Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T₁) and spin-spin (T₂) relaxation times of water in cysts hydrated with D₂O have been measured for hydrations between 1.5 and 0.1 g of D₂O per gram of dry solids. When the relaxation rates (I/T₁, I/T₂) of ²H and ¹⁷O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D₂O (or H₂ ¹⁷O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The ²H and ¹⁷O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.

It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H₂O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T₁ as a function of hydration in the vicinity of 0.6 g H₂O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

     

Counterion binding and hydration of hyaluronate and chondroitin in solution: An 17O, 23Na,and 25Mg nuclear-magnetic-relaxation study

Nuclear magnetic relaxation rates of H₂¹⁷O, ²³Na⁺, and ²⁵Mg²⁺ have been measured in aqueous hyaluronate solutions. The dependence on solution pH of the relaxation rates has been investigated, as well as the competition behavior of Na⁺ with Ca²⁺ and Mg²⁺. H₂¹⁷O and ²³Na⁺ relaxation rates in chondroitin and hyaluronate solutions have been compared in the interval, 2 ? pH ? 12.5. The ion binding of hyaluronate can be fully accounted for by Coulomb interactions, with no need to involve chemical specificity. The hydration is only slighly pH dependent, and is comparable in magnitude to hydration of synthetic polyelectrolytes and monosaccharides. Ion-binding and hydration properties of hyaluronate and chondroitin are quite similar, except at elevated pH. At alkaline pH, an increase in charge density with pH is seen in hyaluronate and, to a much lesser degree, in chondroitin, possibly due to the titration of hydroxy groups. H₂¹⁷O data indicate an alkali-induced transition in both glycosaminoglycans.     

Interrelationships between Water and Cellular Metabolism in Artemia cysts. V. 14CO2 incorporation

The ability of cysts of the brine shrimp, Artemia salina, to incorporate ¹⁴CO₂ into organic compounds soluble in cold-trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H₂O/g dried cysts, the “critical hydration” for CO₂-fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of ¹⁴CO₂ incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO₂-fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H₂O/g is particularly noteworthy since this hydration level is well within the range of the amounts of “bound water” described in the literature for a wide array of cells and tissues.     

Dependence of co-operativity in the reaction of haemoglobin with oxygen on deuterium oxide concentration and temperature

Concentrated deionized solutions of haemoglobin in water were diluted with unbuffered pure ²H₂O and left to stand for 15 to 70 hours. Oxygen equilibrium curves were measured at different temperatures and concentrations of ²H₂O. In 98.5% ²H₂O Hill's constant retained its normal value at temperatures below 11 °C, but was reduced by 0.4 above 11 °C. This temperature effect was reversible. Lowering the ²H₂O concentrations raised the transition temperature between the states of low and high co-operativity of the reaction. The shape of the transition curve remained unchanged. Further experiments allowed a phase diagram to be constructed which shows the boundaries between the two states: one of low co-operativity at high concentration of ²H₂O and high temperature, and another of high co-operativity at low values of these variables. Reversibility of the isotope effect even at ²H₂O concentration of 98.5% excludes a purely steric interpretation. Possible dynamic and co-operative interactions between the protein molecule and its surrounding water molecules are discussed.     

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center

Isotope substitution of H₂O by ²H₂O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P⁺) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P⁺. In the case of H₂O medium these effects are observed at a lower hydration than in ²H₂O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H₂O, the rate of electron transfer from the nearest high-potential cytochrome heme to P⁺ is virtually independent of hydration within the P/P₀ range from 0.1 to 0.5. In samples hydrated in ²H₂O this rate is approximately 1.5 times lower than in H₂O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with ²H₂O is inhibited within this range of P/P₀, whereas in RC samples with H₂O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.     

Different conformations of double-stranded nucleic acid in solution as revealed by circular dichroism

Conformation of two-stranded DNA in H₂O–methanol, H₂O–ethanol, H₂O–isopropanol, and H₂O–dioxane solutions at different concentrations of alkaline ions has been studied with the aid of circular dichroism. The following conclusions are drawn: The conformation of DNA in H₂O and H₂O–methanol belongs to a family of B forms (B, C, T forms are the representatives of the family). The magnitude of the winding angle between adjacent base pairs (θ) is determined by the concentration and type of the cations. In H₂O the cation action is nonspecific and leads to an increase in θ value. In 80% methanol the ions act specifically, Cs⁺ being to stabilize a form with a greater θ value, and Li⁺ being with a lesser one. The total θ change is likely within the limits of 33° ? θ ? 45°. At high content of ethanol, isopropanol, or dioxane (～80%), but not with methanol, and in low ionic strength the conformation of DNA belongs to a family of A forms (A form is one of the members of the family) and is specified by the concentration and type of cation involved. The two-stranded regions of RNA in H₂O are also of A type and winds with the rise of cation concentration. The range of θ variation is not narrower than 30° ? θ 33°. The conformational transitions within the families (induced by ions) are of non-cooperative pattern, wheras the transitions between the families (induced by nonpolar component) are of cooperative pattern. The effect of cations, when specific, is discussed on the basis of steric correspondence between the width of DNA narrow groove and the size of a hydrated cation.     

Thermodynamic functions of biopolymer hydration. II. Enthalpy–entropy compensation in hydrophilic hydration processes

The thermodynamic functions of biopolymer hydration were investigated by multitemperature vapor pressure studies. Desorption measurements were performed that allowed determination of reversible isotherms in the hydration range of 0.1 to 0.3–0.5 g H₂O/g dry polymer. These isotherms are accessible to thermodynamic interpretation and are relevant to the interaction of water with biopolymers in their solution conformation. The results obtained on a series of different biopolymers (lysozyme, α-chymotrypsin, apo-lactoferrin, and desoxyribonucleic acid), show the following common features of interest: (1) The differential excess enthalpies (ΔH^e ) and entropies (ΔS^e ) are negative, and exhibit pronounced anomalies in a well-defined low-humidity range (approx. 0.1 g H₂O/g dry polymer). These initial extrema are interpretable by structural changes, induced in the native biopolymer structures by water removal below a critical degree of hydration. (2) The ΔH^e and ΔS^e terms exhibit statistically significant linear enthalpy–entropy compensation effects in all biopolymer–water systems investigated. The compensation temperatures \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta = \overline {\Delta H} ^e /\overline {\Delta S} ^e $\end{document} are approximately identical for all biopolymers, ranging from 360 to 500 K. The compensation effects are attributable to phase transitions of water molecules between the bulk liquid and the inner-sphere hydration shell of native biopolymers. (3) The negative excess free energies (ΔG^e ) decrease monotonically with increasing water content and are close to zero at 0.3 to 0.5 g H₂O/g polymer. This result indicates that only transitions between the bulk liquid and the inner-sphere hydration shell are associated with significant net free energy effects. The outer-sphere hydration water is thermodynamically comparable to bulk water. The importance of the proportionality factor \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta $\end{document} in the control of the free energy term is discussed.     

Interrelationships between water and cellular metabolism in Artemia cysts. VI. RNA and protein synthesis

Using ¹⁴CO₂ as a labelled precursor the relationship between the initiation of protein and RNA synthesis, and water concentration, has been examined in cysts (encysted embryos) of the brine shrimp, Artemia salina. Although incorporation of radioactivity into amino acids and nucleotides occurred in cysts at hydrations as low as 0.3 g H₂O/g dried cysts, incorporation into proteins and RNA was not measurable until the cysts had achieved a hydration in the range of 0.6–0.6 g/g. In no case was radioactivity detected in DNA of unemerged cysts. Fully hydrated cysts (about 1.3 g/g) that were actively synthesizing proteins and RNA, stopped doing so when dehydrated to levels below the same hydration range: thus, the hydration dependence does not involve appreciable hysteresis. The hydration range required to initiate synthesis of these macromolecules is essentially the same as that previously shown to initiate embryonic development.     

The inhibition of lactate dehydrogenase by hydrated pyruvate

The lactate dehydrogenase-catalyzed reduction of pyruvate by NADH was studied using a spectroscopic method. The inhibitory effect exhibited by high concentrations of pyruvate was investigated in phosphate and 2,2-diethylmalonate buffers. Kinetic studies were carried out in which the rate of the enzyme-catalyzed reaction was monitored at various stages of pyruvate hydration, H₂O + CH₃COCO₂^? ? CH₃C(OH)₂2C0₂^?. Buffered solutions of different initial relative amounts of ketopyruvate and hydrated pyruvate (2,2-dihydroxypropanoic acid) were also preincubated with the enzyme and NAD⁺. Kinetic runs were initiated in the resultant solutions at various stages of incubation by the introduction of NADH. The results of the present investigation indicate that hydrated pyruvate is a major inhibitor of lactate dehydrogenase and forms an inhibitory complex with the enzyme and oxidized coenzyme.     

The effects ofγ-irradiation on the hydration characteristics of DNA and polynucleotides

Summary The effects of-irradiation and changes in the macromolecular structure on the water proton resonance spectra observed in frozen and liquid solutions have been compared for the DNA and polynucleotide solutions, using H₂O or mixed H₂O/D₂O solvents. The results indicate that in order to obtain information concerning the role of hydration water in mediating the overall radiation damage, the NMR studies must be performed in the frozen state.Member of the Euratom biology division     

Ion distributions around left- and right-handed DNA and RNA duplexes: a comparative study

The ion atmosphere around nucleic acids is an integral part of their solvated structure. However, detailed aspects of the ionic distribution are difficult to probe experimentally, and comparative studies for different structures of the same sequence are almost non-existent. Here, we have used large-scale molecular dynamics simulations to perform a comparative study of the ion distribution around (5′-CGCGCGCGCGCG-3′)₂ dodecamers in solution in B-DNA, A-RNA, Z-DNA and Z-RNA forms. The CG sequence is very sensitive to ionic strength and it allows the comparison with the rare but important left-handed forms. The ions investigated include Na⁺, K⁺ and Mg^{2 +}, with various concentrations of their chloride salts. Our results quantitatively describe the characteristics of the ionic distributions for different structures at varying ionic strengths, tracing these differences to nucleic acid structure and ion type. Several binding pockets with rather long ion residence times are described, both for the monovalent ions and for the hexahydrated Mg[(H₂O)₆]²⁺ ion. The conformations of these binding pockets include direct binding through desolvated ion bridges in the GpC steps in B-DNA and A-RNA; direct binding to backbone oxygens; binding of Mg[(H₂O)₆]²⁺ to distant phosphates, resulting in acute bending of A-RNA; tight ‘ion traps’ in Z-RNA between C-O2 and the C-O2′ atoms in GpC steps; and others.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

Theoretical studies on the structures,intra- and inter-molecular hydrogen bonding interactions in HNF and HNF–H2O clusters in the gaseous,aqueous and solid phases

Hydrazimium nitroformate ([N₂H₅]⁺[C(NO₂)₃]^? , HNF) is an ionic oxidiser used in solid propellants. Its properties are easily affected by H₂O because of its hygroscopicity. In this article, density functional theory (DFT) and molecular dynamics (MD) were employed to study the isolated HNF molecule and the HNF–H₂O cluster in gas phase and in the aqueous solution. Three stable conformations were obtained for HNF in the gas phase and in the aqueous solution, respectively, and each conformation can form several different HNF–H₂O clusters. Irrespective of whether it is in gas phase or in solution, intramolecular hydrogen bond interactions and other interactions (e.g. the binding energy, the dispersion energy, the second-order perturbation energy and the energy gap between frontier orbitals) of HNF are weaker in the clusters than in the isolated state. The initial decomposition energy of the cluster is lower than that of the isolated HNF molecule in both gaseous and aqueous phases, while the dissociation processes are the same. Molecular dynamic simulations showed that the clustered H₂O elongates and weakens the C–NO₂ bond in the solid HNF–H₂O cluster compared with that in the solid HNF. H₂O reduces and weakens intramolecular N–HΛO bonds too, and O–HΛN is the dominant intermolecular hydrogen bond between HNF and H₂O.     

Some properties of particles of egg-yolk phosphatidylcholine and cholesterol

Some physicochemical properties of particles of phosphatidylcholine and cholesterol, produced by ultrasonication of their aqueous dispersions in varying proportions and concentrations were investigated. Cholesterol altered the micellar characteristics according to its proportion. S_{20, w} values increased incrementally from 2.53 for phosphatidylcholine alone to 15.65 for particles containing the highest proportion and concentration of solubilized cholesterol (0.28 g/100 ml of each lipid). Similarly, particle weight increased from 3 × 10⁶ to 7.12 × 10⁷, axial ratios from 3.9 (discs) and 3.3 (rods) to 28.0 (discs) and 13.0 (rods), the viscosity shape factor, ν, from 3.9 to 20.7, water of hydration decreased from 0.26 g H₂O/g lipid to 0.22 g H₂O/g lipid and the number of monomers within each particle ranged from 4000 to 84,000.     

Germination of Nosema algerae (Microspora) Spores: Conditional Inhibition by D2O, Ethanol and Hg2+ Suggests Dependence of Water Influx upon Membrane Hydration and Specific Transmembrane Pathways

ABSTRACT. The germination of microsporidian spores under conditions expected to affect water flow across the plasma membrane-wall complex was studied by assessing their responses to in vitro stimulation with Na⁺ or K⁺. Partial or full substitution of common water with D₂O, which more effectively coats ions and electrostatically-charged cell surfaces with relatively stable hydration layers, delayed and inhibited spore germination in a concentration-dependent manner; yet, preincubation in 100% D₂O did not change the normal response to standard stimulation. Water structure-breaking conditions, such as an increase in temperature (within the 15° C to 40° C range) or in ionic strength (2- to 10-fold normal), opposed the inhibition by D₂O and allowed significant stimulation by Li⁺, the monovalent cation with the largest hydration diameter and a usually weak stimulant action on the spores. Ethanol, known to reduce water permeation across cell membranes and phospholipid bilayers, also caused a powerful and dose-dependent (1% to 4% v/v) inhibition of spore germination, but pretreatment with ethanol did not affect the normal response. HgCl₂, an inhibitor of specific water channels, blocked spore germination at just 250 μM in the normal stimulation solution irrespective of the temperature, and permitted only a delayed response in high salt stimulation solutions. However, the inhibition by Hg²⁺ was abolished by the simultaneous presence of 2-mercaptoethanol in the medium. These results suggest (1) that spore germination is keenly dependent upon the hydration states of both the plasma membrane-wall complex and the stimulant ions, and (2) that osmotic water flows into the spores through specific transmembrane pathways with critical sulfhydryl groups, i.e. analogous to the water channels that facilitate water movements across the plasma membranes of highly permeable cells.     

Heme-environment of horseradish peroxidase as observed by oxygen-17 superhyperfine interaction in EPR.

The presence of a water ligand on heme-iron in ferric hemoproteins can, in suitable cases, be detected by observing ¹⁷O superhyperfine interaction in the EPR spectra of solutions in H₂¹⁷O. Although no significant superhyperfine interaction is detectable in the EPR spectra of horseradish peroxidase itself, benzo-hydroxamic acid, which forms an outersphere complex with the enzyme analogous to an enzyme-peracid transition state, stabilizes an innersphere water ligand on the heme, as indicated by a ～1.3 gauss Fe³⁺-¹⁷O superhyperfine interaction in the EPR signal at g = 2, in the presence of 34–39% H₂¹⁷O at 8°K. These results indicate that the predominantly pentacoordinate Fe³⁺ ion in horseradish peroxidase is accessible to the solvent and that it acquires a water or hydroxyl ligand in the presence of benzohydroxamic acid.     

New pulsed field gradient NMR experiments for the detection of bound water in proteins

Summary New H₂O-selective homonuclear and heteronuclear 2D NMR experiments have been designed for the observation of protein hydration (PHOGSY, Protein Hydration Observed by Gradient Spectroscop Y). These experiments utilize selective excitation of the H₂O resonance and pulsed field gradients for coherence selection and efficient H₂O suppression. The method allows for a rapid and sensitive detection of H₂O molecules in labelled and unlabelled proteins. In addition it opens a way to measure the residence time of water bound to proteins. Its application to uniformly ¹⁵N-labelled FKBP-12 (FK-506 binding protein) is demonstrated.     

Medium Acidification by Maize Root Tips and its Inhibition by Heavy Water

Acidification of external solution by maize root tips can apparently proceed by two mechanisms. The first, significant only above pH 6, does not require the presence of salts in the medium and appears to be due to respiratory CO₂ release with subsequent hydration and dissociation into H⁺ + HCO^?₃. The second, much more efficient process, requires permeant ions and proceeds to well below pH 4 under mediation by the plasma membrane H⁺-ATPase. While the first process is affected by acetazolamide (an inhibitor of carbonate dehydratase), the second is blocked by vanadate, erythrosin B, diethylstilbestrol and miconazole, and is strikingly stimulated by fusicoccin. Both mechanisms are markedly inhibited by heavy water, the first by up to 80% with an ID₅₀ of 18–25% D₂O, the second up to 91%, with an ID₅₀ of 20–30% D₂O. The inhibition of the acidification by ATPase can be relieved by replacing D₂O with H₂O, indicating that the effect of D₂O is kinetic rather than on covalently-bound hydrogen atoms. Apparently the H⁺-ATPase uses H⁺ (or possibly H₃O⁺) as substrate which binds to a rather specific site where it cannot be functionally replaced by D⁺ or D₃O⁺.     

Study of effect of molecular mobility in chromatophore membranes of the bacterium <Emphasis Type="Italic">E. shaposhnikovii</Emphasis> on processes of photoinduced electron transport using the NMR-Spin-Echo method with isotope substitution and dehydration

The effect of dehydration and ²H₂O/H₂O isotope substitution on electron transport reactions and relaxation of proton-containing groups was studied in chromatophore membranes of Ectothiorhodospira shaposhnikovii. During dehydration (including isotope substitution of hydrate water) of preliminarily dehydrated isolated photosynthetic membranes there was a partial correlation between hydration intervals within which activation of electron transport from high-potential cytochrome c to photoactive bacteriochlorophyll dimer P890 of photosynthetic reaction center and variation of spin-lattice and spin-spin proton relaxation time was observed. Partial correlation between hydration intervals can be considered as evidence of correlation between mobility of non-water proton-containing groups with proton relaxation frequency ∼10⁸ sec⁻¹ with efficiency of electron transfer at the donor side of the chain.     

The temperature dependence of the redox potential of horse heart cytochrome c in sodium chloride solutions

The E⁰′ values for the conversion of horse heart cytochrome $\text{c}$ from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H₂O and D₂O. In H₂O, the decrease in the E⁰′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D₂O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H₂O above 42°C but not in the more stable D₂O (Kreishman, Foss, Inoue and Leifer (1976) $\text{Biochemistry}\text{,}\text{15}$, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E⁰′.