr spectra of D2O in collagen fibers

The effects of two salts, MgCl₂ and MgSO₄ on the wide-line nmr spectrum of D₂O in oriented, undernatured collagen fibers have been examined at four different D₂O contents. MgCl₂ was found to decrease the nmr doublet splitting, as compared with equal quantities of pure D₂O while the major effect of MgSO₄ was to inhibit the adsorption of D₂O without significantly affecting its nmr spectrum. The results, together with a few observations of KCl and LiCl solutions, indicate that even fairly high concentrations of salt have only small effects on the nmr spectrum of D₂O in fibrous collagen. It is considered unlikely that either “two-state” or “structured-water” models can satisfactorily account for the D₂O-nmr doublet spectrum or the effects of salts on it, over the entire range of observed D₂O content.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.     

The crystal structure of D-glucaro-1,4-lactone monohydrate

In the crystal structure of D-glucaro-1,4-lactone monohydrate, C₆H₈O₇·H₂O, the molecules have the E₃ lactone-ring conformation, with a small distortion of the ring to ₃T². The α-hydroxycarboxylic acid side-chain is axial, with the HO---C---C=O torsion angle within 6° of cis-planar. The orientation of the side-chain is such that the hydroxyl group lies over the lactone ring, which is the same conformation as is reported to preponderate in solution. Calculations of non-bonding repulsion energy show that this conformation corresponds to an energy minimum, although comparable minima can also be obtained with the ring in the alternative ³E conformation. The lactone and water molecules are hydrogen-bonded to form layers two molecules,wide, separated by Van der Waals interactions. One of the water hydrogen atoms is involved in a weak, bifurcated hydrogen-bond.