Role Of Hydration Research Articles

The Role of Hydration on the Mechanism of Allosteric Regulation: In Situ Measurements of the Oxygen-Linked Kinetics of Water Binding to Hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

Role of Hydration in the Binding of lac Repressor to DNA

The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A. This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.

Delirium and dehydration: some fluid for thought?

Delirium is a frequent complication of advanced cancer. It is characterized by cognitive deficits and behavioral disturbance, and therefore can potentially result in severe symptom distress and impeded communication between patient and family and between patient and medical staff. The reversibility of delirium depends on its underlying causes. Delirium is multifactorial in origin, and precipitating or contributory factors include dehydration and hypovolemia. Much of the debate concerning the role of hydration in advanced cancer has centered on symptoms such as thirst, and ethical issues such as parenteral hydration and its association with prolongation of life, with the association between hydration status and delirium largely excluded. This review examines the latter association in the light of recent studies. It also provides an outline of relevant pathophysiology and clinical assessment, a decision-making framework and a practical approach to the techniques of assisting hydration. This will hopefully help physicians and families in weighing up the merits of assisted hydration in an individual context and help them to achieve a consensus on the role of hydration that is consistent with the goals of care.

NMR local range investigations in amorphous starchy substrates I. Structural heterogeneity probed by 13C CP–MAS NMR

The 13C CP–MAS (Cross Polarization and Magic Angle Spinning) NMR signatures of a series of amorphous and semi-crystalline samples prepared from various starchy substrates (native potato starch, amylopectin, amylose) following different techniques of preparation (casting, freeze drying, solvent exchange) are compared. Decompositions of the C1 resonance spectra reveal the existence of four or five main types of α(1–4) linkages, which can be quantified. The influence of the intrinsic primary structure (linear or branched) and of the preparation procedure on conformational changes and resulting crystallinity are interpreted in terms of distributions of average glycosidic linkages dihedral angles ( Φ, Ψ). The role of hydration is also considered. An improved understanding at different structural levels is obtained in relation to local and intermediate range orders. Such information may be useful for the understanding of the structural evolution of a large variety of starchy substrates before or after treatments widely used in industrial processes.

Effect of Cardiolipin on Proton Permeability of Phospholipid Liposomes: The Role of Hydration at the Lipid–Water Interface

The effect of cardiolipin on the proton permeability of dipalmitoyl-phosphatidylcholine small unilamellar vesicles was examined by utilizing the pH-dependent fluorescence emission of 5- (and 6-) carboxyfluorescein. It has been found that the proton permeability of the phospholipid bilayer was greatly enhanced in the presence of cardiolipin, an acidic phospholipid mainly found in the inner mitochondrial membranes. In the presence of bovine heart cardiolipin, the bilayer surface hydration, as assessed with the fluorescence lifetime of 1-anilinonaphthalene-8-sulfonic acid, was increased, while hydration in the acyl chain region was not altered. In addition, the bilayer fluidity was also not affected. Taken together, these results suggest that the lipid–water interface is the major energy barrier for proton permeation of the bilayer vesicles, and alteration to properties of this interface by cardiolipin headgroup appears to be responsible for the enhanced proton permeability.

Modulation of pig kidney Na+/K+-ATPase activity by cholesterol: role of hydration.

Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.

The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The adsorption of water by alcohol dehydrogenase from baker's yeast (YADH) has been measured in a continuous-flow gas reactor at varying temperatures. Adsorption isotherms in the presence of gaseous organic substrates are compared to those from organic-free gas mixtures. Almost no effect of the hydrophobic molecule on total water adsorption was observed. A rarely mentioned multilayer isotherm model from the 1930s, the Huttig's isotherm, has been found to fit the experimental data with extremely good accuracy. The model enables the calculation of both the heat of adsorption of water to the enzyme and the total amount of water necessary for monolayer coverage. The heat of adsorption of water in the first layer is approximately -16 kcal/mol. This tight binding of water, which is much higher than the heat of condensation of pure water, helps to explain the kinetic properties of YADH-catalyzed reactions on vapor phase substrates. While the monolayer coverage is temperature independent, the enzyme demonstrates hysteresis when transitioning between adsorption and desorption. The hysteresis observed in water sorption studies may also explain previously reported properties of the enzyme. (c) 1996 John Wiley & Sons, Inc.

The role of hydration in enzyme activity and stability: 2. Alcohol dehydrogenase activity and stability in a continuous gas phase reactor

The degree of enzyme hydration is the one of the most important factors which can affect enzyme activity and stability in water-limited environments. Alcohol dehydrogenase from baker's yeast (YADH) has been used as a model enzyme to study the effects of hydration on activity, stability, and cofactor stability with gas phase substrates. In all cases, the enzyme is essentially inactive until a temperature-independent degree of surface coverage by water molecules has been reached. The critical water content corresponds to 40-50% of a single monolayer. Careful control of the degree of hydration, by adjustments to gas humidity and temperature, enables the enzyme to be stabilized for periods exceeding 1 month, whereas in water the half-life of the enzyme is 30 min. The reaction with gas phase substrates follows a pseudo-first-order mechanism with an activation energy of 7.5 +/- kcal/mol, which is almost half of that in aqueous solution. (c) 1996 John Wiley & Sons, Inc.

A Thermodynamic Study of Complexation of Ciprofloxacin and Lomefloxacin with Aluminium Ion

Gibbs energy, enthalpy and entropy for the reactions of ciprofloxacin and lomefloxacin with aluminium ion were determined. No significant differences were observed for Gibbs energy values for these fluorquinolones while significant differences were found for enthalpy and entropy. The highly positive values observed for the entropy give evidence for a strong cation-ligand interaction and point out the role of hydration in the complex formation. As the value of the entropy for the reaction with ciprofloxacin is higher than that observed for lomefloxacin one concludes that the former ligand interacts more strongly with the metal ion than the latter.

Thermodynamic analysis of biomolecules: a volumetric approach

Fundamental thermodynamic relationships reveal that volumetric studies on molecules of interest can yield useful new information. In particular, appropriately designed volumetric studies can characterize the properties of molecules as a function of solution conditions, including the role of solvation. Until recently, such studies on biologically interesting molecules have been limited because of the lack of readily available instrumentation with the requisite sensitivity; however, during the past decade, advances in the development of highly sensitive, small-volume densimetric, acoustic and high-pressure spectroscopic instrumentation have enabled biological molecules to be subjected to a wide range of volumetric studies. In fact, the volumetric methods used in these studies have already provided unique insights into the molecular origins of the intramolecular and intermolecular recognition events that modulate biomolecular processes. Of particular note are recent volumetric studies on globular proteins and nucleic acid duplexes. These studies have provided unique insights into the role of hydration in modulating the stabilities of these biopolymers, as well as their conformational transitions and ligand-binding properties.

Role of Hydration and Conformational Changes in Adsorption Dynamics of Ethyl(Hydroxyethyl)cellulose at the Air/Solution Interface

Previous studies have discussed the kinetics involved in the reduction in surface tension of ethyl(hydroxyethyl)cellulose (EHEC) at the air/aqueous solution interface in terms of configuration changes involved in the transfer of individual polymer segments between soluted and adsorbed states. It was suggested that the process kinetics were governed by a substantially high activation barrier. In the present study, based on molecular mechanic simulations, it is shown that a nearly activation-free path between configurations exists for EHEC due to the extreme flexibility of the polymer chain. Hence, the relatively slow kinetics of the process should rather be attributed to a stepwise transformation between conformations which is expressed as a sequence of elementary processes involving a considerable number of intermediate isomers. This occurs over an extended time period. Also, the configuration changes involved in the uncoiling of the polymer at the interface are related to the hydration effects.

Roles of hydration, sodium, and chloride in regulation of canine mucociliary transport system.

To gain insight into the homeostatic mechanisms regulating airway ion/water fluxes and mucociliary transport, the canine tracheobronchial airway fluid was perturbed by deposition of hypo- and hyperosmotic aerosols for >1 h. Tracheal ciliary beat frequency (CBF) was measured by using heterodyne laser light scattering. Tracheal mucus velocity (TMV) and bronchial mucociliary clearance (BMC) were measured by using radioaerosols and nuclear imaging. Respiratory tract fluid output (RTFO) was collected by using a secretion-collecting endotracheal tube. In six dogs, CBF increased during water deposition in the airways to 180 +/- 30 mg/min and RTFO increased from 2.2 +/- 0.5 to 18.3 +/- 1.6 mg/min, accounting for <10% of the fluid deposition. TMV and BMC were unchanged. CBF, TMV, and BMC were markedly increased by inhalation of aerosolized 3.4 M NaCl. Aerosolized 0.85 M NaCl, in contrast, decreased BMC. In this case, RTFO represented 24% of aerosol deposition. Aerosolized 0.85 M choline chloride and 0.85 M sodium gluconate enhanced BMC and TMV concurrent with a decrease in CBF. RTFO of sodium gluconate studies exceeded 50% of aerosol deposition. Thus the airways appear to have transepithelial compensatory mechanisms that reduce the impact of a moderate increases in NaCl and hydration load, but when these responses cannot adequately respond because of the delivery of impermeable ions or very high tonicity, removal of the challenges are affected by a stimulation of mucociliary transport.

Role of Hydration via Alkaline Wash Liquor in the Removal of Starchy Soil during Machine Dishwashing

A materials science approach is applied to understanding the removal of starchy soils from crockery in a dishwasher. Specifically, the action of hydration and the role of alkalinity in reducing the cohesion of a retrograded Cream-of-Wheat soil are studied using the rigidity modulus of the soil as a measure of cohesive strength. Cream-of-Wheat gels prepared undersaturated with water were exposed to simple wash liquors buffered in the alkaline pH range. The gain in water content by the gel was analyzed via a differential and an integral approach, both treating the process of swelling with wash liquor as a one-dimensional diffusion problem. A phenomenological diffusion coefficient describing the swelling process is determined as 3x10 -11 m 2 /s at pH 10 and 10 -10 m 2 /s at pH 12. The ingress of alkalinity into the soil was followed by having an acid/base color indicator uniformly distributed throughout the gel. Treating the progress of the color change into the soil as one-dimensional diffusion, diffusion coefficients were obtained for the ingress of alkalinity: 10 -9 m 2 /s at pH 10 and 3x10 -9 m 2 /s at pH 12. The decay in the soil layer cohesion with exposure to wash liquor occurs on a time scale too rapid to be related to the swelling process but in keeping with that of alkaline ingress. The penetrating hydroxyl ions deprotonate a portion of the hydroxyl groups which cross-link starch, making the soil more easily removed by mechanical forces in a dishwasher.

Role of water in protein kinase C catalysis and its binding to membranes.

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.

X-ray diffraction analysis of tendon collagen at ambient and cryogenic temperatures: role of hydration

Equatorial (d) and meridional (D) spacings of native rat tail tendon (RTT) and unmineralized native turkey leg tendon (UTLT) were measured at ambient and liquid-nitrogen temperatures, using X-ray diffraction. Cooling of air-dried RTT or UTLT caused little change in d, which was approximately equal (1.1 nm) in the two tissues before and after cooling. For fully hydrated RTT or UTLT, cooling caused the familiar broad equatorial diffraction pattern to increase in sharpness to more resemble the pattern seen in dehydrated tissue. The d-spacings of hydrated RTT and UTLT fell by 0.12 nm (8.5%) and 0.19 nm (13%), respectively, to values seen (at ambient temperature) when the tissue water content is the maximum possible in the absence of unbound water (0.5g water/g dry collagen). These results can be explained by the movement of water within the fibril. In tissue with a water content of greater than 0.5 g/g dry collagen the spacings reflecting the lateral packing of the axially-linked tropocollagen molecules comprising a collagen fibril are determined partly by the unbound component of intermolecular hydration. As the bulk water between the fibrils freezes, this mobile component remains initially unfrozen as the tissue is cooled below 0°C. It diffuses from the intermolecular spaces into the interfibrillar spaces where it also rapidly freezes. This allows the d-spacing to decrease to the value appropriate for the presence of bound intermolecular water only. The mechanism is likely to be an energetically favourable relaxation of the lateral positions of the tropocollagen molecules from a quasi-hexagonal arrangement to hard-disk liquid-like packing. The results and methods of this study may be applicable in the elaboration of more complex collagenous systems.

Hydration as a control mechanism in membrane proteins: the case of cytochrome c oxidase

Variable hydration and volume exclusion are postulated for the deviations from osmotic ideality of macromolecules. Interactions between different macromolecules [e.g. polyethyleneglycols (PEGs) and proteins] in solution may involve both hydration–dehydration and direct binding. Membrane enzymes such as cytochrome c oxidase undergo functionally significant conformational changes with osmotic consequences during their catalytic cycles and in reactions with inhibitors such as cyanide. Osmotic stress identifies three processes in the oxidase involving water binding and release: (i) uptake of water on binding of the substrate cytochrome c when bulk osmolarity is adjusted with sucrose. With glucose or glycerol, a smaller number of water molecules are taken up. Dextran increases cytochrome c binding, suggesting multiple regions in the cytochrome c binding cleft on the oxidase with differing osmolyte accessibilities. (ii) diminution of maximal enzyme activity by high osmolarity: water-stressed states are less active than the fully hydrated state. (iii) modulation of the transitions from resting to pulsed states and from free to cyanide-ligated states by high osmolarity, indicating a role for hydration in conformational changes. In each type of process water may play a mechanistically distinct role.

The activation volume of a DNA helix-coil transition.

The role of hydration in the kinetics of a DNA helix-coil equilibrium is investigated by studying the effect of hydrostatic pressure on the rate constants describing the reaction. The kinetics were measured using the thermal hysteresis between the denaturation and renaturation curves of the triplex-forming oligonucleotides: 5'd[AAA-GGAGGAGAAGAAGAAAAAA] (sequence of purine strand) and 5'd[TTTCCTCCTCTTCTTCTTTTTT] (third strand). The kinetics at atmosphere pressure for this system have been recently reported [Rougée et al. (1992) Biochemistry 31, 9269-9278]. At all pressures the data are consistent with a single-step bimolecular reaction under the conditions of our experiments (100 mM NaCl, 10 mM cacodylate, pH 6.5). The rate of formation of the triplex from the duplex + single strand is accelerated by pressure. At the midpoint of the helix-coil transition (32.5 degrees C), the activation volume for helix formation, V*1, equals -11.8 (+/- 2.4) cm3 mol-1 at atmospheric pressure. At the same temperature, the activation volume for helix dissociation, V*-1, equals +39.9 (+/- 5.0) cm3 mol-1; that is, the rate of strand separation is slowed by pressure. These findings emphasize the importance of solvent interactions in the stabilization and formation of DNA helices. It is proposed that the activation volume of the forward reaction may arise from the volume change due to charging the cytosine residues and the formation of base-stacking interactions in the third strand. The positive activation volume of strand separation may be a consequence of poor solvent packing of the DNA duplex major groove during dissociation of the third strand.

Role of hydration and water structure in biological and colloidal interactions.

The conventional explanation of why hydrophilic surfaces and macromolecules remain well separated in water is that they experience a monotonically repulsive hydration force owing to structuring of water molecules at the surfaces. A consideration of recent experimental and theoretical results suggests an alternative interpretation in which hydration forces are either attractive or oscillatory, and where repulsions have a totally different origin. Further experiments are needed to distinguish between these possibilities.

Some problems in PTC theory: solvation, crystalline lattice strength, and topology

It was shown thatSN2-type reactions both in solid-liquid and liquid-liquid systems can proceed at the interface through the formation of cyclic adsorption complexes. The conditions for the formation of the latter were formulated, and the kinetic and thermodynamic parameters of reaction with solid ionophoric salts were estimated. A linear relation between the energy of the crystalline lattice of the salt and its melting point was estimated, the latter being shown to be useful as a simplified estimation of the reactivity of solid salts in reactions where the crystalline lattice is destroyed. In solid-liquid systems the “benzene” effect of solvents was discussed. In highly basic nitrogen-containing solvents (pyridine,etc.), a substitution reaction proceeds in the absence of a PT catalyst. The role of hydration in liquid-liquid systems was discussed.

Kinetic effects in protein crystals. I. The role of hydration in protein aggregation

Crystallizing proteins presents long-standing problems with sluggish kinetics and competing growth-limiting factors in solution. This work examines potential limiting factors arising from protein-water interactions and protein-protein aggregation. Since a hydrated shell shields bare protein contacts, multiple layers of highly viscous water impose restrictions on protein addition. If proteins attach to the crystal as aggregates, they are found to be particularly sensitive to misalignments and denaturation. The modelling extends to include the protein's shape as a fractal, the number of its binding sites and possible effects of flow-induced shear. It concludes that: (i) when protein-water interactions are taken into account convective shear approaches velocities of the order of magnitude that alter protein kinetics; (ii) protein aggregation puts narrow tolerances on correct molecular alignments; and (iii) a protein's characteristic 'stringy' shape as a fractal can favour aggregates compared with the monomer as a growth unit. A model for protein addition as an extensible, tethered molecule is proposed and examined against flow strengths and random thermal vibrations. Comparison of the protein model with experimental lysozyme results suggests that forced fluid flows as small as 100 mu m s-1 can disrupt protein attachment and thus detrimentally limit crystal growth on a microscopic scale.