The hydration response of poly (L-lysine) dynamics measured by 13C-NMR spectroscopy.

13C-nmr measurements are reported for samples of poly (L-lysine) both static and spinning at the magic angle in the beta-sheet form as a function of water content. The addition of water decreases the side-chain line widths considerably. Measurements of the cross-polarization time constants indicate that hydration by either H2O or D2O increases the time constant. Measurements of spin-lattice relaxation times in the laboratory frame and the rotating frame indicate that hydration does not change the dynamics of the backbone carbon atoms in the beta-sheet structure appreciably, but the side-chain atoms experience considerable increase in local mobility with increasing hydration. Deuteration of the exchangeable protons or the water has only small effects on the carbon relaxation times, indicating that relaxation is driven by intramolecular dipole-dipole interactions.     

Hydration effects on dynamics of polyglycine and sodium poly(L-glutamate).

Solid state nmr methods were applied to the study of the motions and structural heterogeneity in polyglycine, sodium poly(L-glutamate), and poly(L-alanine). The response of both the main-chain and side-chain resonances to the addition of water was studied using static and magic-angle sample-spinning line shapes as well as the carbon spin-lattice relaxation times, the proton spin-lattice relaxation time in the rotating frame, and the proton-carbon cross-polarization time. The polyglycine motions are not drastically affected by the addition of water when the polymer is in the 3(1)-helix or the beta-sheet structure. The sodium poly(L-glutamate), however, responds to increased hydration with little motion in the main-chain carbon atoms, but considerable flexibility of the side-chain atoms. The greatest motions are reported for the C5 carbon with rotational amplitudes about the C4-C5 bond of about of 50 degrees. In addition, motions somewhat less than half this size are required closer to the main chain.     

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.     

Pulsed NMR studies of water in striated muscle non-freezing factor of water II. Spin-lattice relaxation times and the dynamics of the non-freezing fraction of water

Spin-lattice relaxation times for the water protons in frog gastrocnemius muscle are reported over the temperature range 193 to 283 °K at Larmor frequencies of 30 and 60 MHz. Results of measurements under similar conditions of the transverse relaxation times are also reported. The relaxation times of the non-freezing 20% of the muscle water are interpreted in terms of water molecules, absorbed on or interacting with the proteins, and which are undergoing anisotropic motion, probably with a distribution of correlation times. Proton spin-lattice relaxation times are also reported for muscles under tension, the tension being produced by loading of the muscles with varying weights.     

Relaxation of water protons in highly concentrated aqueous protein systems studied by 1H NMR spectroscopy

Concentrated Aqueous Protein Systems, Proton Relaxation Times, Slow Chemical Exchange In this paper we present proton spin-lattice (T1) and spin-spin (T2) relaxation times measured vs. concentration, temperature, pulse interval (tauCPMG) as well as 1H NMR spectral measurements in a wide range of concentrations of bovine serum albumin (BSA) solutions. The anomalous relaxation behaviour of the water protons, similar to that observed in mammalian lenses, was found in the two most concentrated solutions (44% and 46%). The functional dependence of the spin-spin relaxation time vs. tauCPMG pulse interval and the values of the motional activation parameters obtained from the temperature dependencies of spin-lattice relaxation times suggest that the water molecule mobility is reduced in these systems. The slow exchange process on the T2 time scale is proposed to explain the obtained data. The proton spectral measurements support the hypothesis of a slow exchange mechanism in the highest concentrated solutions. From the analysis of the shape of the proton spectra the mean exchange times between bound and bulk water proton groups (tauex) have been estimated for the range of the highest concentrations (30%-46%). The obtained values are of the order of milliseconds assuring that the slow exchange condition is fulfilled in the most concentrated samples.     

Low-frequency Raman spectra of lysozyme crystals and oriented DNA films: dynamics of crystal water.

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.     

Boat conformations. Analysis and simulation of the complex 1H NMR spectrum of methyl 2,6:3,4-dianhydro-alpha-D-altropyranoside

The complex 1H NMR spectrum of methyl 2,6:3,4-dianhydro-alpha-D-altropyranoside (1) has been analyzed and simulated in detail by using input parameters derived from experimental 1H chemical shifts, long- and short-range coupling constants, spin-lattice relaxation times, and effective, spin-spin relaxation times obtained by trial and error matching of the experimental and simulated spectra. The 13C spin-lattice relaxation times of 1 have also been measured, and along with the 1H-1H long- and short-range coupling constants, have been interpreted in terms of the geometry of 1 defined by molecular dynamics with simulated annealing.     

A 13C-NMR study on the conformational and dynamical properties of a cereal seed storage protein,C-hordein,and its model peptides

We have recorded the ¹³C CP-MAS and DD-MAS nmr spectra of dry and hydrated barley storage protein, C-hordein, as a model for wheat S-poor prolamins, together with those of model synthetic peptides (Pro)₂(Gln)₆(I) and (Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln)₃(II) under dry or hydrated conditions. The spectral features of C-hordein as well as these peptides were appreciably different from each other depending on the extent of hydration, reflecting different domains that adopt different types of conformations as well as dynamics. In particular, considerable proportions of the peak intensities were lost in the CP-MAS spectra, and well-resolved ¹³C-nmr signals emerged in DD-MAS nmr spectra owing to acquisition of molecular motions by swelling. It was shown that local β-turn or (Pro)_n type II conformation is more preferable for individual Pro residues and β-sheet type conformation is dominant for individual Gln residues in the dry and hydrated systems. In addition, two types of Gln environments are originated in C-hordein that differ in their mobility. Further, ¹³C spin-lattice relaxation times (T₁'s) of C- hordein and peptide II were reduced by more than one order of magnitude by hydration, reflecting the presence of well-swollen molecular chains. In contrast, theT₁ values of peptide I upon hydration remained one third of those in the dry state. Carbon-resolved proton spin-lattice relaxation times in the rotating frame (T_1ρ's) were also decreased by about 50% upon hydration, although these parameters were less sensitive as compared to T₁ values. In addition, the ¹³C-nmr signals of the aromatic side chain of Phe residues disappeared on hydration owing to interference between the frequency of the acquired flip-flop motion and the proton decoupling frequency. This information gives a new insight into establishing the structural properties of the studied protein system. A model may be put forward for a gel-type structure in which the more rigid part of the system involves intermolecular hydrogen-bonded Gln side chains as well as some hydrophobic “pockets” involving Pro and Phe residues. The liquid-like domain is characterized by considerable backbone and side-chain motion as well as rapid ring-puckering motion in Pro residues. © 1997 John Wiley & Sons, Inc.     

Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance

Natural-abundance 13C NMR signals from glycogen are observable in situ within the perfused livers of rats. The nuclear magnetic relaxation properties (T1, T2, eta + 1) of glycogen were measured for glycogen in situ and in vitro and were found to be identical. All of the carbon nuclei in glycogen contribute to the high-resolution NMR spectrum, in spite of glycogen's very large molecular weight. The metabolism of glycogen in situ in the perfused rat liver was followed by 13C NMR. Stimulation of the fed rat liver by physiological glucagon levels led to rapid glycogenolysis. Perfusion of the liver with [1-13C]glucose led to net glycolysis, with concomitant scrambling of the label from C1 to C6 due to triosephosphate isomerase activity.     

H and C NMR studies on the temperature-dependent water and protein dynamics in hydrated elastin,myoglobin and collagen

²H NMR spin-lattice relaxation and line-shape analyses are performed to study the temperature-dependent dynamics of water in the hydration shells of myoglobin, elastin, and collagen. The results show that the dynamical behaviors of the hydration waters are similar for these proteins when using comparable hydration levels of h = 0.25–0.43. Since water dynamics is characterized by strongly nonexponential correlation functions, we use a Cole–Cole spectral density for spin-lattice relaxation analysis, leading to correlation times, which are in nice agreement with results for the main dielectric relaxation process observed for various proteins in the literature. The temperature dependence can roughly be described by an Arrhenius law, with the possibility of a weak crossover in the vicinity of 220 K. Near ambient temperatures, the results substantially depend on the exact shape of the spectral density so that deviations from an Arrhenius behavior cannot be excluded in the high-temperature regime. However, for the studied proteins, the data give no evidence for the existence of a sharp fragile-to-strong transition reported for lysozyme at about 220 K. Line-shape analysis reveals that the mechanism for the rotational motion of hydration waters changes in the vicinity of 220 K. For myoglobin, we observe an isotropic motion at high temperatures and an anisotropic large-amplitude motion at low temperatures. Both mechanisms coexist in the vicinity of 220 K. ¹³C CP MAS spectra show that hydration results in enhanced elastin dynamics at ambient temperatures, where the enhancement varies among different amino acids. Upon cooling, the enhanced mobility decreases. Comparison of ²H and ¹³C NMR data reveals that the observed protein dynamics is slower than the water dynamics.     

Conformation of a heptadecapeptide comprising the segment encephalitogenic in rhesus monkey

The 17-residue peptide FKLGGRDSRSGSPMARR derived from myelin basic protein, containing an epitope encephalitogenic in rhesus monkey, has been studied in aqueous solution by high-resolution one- and two-dimensional carbon and proton nuclear magnetic resonance spectroscopy. The resonances of the spectra from both nuclei were assigned with the aid of two-dimensional correlated spectroscopy, pH and solvent titrations, and one-dimensional spin-decoupling techniques and by comparison of the spectra of the heptadecapeptide with those of a phosphorylated form of the peptide, the pentadecapeptide FKLGGRDSRSGSPMA, and the nonapeptide FKLGGRDSR. Amide proton temperature coefficients, coupling constants, 13C- spin-lattice relaxation times, and nuclear Overhauser effect data suggest the existence of three structured regions comprising residues 3-6, 7-12, and 12-14 in the solution conformations of the encephalitogenic heptadecapeptide.     

A high-resolution solid-state 13C-NMR study on crystalline bovine heart cytochrome-c oxidase and lysozyme. Dynamic behavior of protein and detergent in the complex.

We have recorded 100.6-MHz high-resolution solid-state 13C-NMR spectra of crystalline cytochrome-c oxidase from bovine heart muscle and hen egg-white lysozyme, to compare conformation and dynamics of a typical membrane-protein complex with those of lysozyme. The absence of severe interference with the solid-state 13C-NMR spectra, from both the line broadenings from paramagnetic centers and overlapping of intense detergent signals, provided spectral resolution of 13C-NMR feature of cytochrome-c oxidase crystals comparable to that of lysozyme crystal and better than that of dissolved or lyophilized samples. In fact, the observed peak intensities of the polar heads of the detergents BL8SY and Brij 35 were only about 10% and 3% of the anticipated values, respectively. The dynamic behavior of the backbone and side chains of cytochrome-c oxidase was compared with that of lysozyme on the basis of the 13C spin-lattice relaxation times (T1): the backbone of the cytochrome-c oxidase turned out to be more flexible than that of lysozyme. Molecular motions of the detergent molecules attached to the proteins are found to be highly heterogeneous. Detergent molecules undergo rapid tumbling motions in the crystals in about 10 ns as detected by T1. In addition to rapid motions, slow motions were detected by 1H spin-lattice relaxation time in the rotating frame (TH1 rho) and cross-polarization time (TCH), together with data from static spectra, indicating that the aliphatic portion of the detergent interacts more strongly with hydrophobic protein surfaces than do the polar heads.     

Dynamic properties of water at phosphatidylcholine lipid-bilayer surfaces as seen by deuterium and pulsed field gradient proton NMR

The dynamic properties of water in phosphatidylcholine lipid/water dispersions have been studied, applying a combination of ²H-NMR techniques (quadrupole splitting and spin-lattice relaxation time) and self-diffusion measurements using pulsed field gradient (PFG) ¹H-NMR. The hydration properties of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) were compared with those of DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) and EYL (egg yolk phosphatidylcholine (lecithin)). A model is presented that assumes an exponentially decaying influence of the bilayer surface on water dynamics as well as on water orientation with increasing hydration. This assumption is based on an exponentially decaying hydration potential which results from direct lipid-water and water-water interactions. The model describes successfully the experimental data for a large water concentration range, especially at low hydration, where other models failed. With the exception of a small fraction of water which is significantly influenced by the surface in slowing down the mobility, the interbilayer water has isotropic, free water characteristics in terms of correlation times and molecular order. Hydration properties of POPC are comparable with those of EYL but differ from DOPC. At very low water content the correlation times of headgroup segmental reorientation and water are similar, indicating a strong coupling of this water to the lipid lattice. The hydration properties of the three lipids studied are explained in terms of slightly different headgroup conformations due to different lateral packing of the molecules by their fatty-acid chain composition.     

Nuclear magnetic resonance studies of lipid hydration in monomethyldioleoylphosphatidylethanolamine dispersions.

Solid-state proton nuclear magnetic resonance has been used to examine surface hydration in suspensions of monomethyldioleoylphosphatidylethanolamine (MeDOPE). The magic-angle spinning (MAS) 1H spectra for aqueous suspensions of MeDOPE in the L alpha phase exhibited two resonances of roughly equal intensity that could be ascribed to water protons, but both their spin-lattice relaxation times and chemical shifts converged upon conversion to the hexagonal phase. Only a single water peak was observed for analogous samples of dioleoylphosphatidylcholine (DOPC). MAS-assisted two-dimensional nuclear Overhauser effect spectroscopy (NOESY) was conducted for multibilayers of both MeDOPE and DOPC. Through-space interactions were identified between pairs of lipid protons, as expected from their chemical structure. For lamellar suspensions of MeDOPE, positive NOESY cross-peaks were observed between the downfield-shifted water resonance (only) and both CH2N and NH2CH3+ protons of the lipid headgroup. These cross-peaks were not observed in the NOESY spectra of MeDOPE in its hexagonal or cubic phases or for lamellar DOPC reference samples. Taken together, the observation of two water peaks, spin-lattice relaxation behavior, and NOESY connectivities in MeDOPE suspensions support the interpretation that the low-field water peak corresponds to hydrogen-bonded interlamellar water interacting strongly with the lipid. Such a population of water molecules exists in association with MeDOPE in the lamellar phase but not for its inverted phases or for lamellar dispersions of DOPC.     

Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs

Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart.     

Determination of the degree of acetylation of chitin materials by 13C CP/MAS NMR spectroscopy

¹³C CP/MAS NMR spectroscopy has been shown to be a powerful tool to quantify the degree of acetylation of chitin and chitosan. In order to optimise the parameters which afford quantitative ¹³C cross-polarisation magic-angle spinning NMR spectra, a detailed relaxation study has been carried out on selected chitin and deacetylated chitin samples. A relaxation delay of 5 s and a contact time of 1 ms have been found to yield quantitative NMR spectra of samples with deacetylation degree values of 0.68 and 0.16. The measured spin-lattice relaxation times in the rotating frame, T_1ρH, are in the range 6.4–8.9 ms for chitin and 4.3–7.3 ms for deacetylated chitin, while T_CH values for both samples are very similar and range from 0.03 to 0.19 ms. Spin-counting experiments indicate that, within experimental error, all carbon is detected by NMR indicating that the samples studied contain no (or very few) paramagnetic centres.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.     

Deuterium NMR investigation of backbone dynamics in the synthetic oligonucleotide [d(CGCGAATTCGCG)]2

Backbone dynamics in the [5',5"-2H2]2'-deoxythymidine labeled duplex dodecamer [d-(CGCGAAT*T*CGC)]2 have been investigated by solid-state 2H NMR. Quadrupolar echo line shapes, spin-lattice relaxation, and quadrupolar echo decay times were obtained over hydration levels ranging from W = 0.0 to 25.2 (moles of H2O/mole of nucleotide). Variation of the line shape with changing hydration level was analyzed by using models employed in previous investigations of dodecamer base and sugar dynamics. Both fast local motions and a slower helix motion were present within the oligonucleotide. The fast motion was modeled as a four-site libration whose amplitude increased with hydration level. The root mean square amplitude of this librational model was 2-6 degrees larger than the amplitude observed in either the furanose ring or base labeled material for the entire range of hydration levels investigated. The observed line shape was inconsistent with a rapid three-site trans-gauche isomerization. A slow motion about the helix axis was observed at low water levels and increased in rate and amplitude with hydration. This motional model is in agreement with previous oligonucleotide studies.     

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.     

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.