Piezoelectric and related properties of hydrated collagen.

Two piezoelectric constants (polarization per unit stress, d=d'-id', and polarization per unit strain, e=e'-ie'), the elastic constant, and dielectric constant are determined for oriented collagen at different hydration levels at 10 Hz from -150 to 50 degrees C. With no hydration (approximately 0% H2O), d' increases slightly with higher temperatures, while e' decreases slightly. Near 11 wt% H2O, both d' and e' increase then decrease around 0 degrees C, and is probably caused by an increase of the dielectric constant and the ionic conductivity in the nonpiezoelectric phase. Hydration greater than 25 wt%, d' and e' decrease above -50 degrees C which is considered to be due to a greater ionic conductivity surrounding the piezoelectric phase.     

Electro-biorheology

Studies on piezoelectricity in biopolymers are reviewed with some examples. By measuring anisotropy of elastic and piezoelectric constants in fish skin, the direction of preferred orientation of collagen fibers is determined. Temperature and moisture dependence of real and imaginary parts of the piezoelectric constant observed for oriented film of fibrin gel indicates that temperature-time equivalence holds for the piezoelectric relaxation. The piezoelectric constant for oriented film of polyhydroxybutyrate decreases above glass transition temperature, which is related to the elastic and dielectric relaxations. When alpha-helical molecules of poly-gamma-methyl-L-glutamate are electrically oriented in ethylene dichloride solution and shear stress is given by ultrasound, the piezoelectric polarization is observed. When the film of poly-gamma-methyl-glutamate is wrapped around the femur of rat, the formation of new bone is produced.     

Temperature-dependent dielectric properties of slightly hydrated horn keratin

With an aim to reveal the mechanism of protein-water interaction in a predominantly two phase model protein system this study investigates the frequency and temperature dependence of dielectric constant epsilon' and loss factor epsilon' in cow horn keratin in the frequency range 30 Hz to 3 MHz and temperature range 30-200 degrees C at two levels of hydration. These two levels of hydration were achieved by exposing the sample to air at 50% relative humidity (RH) at ambient temperature and by evacuating the sample for 72 h at 105 degrees C. A low frequency dispersion (LFD) and an intermediate frequency alpha-dispersion were the two main dielectric responses observed in the air-dried sample. The LFD and the high frequency arm of the alpha-dispersion followed the same fractional power law of frequency. Within the framework of percolation cluster model these dispersions, respectively have been attributed to percolation of protons between and within the clusters of hydrogen-bonded water molecules bound to polar or ionizable protein components. The alpha-dispersion peak, which results from intra-cluster charge percolation conformed to Cole-Cole modified Debye equation. Temperature dependence of the dielectric constant in the air-dried sample exhibited peaks at 120 and 155 degrees C which have been identified as temperatures of onset of release of water bound to polar protein components in the amorphous and crystalline regions, respectively. An overall rise in the permittivity was observed above 175 degrees C, which has been identified as the onset of chain melting in the crystalline region of the protein.     

Effect of water on the molecular mobility of elastin

Purified and hydrated elastin is studied by both thermal and dielectric techniques to have insight into the chain dynamics of this protein. By differential scanning calorimetry, the glassy behavior of elastin is highlighted; the glass transition temperature (T(g)) of elastin is found to be widely dependent on hydration, falling from 200 degrees C in the dehydrated state to 30 degrees C for 30% hydration. A limit of T(g) at around 0 degrees C is found when crystallizable water is present in the system, that is, when the formation of ice prevents motions of some 10 nm along the polypeptidic chains. The technique of thermally stimulated currents, carried out in the -180 to 0 degrees C temperature range, is useful to detect localized motions. In this case, too, the localized motions vary considerably according to hydration: a first relaxation mode is observed at -145 degrees C and it is associated with the reorientation of crystallizable water in ice I; a second relaxation mode, more complex and cooperative, occurs at around -80 degrees C and could be attributed to the complex constituted by the dipolar groups of the polypeptidic chain and noncrystallizable water, behaving as a glassy system.     

Solid-state (13)C NMR reveals effects of temperature and hydration on elastin.

Elastin is the principal protein component of the elastic fiber in vertebrate tissue. The waters of hydration in the elastic fiber are believed to play a critical role in the structure and function of this largely hydrophobic, amorphous protein. (13)C CPMAS NMR spectra are acquired for elastin samples with different hydration levels. The spectral intensities in the aliphatic region undergo significant changes as 70% of the water in hydrated elastin is removed. In addition, dramatic differences in the CPMAS spectra of hydrated, lyophilized, and partially dehydrated elastin samples over a relatively small temperature range (-20 degrees C to 37 degrees C) are observed. Results from other experiments, including (13)C T(1) and (1)H T(1 rho) measurements, direct polarization with magic-angle spinning, and static CP of the hydrated and lyophilized elastin preparations, also support the model that there is significant mobility in fully hydrated elastin. Our results support models in which water plays an integral role in the structure and proper function of elastin in vertebrate tissue.     

Piezoelectric properties of poly-β-hydroxybutyrate and copolymers of β-hydroxybutyrate and β-hydroxyvalerate

The shear piezoelectricity was observed in oriented films of poly-β-hydroxybutyrate (PHB) and copolymers of β-hydroxybutyrate (HB) and β-hydroxyvalerate (HV). The piezoelectric stress constant 3₁₄ = e′₁₄ − ie″₁₄ (polarization/strain), the piezoelectric strain constant d₁₄ = d′₁₄ − id″₁₄ (polarization/stress), the elastic constant c = c′ + ic″ and the dielectric constant = ′ − i″ were determined at a frequency of 10 Hz over a temperature range from −150° to +150°C. Piezoelectric relaxations as well as elastic and dielectric relaxations were clearly observed at the glass transition temperature of about 15°C. In order to evaluate the piezoelectric constants (e₂ and d₂) for the piezoelectric phase which consists of the crystalline region and the oriented non-crystalline region, a spherical dispersion two phase model was utilized. Assuming the appropriate fixed values for the elastic and dielectric constants in the piezoelectric phase, d₂ and d₂ were calculated as a function of temperature. For a PHB and a copolymer (17 HV/83 HB), e₂ and d₂ showed relaxations, leading to a conclusion that the instantaneous piezoelectric constant in the crystalline phase is constant independent of temperature but the piezoelectric constant in the oriented non-crystalline phase is relaxational and has the opposite sign. For a copolymer (25 HV/75 HB) and a chloroform treated copolymer (17 HV/83 HB), e₂ and d₂ were constant independent of temperature, indicating that the oriented non-crystalline phase has disappeared owing to the increased molecular flexibility due to copolymerization or annealing in chloroform vapour.     

Stratum corneum hydration: phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes

The outermost layer of skin, stratum corneum (SC), functions as the major barrier to diffusion. SC has the architecture of dead keratin filled cells embedded in a lipid matrix. This work presents a detailed study of the hydration process in extracted SC lipids, isolated corneocytes and intact SC. Using isothermal sorption microcalorimetry and relaxation and wideline (1)H NMR, we study these systems at varying degrees of hydration/relative humidities (RH) at 25 degrees C. The basic findings are (i) there is a substantial swelling both of SC lipids, the corneocytes and the intact SC at high RH. At low RHs corneocytes take up more water than SC lipids do, while at high RHs swelling of SC lipids is more pronounced than that of corneocytes. (ii) Lipids in a fluid state are present in both extracted SC lipids and in the intact SC. (iii) The fraction of fluid lipids is lower at 1.4% water content than at 15% but remains virtually constant as the water content is further increased. (iv) Three exothermic phase transitions are detected in the SC lipids at RH=91-94%, and we speculate that the lipid re-organization is responsible for the hydration-induced variations in SC permeability. (v) The hydration causes swelling in the corneocytes, while it does not affect the mobility of solid components (keratin filaments).     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Supercooling capacity of seeds and seedlings in Arabidopsis thaliana.

The influence of the water content of seeds and seedlings of Arabidopsis thaliana (Ecotype Columbia:2) on their supercooling capacity was investigated. Equilibration of the seeds to various air relative humidities resulted in final moisture contents ranging from 8 to 82% (dry weight basis). No supercooling point could be detected when the water content remained below 32.5%, and in seeds at just above this moisture level ice crystals started to form at -26 degrees C. However, cooling partly affected the germination of seeds down to a water content of 26.5%. Upon imbibition, the supercooling point of the seeds remained around -21.6 degrees C and rose sharply to -14.7 degrees C when visible germination started. It remained around -13 degrees C during the following 96 h while the water content of the seedlings increased from 155 to 870%. Hydrated seeds (above 32.5% water content), germinated seeds, and seedlings of Arabidopsis cannot survive being frozen.     

Dielectric properties of developing rabbit brain at 37 degrees C

The dielectric properties of developing rabbit brain were measured at 37 degrees C between 10 MHz and 18 GHz using time domain and frequency domain systems. The results show a variation with age of the dielectric properties of brain. An analysis of the data suggests that the water dispersion in the brain of newly born animals can be represented by a Debye equation. This dispersion increases in complexity with age, and there is evidence of a smaller additional relaxation process centered around 1 GHz. It is concluded that the principal contribution to this subsidiary dispersion region arises from water of hydration.     

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).     

Effects of small neutral osmolytes on the supercoiling free energy and intrinsic twist of p30delta DNA

Both theory and experiments are employed to investigate the effects of small neutral osmolytes on the average intrinsic twist (l0), the torsion and bending elastic constants, and the twist energy parameter (ET) that governs the supercoiling free energy. The experimental data for ethylene glycol and acetamide at 37 degrees C suggest, and are interpreted in terms of, a model wherein the DNA exhibits an equilibrium between two distinct conformational states that possess different numbers of bound water molecules and exhibit different intrinsic twists and torsion and bending elastic constants. Expressions are derived to relate the effective ET and l0 to the equilibrium constant, water activity (aw), and number (n) of bound water molecules released per cooperative domain undergoing the two-state transition. The variations of l0 and ET with -ln(aw) are similar for acetamide and ethylene glycol at 37 degrees C. Fitting the theory to those data yields the range n = 103-125 for ethylene glycol and n = 71-113 for acetamide, depending on the assumed value of ET for the dehydrated state. The cooperative domain size of the two-state transition is estimated to exceed about 25-30 base pairs (bp). Between 0 and 19.4 w/v % ethylene glycol, the torsion elastic constant, measured by time-resolved fluorescence polarization anisotropy (FPA), increases by 1.37-fold, whereas the measured ET decreases by 1.15-fold over that same range. The implied decrease in bending rigidity over that range is by a factor of about 0.7. The variations of l0 and ET with increasing -ln(aw) due to added ethylene glycol at 37 degrees C are far smaller than the corresponding variations observed previously at 14 and 15 degrees C. However, at 21 degrees C, upon adding either ethylene glycol or acetamide, l0 and ET initially decline steeply with increasing -ln(aw), with slopes possibly comparable to those seen at 14 and 15 degrees C, but then flatten out and follow curves similar to those at 37 degrees C. Possible origins of such mixed behavior are discussed. The effects of betaine at both 37 and 21 degrees C differ qualitatively and quantitatively in various respects from those of ethylene glycol and acetamide. Upon adding sucrose, l0 initially jumps to higher plateaus at both 37 and 21 degrees C, but its effects on ET cannot be reliably assessed, due to the limited range of -ln(aw).     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. IV. Enthalpies of hydration of 5-alkyluracils

Enthalpies of sublimation, DeltaH degrees (subl) and of solution in water, DeltaH degrees (sol) were determined for a series of crystalline 1,3-dimethyl-uracil derivatives substituted at the C5-ring carbon atom with alkyl groups (-C(n)H(2n+1), n = 2-4) and some of their C(5.6)-cyclooligomethylene analogues (-(CH2)(n)-, n = 3-5). From these data. enthalpies of hydration DeltaH degrees (hydr)= DeltaH degrees (sol) - DeltaH degrees (subl) were calculated and corrected for energies of cavity formation in pure liquid water in order to obtain enthalpies of interaction, DeltaH degrees (int) of the solutes with their hydration shells. The latter are discussed together with the recalculated DeltaH degrees (int) for variously methylated uracils, obtained previously according to a simplified correction procedure, in terms of perturbations in the energy and scheme of hydration of the diketopyrimidine ring brought about by alkyl substitution. It was found that each -CH2-group added with an alkyl substitution contributes favorably about -20 kJ mol(-1) toDeltaH degrees (int).This contribution is partially cancelled by the unfavorable contribution to DeltaH degrees (int) connected with removal of some water molecules bound in the first and subsequent hydration layers by an alkyl substituent. This is particularly evident on substitution at the polar side of the diketopyrimidine ring on which water molecules are expected to be bound specifically.     

IR spectroscopic studies of major cellular components. III. Hydration of protein,nucleic acid,and phospholipid films

The IR absorption spectra of protein, DNA, RNA, and phospholipid films as a function of the water content are reported. We find that the hydration of protein films affects the peak intensity of amide I and amide II bands and the shape of the amide III band. For nucleic acids, the symmetric (nu(S) PO(2) (-)) and antisymmetric (nu(AS) PO(2) (-)) stretching vibrations of the phosphate linkage are the most affected by hydration, because both intensity changes and frequency shifts are observed. The spectra of phospholipid films are also sensitive to hydration, and they exhibit changes in the peak intensities and frequencies of both nu(S) PO(2) (-) and nu(AS) PO(2) (-) vibrations. We interpret the spectral differences between water saturated and dried films both in terms of structural changes and the change in the local dielectric in the vicinity of the polar and solvent exposed groups. In addition, we observe that the most significant change in the absorption intensity, frequency, and shape of the water sensitive vibrations occurs at high hydration levels. The principal component analysis of hydration results and the kinetics of water removal from sample films are also discussed. In addition, protein spectra acquired using film and KBr pellet sampling techniques are compared.     

Thermodynamic, thermomechanical, and structural properties of a hydrated asymmetric phosphatidylcholine.

1-Behenyl-2-lauryl-sn-glycero-3-phosphocholine (22/12 PC) belongs to a unique group of phospholipids in which the molecule has one acyl chain almost twice as long as the other. The temperature-composition phase diagram for this lipid in the range of 25-65 degrees C, and 0 to 84.3% (w/w) water has been constructed by using the isoplethal method in the heating direction and x-ray diffraction for phase identification and structure characterization. At water contents between 10.3 and 34% (w/w) and at temperatures below 43 degrees C, a single mixed interdigitated lamellar gel phase (Lm beta, [symbol: see text]) of the type described by Hui et al. (1984. Biochemistry. 23:5570-5577) and McIntosh et al. (1984. Biochemistry. 23:4038-4044) was found. A second phase consisting of bulk aqueous solution coexists with the Lm beta phase at hydration levels above 34% (w/w) water in the temperature range between 25 and 43 degrees C. Above 43 degrees C, a partially interdigitated lamellar liquid crystalline (Lp alpha) phase ([symbol: see text]) is seen in the water concentration range extending from 0 to 84.3% (w/w). The pure Lp alpha phase is found below 43% (w/w) water, while coexistence of the Lp alpha phase and the bulk aqueous solution is observed above this water concentration which marks the hydration boundary. Interestingly, the latter boundary for both Lm beta and Lp alpha phases is nearly vertical in the temperature range studied. Furthermore, the lamellar chain-melting transition temperature appears to be relatively insensitive to hydration in the range 0-85% (w/w) water. We have confirmed the identify of the Lm beta phase by constructing a 5.7-A resolution electron density profile on oriented samples by the swelling method. Temperature-induced chain melting effects an increase in lipid bilayer thickness suggesting that the Lp alpha phase has chains packed in the partially as opposed to the mixed interdigitated configuration. Unlike the symmetric phosphatidylcholines a ripple (P beta') phase was not found as an intermediate between the low and high temperature lamellar phases of 22/12 PC. The specific volume of 22/12 PC is 940 (+/- 1) microliter/g and 946 (+/- 1) microliter/g in the hydrated lamellar gel state at 28 (+/- 2) and 40 (+/- 2) degrees C, respectively, from neutral buoyancy experiments. Based on measurements of the temperature dependence of the various lattice parameters of the different phases encountered in this study the corresponding lattice thermal expansion coefficients have been measured. These are discussed and their dependence on lipid hydration is reported.     

Stratum corneum hydration: Phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes

The outermost layer of skin, stratum corneum (SC), functions as the major barrier to diffusion. SC has the architecture of dead keratin filled cells embedded in a lipid matrix. This work presents a detailed study of the hydration process in extracted SC lipids, isolated corneocytes and intact SC. Using isothermal sorption microcalorimetry and relaxation and wideline ¹H NMR, we study these systems at varying degrees of hydration/relative humidities (RH) at 25 °C. The basic findings are (i) there is a substantial swelling both of SC lipids, the corneocytes and the intact SC at high RH. At low RHs corneocytes take up more water than SC lipids do, while at high RHs swelling of SC lipids is more pronounced than that of corneocytes. (ii) Lipids in a fluid state are present in both extracted SC lipids and in the intact SC. (iii) The fraction of fluid lipids is lower at 1.4% water content than at 15% but remains virtually constant as the water content is further increased. (iv) Three exothermic phase transitions are detected in the SC lipids at RH = 91-94%, and we speculate that the lipid re-organization is responsible for the hydration-induced variations in SC permeability. (v) The hydration causes swelling in the corneocytes, while it does not affect the mobility of solid components (keratin filaments).     

Potentiometric, spectrophotometric and calorimetric study on iron(III) and copper(II) complexes with 1,2-dimethyl-3-hydroxy-4-pyridinone

The iron(III)-1,2-dimethyl-3-hydroxy-4-pyridinone (Deferiprone) system is carefully characterized by a combined potentiometric-spectrophotometric procedure at 25 and 37 degrees C at different ionic strengths, and by thermochemical and quantum-chemical studies. The main purpose of this work was to determine how the temperature dependence of both complex-formation and protonation constants can affect the pFe values on going from 25 degrees C (pFe is normally calculated using 25 degrees C stability constants) to the physiological temperature of 37 degrees C at which chelating agents are active in vivo. The copper(II)-Deferiprone system is also studied and the iron(III)-Deferiprone distribution diagrams in presence of variable copper(II) amounts are shown so as to explain possible side effects due to a competing metal ion during the chelating therapy of iron overload.     

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.     

Protein--water interactions. Heat capacity of the lysozyme--water system.

Calorimetric measurements of the heat capacity of the lysozyme-water system have been carried out over the full range of system composition at 25 degrees C. The partial specific heat capacity of the protein in dilute solution is 1.483 +/- 0.009 J K-1 g-1. The heat capacity of the dry protein is 1.26 +/- 0.01 J K-1 g-1. The system heat capacity responds linearly to change in composition from dilute solution to 0.38 g of water per g of protein (h) and is an irregular function at lower water content. The break in the heat capacity function at 0.38 h defines the amount of water needed to develop the equilibrium solution properties of lysozyme as being 300 molecules of water/protein molecule, just sufficient for monolayer coverage. The heat capacity behavior at low water content describes three hydration regions. The most tightly bound water (0-0.07 h), probably principally bound to charged groups, is characterized by a partial specific heat capacity of 2.3 J K-1 g-1, a value close to that for ice. A heat of reaction associated with proton redistribution is reflected in the heat capacity function for the low-hydration region. Between 0.07 and 0.25 h the heat capacity increases strongly, which is understood to reflect the growth of patches of water covering polar and adjacent nonpolar portions of the protein surface. The hydration shell is completed by condensation of solvent over the weak-interacting portions of the surface, in a process displaying a transition heat.     

The kinetics of interactions of bilirubin with lipid bilayers and with serum albumin.

Rate constants for the hydration of bilirubin bound to unilamellar bilayers of dioleoylphosphatidylcholine and albumin were measured by stopped-flow methods. Rate constants for association of bilirubin with these vesicles and albumin were calculated from measured rate constants for dissociation and the equilibrium binding constants of bilirubin and lipids or albumin. Rate constants for hydration (dissociation) for bilirubin bound to dioleoylphosphatidylcholine and albumin were 71 s-1 and 1.8 s-1 respectively. Rate constants for association were 4.0 10(7) s-1 and 1.1 10(9) M-1 s-1, respectively. Both rates for interactions of bilirubin with bilayers were essentially independent of temperature in the range 0-40 degrees C, indicating that barriers to entry and exit of bilirubin from bilayers were entropic. Rates of transbilayer movement of bilirubin in dioleoylphosphatidylcholine were too fast to resolve by measuring rates of hydration of bilirubin. Rate constants for hydration of bilirubin bound to bilayers with less avidity for bilirubin as compared with dioleoylphosphatidylcholine also were too fast to measure with stopped-flow methods. In addition to providing details of the energetic basis for interactions between bilirubin and membranes, the data allow for calculating the maximal rates at which bilirubin could transfer spontaneously from sites on albumin in blood to the interior of cells. The data show, in this regard, that this rate is 10-50 fold faster than measured rates of uptake of bilirubin by intact liver.