An NMR method to characterize multiple water compartments on mammalian collagen

A molecular model is proposed to explain water 1H NMR spin-lattice relaxation at different levels of hydration (NMR titration method) on collagen. A fast proton exchange model is used to identify and characterize protein hydration compartments at three distinct Gibbs free energy levels. The NMR titration method reveals a spectrum of water motions with three well-separated peaks in addition to bulk water that can be uniquely characterized by sequential dehydration. Categorical changes in water motion occur at critical hydration levels h (g water/g collagen) defined by integral multiples N = 1, 4 and 24 times the fundamental hydration value of one water bridge per every three amino acid residues as originally proposed by Ramachandran in 1968. Changes occur at (1) the Ramachandran single water bridge between a positive amide and negative carbonyl group at h1 = 0.0658 g/g, (2) the Berendsen single water chain per cleft at h2 = 0.264 g/g, and (3) full monolayer coverage with six water chains per cleft level at h3 = 1.584 g/g. The NMR titration method is verified by comparison of measured NMR relaxation compartments with molecular hydration compartments predicted from models of collagen structure. NMR titration studies of globular proteins using the hydration model may provide unique insight into the critical contributions of hydration to protein folding.     

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 line-width, 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 deoxygenation 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 are affected 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 degrees C to 500 Hz at -36 degrees 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 degrees C for measurements at 44.4 MHz and -50 degrees 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 irrotationally bound water, is altered during the sickling process.     

Characterization of water in unfertilized and fertilized sea urchin eggs

The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 X 10(-12) sec (bulk water), 1 X 10(-10) sec (structured water) and about 2 X 10(-9) sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane.     

On the osmotically unresponsive water compartment in cells

Differences in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) between water in living cells and pure bulk water were investigated by re-evaluating reports of the osmotic behavior of mammalian cells. In five different animal cells, osmotically unresponsive water (OUW) values ranged from 1.1 to 2.2 g per g dry mass. Detailed analysis of human red blood cell (RBC) data indicates a major role for hemoglobin OUW-values, aggregation and packing in cell volume regulation that can be explained for the first time in relevant molecular terms.     

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.     

A study on the reaction of human erythrocytes with hydrogen peroxide

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.     

Extent and properties of nonbulk "bound" water in crystalline lens cells

Crystalline lenses provided good material to study and measure the properties of cellular water. Different methods were used to establish the extent and properties of nonbulk water in mammalian lenses. These methods include: NMR titration analysis, a test of the osmotic properties, a test of dye exclusion In lenses with intact cell membranes and in lenses with disrupted cell membranes, and the water-holding capacity of lenses subjected to 40,000 x g for 1 hour with intact cell membranes and in lenses with disrupted cell membranes. The data from these methods, as well as other data from the literature, lead to the conclusion that most, if not all, of the water in lens cells (up to 2.2 g water/g dry mass) has motional and osmotic properties that distinguish it from bulk water. These findings call into question the common and convenient assumption that all but a small proportion of cellular water is like that in dilute solution.     

Oxygen-17 and deuterium nuclear magnetic relaxation studies of lysozyme hydration in solution: field dispersion, concentration, pH/pD, and protein activity dependences

A comparison of 17O and 2H NMR relaxation rates of water in lysozyme solutions as a function of concentration, pH/pD, and magnetic field suggests that only 17O monitors directly the hydration of lysozyme in solution. NMR measurements are for the first time extended to 11.75 T. Lysozyme hydration data are analyzed in terms of an anisotropic, dual-motion model with fast exchange of water between the "bound" and "free" states. The analysis yields 180 mol "bound" water/mol lysozyme and two correlation times of 7.4 ns ("slow") and 29 ps ("fast") for the bound water population at 27 degrees C and pH 5.1, in the absence of salt, assuming anisotropic motions of water with an order parameter value for bound water of 0.12. Under these conditions, the value of the slow correlation time of bound water (7.4 ns) is consistent with the value of 8 ns obtained by frequency-domain fluorescence techniques for the correlation time associated with the lysozyme tumbling motion in solutions without salt. In the presence of 0.1 M NaCl the hydration number increases to 290 mol/mol lysozyme at pD 4.5 and 21 degrees C. The associated correlation times at 21 degrees C in the presence of 0.1 M NaCl are 4.7 ns and 15.5 ps, respectively. The value of the slow correlation time of 4.7 ns is consistent with the calculated value (4.9 ns) for the lysozyme monomer tumbling in solution. The systematic deviations of the relaxation rates, estimated with the single-exponential approximation, from the theoretical, multiexponential nuclear (I' + 1/2) spin relaxation are evaluated at various frequencies for 17O (I = 5/2) with the first-order, linear approximation (25). All NMR relaxation data for hydrated lysozymes are affected by protein activity and are sensitive both to the ionization of protein side chains and to the state of protein aggregation.     

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.

     

Role of hydration of polyhydroxy compounds in biological systems.

The spin-lattice relaxation times, T1, of H2(17)O have been measured for aqueous solutions of 9 polyols as a function of the concentration at 25 degrees C. The dynamic hydration number, nDHN, for polyols were obtained. The values of nDHN increased with an increase in the number of OH groups and depended on the conformation of isomers. The value of nDHN for inositol was the largest. This means that the thermal motion of water molecules around inositol is most inhibited among the polyols studied. The increment of thermal stability of proteins by polyols and sugars, the equilibrium distribution coefficients of polyols and sugars in the denatured hemoglobin solution, and the effect of sugars on the osmotic flow of water were expressed by linear relation of nDHN. On the bases of these relations, a systematic treatment for the effect of hydration of polyols and sugars on these phenomena is discussed.     

Maintenance and mobility of hemoglobin and water within the human erythrocyte after detergent disruption of the plasma membrane.

Is an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water-holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D₂O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D₂O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin-sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij-permeabilized erythrocytes washed in D₂O saline or in a D₂O K buffer maintained a higher heavy water-holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij-treated and washed erythrocyte “shells” revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water-holding capacity of the erythrocyte can be accounted for by the water-holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the submembrane lamina.     

Augmented water binding and low cellular water content in erythrocytes of camel and camelids.

We investigated a link between hemoglobin primary structure, hemoglobin hydrophobicity-hydrophilicity, and erythrocyte water content in various mammalian species. Some hemoglobin molecules, particularly those of the camel and camelids, contain more charged amino acid residues and are more hydrophilic than the hemoglobins of human and a number of other mammalian species. To test the in vivo significance of these alterations of hemoglobin primary structure, we determined the osmotically unresponsive erythrocyte water fractions in mannit solutions of various osmolarities at 4 degreesC. Among the species investigated, the size of the osmotically unresponsive erythrocyte water fraction relates in a positive linear way to hemoglobin hydrophilicity. The extreme low total erythrocyte water content of camel erythrocytes (1.1-1.3 g water/g dry mass) may be explained by a comparatively high osmotically unresponsive erythrocyte water fraction. It is proposed that alterations of hemoglobin sequences of camel and camelids may be the part of a natural selection process aimed at protecting these animals against osmotic dehydration in arid environments.     

Verification of simple hydration/dehydration methods to characterize multiple water compartments on tendon type 1 collagen

A molecular model of collagen hydration is used to validate centrifugal dehydration force (CDF) and re-hydration isotherm (RHI) methods to measure and characterize hydration compartments on bovine tendon. The CDF method assesses fluid flow rate from flexor and extensor tendons expressed in (g-water/g-dry mass-minute) and hydration capacity of compartments in (g-water/g-dry mass). Measured water compartment capacities agree with the molecular model of collagen hydration [Fullerton GD, Rahal A. Collagen structure: the molecular source of tendon magic angle effect. J Mag Reson Imag 2007;25:345-361; Fullerton GD, Amurao MR. Evidence that collagen and tendon have monolayer water coverage in the native state. Cell Biol Int 2006;30(1):56-65]. Native tendon hydration has monolayer coverage on collagen h(m)=1.6 g/g which divides into primary hydration on polar surfaces h(pp)=0.8 g/g and secondary hydration h(s)=0.8 g/g bridging over hydrophobic surfaces. Primary hydration is hydrogen bonded to collagen polar side chains h(psc)=0.54 g/g with small free energy or to the protein main chain hydration h(pmc)=0.26 g/g with greater free energy of binding. The CDF method replaces the more time consuming water proton NMR spin-lattice dehydration (NMR titration) method, confirms the presence of three non-bulk water compartments on collagen (h(pmc)=0.26 g/g, h(pp)=0.8 g/g and h(m)=1.6 g/g). This CDF method provides the most reproducible experimental measure of total tissue non-bulk water (TNBW). The re-hydration isotherm method, on the other hand, provides the most accurate measure of the Ramachandran water-bridge capacity h(Ra)=0.0656 g/g. The only equipment needed are: microfilterfuge tubes, a microcentrifuge capable of 14,000 x g or 4MPa, a vacuum drying oven, an accurate balance and curve fitting ability. The newly validated methods should be useful for characterizing multiple water compartments in biological and non-biological materials by allowing direct measurement of water compartment changes induced by pH, co-solute salt, glycation and protein cross-linking.     

Organic phosphate binding to hemoglobin in intact human erythrocytes determined by 31P nuclear magnetic resonance spectroscopy.

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to estimate the percent of 2,3-diphosphoglycerate and ATP bound to hemoglobin in intact human erythrocytes at 37 degrees C. Binding was assessed by comparing the chemical shifts (delta) of 2,3-diphosphoglycerate and of ATP observed in intact cells with the delta values of these organic phosphates determined in model solutions closely simulating intracellular conditions, in which percent binding was directly evaluated by membrane ultrafiltration. The results showed that the percent of bound 2,3-diphosphoglycerate in intact cells varied with pH, the state of oxygenation, and 2,3-diphosphoglycerate concentration. The values ranged from 33% in cells incubated with glucose in air at an intracellular pH of 7.2 to 100% in cells incubated with inosine in N2 at a pH of 6.75. At the same 2,3-diphosphoglycerate concentration, a greater percentage of the compound appeared to be bound in erythrocytes than in the closely simulated model system. ATP was not significantly bound to hemoglobin under any condition examined, but appeared to be strongly complexed to Mg2+ inside the erythrocyte. The binding percentages for both 2,3-diphosphoglycerate and ATP in intact cells estimated by 31P NMR spectroscopy were lower than those calculated by others from individual association constants determined for the binding of different ligands to hemoglobin.     

Phosphorus-31 relaxation times of 2,3-diphosphoglycerate in intact human erythrocytes

The reciprocals of the spin-lattice relaxation times (T₁s) of the 2-P and 3-P nuclei of 2,3-diphosphoglycerate (DPG) increased linearly as percent DPG bound was raised in model hemoglobin solutions. The 2-P T₁ was slightly greater in intact erythrocytes than in model solutions under similar experimental conditions. The change in the 3-P T₁ with cellular deoxygenation was anomalous indicating that this nucleus should not be used to estimate DPG binding inside intact erythrocytes.     

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of acatalasemic erythrocytes treated with low and high dose hydrogen peroxide. Hemolysis and aggregation

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.     

Water in barnacle muscle. IV. Factors contributing to reduced self-diffusion.

The relative self-diffusion coefficients D/Do, of water in various solutions, in fresh barnacle muscle fibers, and in membrane-damaged fibers equilibrated with several media have been estimated from NMR relaxation rates in the presence of applied field gradients. A model has been developed to account for the contributions to the observed reduction in D/Do from small organic solutes, and from the hydration and obstruction effect of both soluble macromolecules and myofilament proteins. Intracellular ions do not affect D/Do, but all tested organic solutes do. Solute effects are additive. When artificially combined in the proportions found in barnacle muscle ultracentrifugate (measured D/Do = 0.77), organic acids, small nitrogenous solutes, and proteins give D/Do = 0.77. After correcting the D/Do measured in fibers for this value, we calculate the myofilament hydration, Hm, in fresh muscle to be 0.65 g H2O/g macromolecule. Only in membrane-damaged fibers, highly swollen by salt-rich media, was this significantly increased. Because our earlier NMR relaxation measurements indicate only 0.07 g H2O bound/g myofilament protein, we conclude that the "hydration" water measured by reduction of D/Do cannot be described by stationary layers of water molecules; instead, we propose that nonpolar groups on the proteins cause extensive, hydrophobically-induced interactions among a large fraction of solvent molecules, slowing their translational motion.     

Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites.

We used static and dynamic light scattering for comparing the mass (MW) and hydrodynamic radius (R(h)) of several hemoglobin systems, namely human hemoglobin, bovine hemoglobin, human hemoglobin cross-linked with a sebacyl residue, and bovine hemoglobin cross-linked with an adipoyl residue. We measured the MW and R(h) of these systems in 0.1M phosphate buffer at pH 7.0 in the absence and in the presence of either betaine or glycerol up to 1.7 molal concentrations. The 90 degrees scattering was measured with a photon counting machine equipped with a diode laser at 783 nm. The Rayleigh ratio [R(theta)] of the instrument was estimated using R(theta) = 7.19E-6 cm(-1) for toluene at 783 nm. The refractive index increment of hemoglobin solutions was measured using a laser beam at 750 nm. We estimated a value dn/dc = 0.210 cm3/g in the absence and dn/dc = 0.170 in the presence of 1.7 molal osmolites. For all systems both in liganded and unliganded form, the static light scattering data showed a 16% mass increase with increasing concentration of osmolites. The hydrodynamic radii of all investigated systems in the presence and absence of osmolites were close to 3.17 nm. Assuming a partial specific volume nu = 0.739 for hemoglobin, and using spherical geometry, the estimated average hydration volume of hemoglobin was 32.6 L/mole in the absence of osmolites. It decreased to 23.5 L/mole in the presence of 1.7 molal osmolites. Assuming that the density of water in the hydration volume is D = 1.0 g/cm3, the hydration of Hb was 0.51 gH2O/gHb, with a surface density of 0.20 molH2O/A2. The hydration decreased to 0.33 gH2O/gHb and 0.14 molH2O/A2 in the presence of 1.7 molal osmolites. The decreased hydration was compensated by the increased mass (i.e., decreased surface area per unit volume) so that the thickness of the water shell around these proteins remained close to a single layer of water molecules. These findings indicate that the combination of static and dynamic light scattering offer unique means for investigating the relevance of water activity on the structure and function of biological macromolecules. In the case of hemoglobin, the data suggest that the decreased oxygen affinity in the presence of osmolites reported by Colombo et al. (M. F. Colombo, D. C. Rau, and V. A. Parsegian Science, 1992, Vol. 256, pp. 655-659), as due to ligand linked water binding on hemoglobin surface, is part of a complex phenomenon involving the hydration shell of hemoglobin and the formation of low affinity supertetrameric molecules.     

Freezing stresses and hydration of isolated cell walls

The hydration of the cell walls of the giant alga Chara australis was measured as a function of temperature using quantitative deuterium nuclear magnetic resonance (NMR) of samples hydrated with D2O. At temperatures 23-5K below freezing, the hydration ratio (the ratio of mass of unfrozen water in microscopic phases in the cell wall to the dry mass) increases slowly with increasing temperature from about 0.2 to 0.4. It then rises rapidly with temperature in the few Kelvin below the freezing temperature. The linewidth of the NMR signal varies approximately linearly with the reciprocal of the hydration ratio, and with the freezing point depression or water potential. These empirical relations may be useful in estimating cell wall water contents in heterogeneous samples.