Effect of oxygen concentration on trasverse water proton relaxation times in erythrocytes homozygous and heterozygous for hemoglonin S.

The transverse water proton relaxation times (T₂) of erythrocytes homozygous and heterozygous for hemoglobin S have been measured as a function of oxyhemoglobin concentration at 37 °C. An immediate decrease in T₂ is observed in S/S erythrocytes as the amount of oxyhemoglobin is decreased and the maximum change is observed at 50% deoxyhemoglobin S. In heterozygous erythrocytes, the T₂ remains unchanged until a critical level of deoxyhemoglobin is attained. The critical level of deoxyhemoglobin is a function of the percentage of hemoglobin S in the heterozygous erythrocytes. A Hill plot of the data obtained from S/S erythrocytes gives an n value of around 2.4. These results suggest that the measurement of T₂ is sensitive to the very early stages of the polymerization process. This suggestion is supported by calculations; our T₂ measurements are sensitive to a range of correlation times expected for hemoglobin monomers at one extreme and linear polymers of seven hemoglobin molecules at the other extreme.     

Thermodynamics of gelation of sickle cell deoxyhemoglobin.

This paper describes the thermodynamic behavior of gels of deoxyhemoglobin S. The solubility of the protein with respect to assembled hemoglobin fibers has been measured using a sedimentation technique. The solubility in 0.15 m-potassium phosphate buffer (pH 7.15) is found to decrease with increasing temperature, attain a minimum value of 0.16 g cm^?3 at 37 °C, and then increase at higher temperatures. The amount of polymer present at various hemoglobin concentrations and temperatures is presented as part of a phase diagram that may be useful for the calibration of other measurement techniques. The effects of varying pH and urea concentration upon the solubility have also been studied.The heat absorption accompanying gelation has been measured by scanning calorimetry. Using sedimentation data on the amount of polymer formed, molar enthalpy changes are obtained. There is a large negative heat capacity change of ? 197 cal deg. mol^?1 and ΔH = 0 near 37 °C. Calorimetric molar enthalpy changes are found to agree with those calculated from the temperature dependence of the solubility by the van't Hoff equation.Our previous two-phase, two-component thermodynamic model of gelation is extended to include the effects of solution non-ideality. A large contribution to the activity of the hemoglobin in the solution phase results from the geometric effect of excluded volume. Incorporating solution phase non-ideality permits the calculation of standard state thermodynamic quantities for the gelation process at 37 °C: ΔG^O ? ?3 k cal mol^?1, ΔH^O ~ 0, ΔS^O ~ 10 cal deg.^?1 mol^?1. The excluded volume effect is also capable of explaining observations of the minimum gelling concentrations of hemoglobin mixtures containing deoxyhemoglobin S without requiring copolymerization of the non-S hemoglobin.     

Inhibitory effect of deoxyhemoglobin A2 on the rate of deoxyhemoglobin S polymerization.

From a consideration of the primary sequence of hemoglobin A₂ and the reported 5 å molecular contacts between deoxyhemoglobin S molecules in a crystal, it is predicted that hemoglobin A₂ might act as an inhibitor of the polymerization of deoxyhemoglobin S in a manner similar to hemoglobin. F. This has been tested experimentally by measuring the rate of change of the transverse water proton relaxation times (T₂) in equimolar mixtures of hemoglobin S and one of the non-gelling hemoglobins A, F or A₂. Hemoglobins A₂ and F have far more pronounced inhibitory effects on the rate of polymerization than does hemoglobin A. These molecules contain several amino acid differences from hemoglobin A beta chains which are located in the 5 Å molecular crystal contacts and these altered crystal contacts result in a much stronger inhibition of the rate of polymerization. Since hemoglobin A₂ is a normal hemoglobin found in small amounts in all adult red cells, increased delta chain synthesis may have potential importance in therapy for sickle cell disease.     

Tracer diffusion coefficients of oxyhemoglobin a and oxyhemoglobin s in blood cells as determined by pulsed field gradient NMR

It is demonstrated that tracer diffusion coefficients can be determined for oxyhemoglobin A (HbA-O₂) and oxyhemoglobin S (HbS-O₂) in intact blood cells by means of pulsed field gradient NMR (PFG-NMR). This is possible because the method discriminates between both rapidly moving water molecules and molecules having small proton transverse relaxation times (T₂). The results indicate that only hemoglobin molecules contribute to the echo signals when large field gradients are used. The dependence of the measured diffusion coefficients on osmolarity and pH are attributed to changes in hemoglobin concentration resulting from changes in cell volume.     

Evaluation of the water environments in deoxygenated sickle cells by longitudinal and transverse water proton relaxation rates

The longitudinal and transverse water proton relaxation rates of oxygenated and deoxygenated erythrocytes from both normal adults and individuals with sickle cell disease were measured as a function of temperature at two different frequencies. The simplest model which fits all of the data consists of three different environments for water molecules. The majority of the water (98%) has a correlation time indistinguishable from bulk water (3 × 10^?11 sec). Secondly, there is a small amount of water (1.3–1.5%) present which has a correlation time of 2–4 × 10 ^?9 sec and is apparently independent of the erythrocyte sample studied. Presumably this water is the hydration sphere around the hemoglobin molecules and its correlation time is significantly slower than bulk water. The third environment contains approximately 0.2% of the water present and has a correlation time≥ 10^?7 sec. This third environment is considered tightly bound to the hemoglobin because the water proton correlation time is very similar to the expected rotational correlation time for the hemoglobin molecules. The value of the transverse relaxation rate, f_b(T_2b)^?1, for the tightly bound water fraction decreases in oxy ($\text{S}\text{S}$), deoxy ($\text{A}\text{A}$), and oxy ($\text{A}\text{A}$) erythrocyte samples as the temperature is increased as expected for a rotational correlation time process. In dramatic contrast,f_b (T_2b)^?1 increases almost linearly as the temperature is increased over the whole 4 ° to 37 °C temperature range in samples of deoxy ($\text{S}\text{S}$) erythrocytes. The observation suggests a continual increase in the formation of deoxyhemoglobulin S polymers rather than a sudden transition from a homogeneous solution of deoxyhemoglobin S molecules to a solid gel.     

The Binding of Asparagine,Glutamine and Homoserine to Human Erythrocytes Containing Hemoglobins S and CS and to Soluble Hemoglobins S and CS

Abstract

Tritium labeled asparagine binds to oxyhemoglobin S and to a mixture of hemoglobins C and S in the molar ratio of 3.38:1 and 8.2:1 respectively. From the dialysis equilibrium studies it appears that labeled asparagine does not bind to oxy- or deoxy- hemoglobin A nor to deoxyhemoglobin S. The constant for equilibrium association of asparagine for oxyhemoglobin S is 7.38 × 10⁷ M^?1 and for'oxyhemoglobin CS 4.8 × 10⁴ M^?1 at 23°C. Tritium labeled asparagine is bound to oxyhemoglobin S and CS sufficiently strongly to prevent dissociation under the conditions of gel electrophoresis at pH 9.50. The protein with and without bound asparagine, gluta-mine or homoserine, is indistinguishable in molecular net charge and size by the criteria of quantitative polyacrylamide gel electrophoresis (PAGE). Also there were no significant differences in mobility between hemoglobin S and hemoglobin C in the presence and absence of asparagine, glutamine and homoserine as detectable in agar coated cellulose acetate electrophoresis at pH 6.3. Erythrocytes containing hemoglobin S and CS, after incubation with tritium labeled asparagine and lysis under the conditions of gel electrophoresis at pH 9.5, release hemoglobin S and C with bound tritiated asparagine. No tritiated asparagine remains bound to the ghost.     

Calorimetric and optical characterization of sickle cell hemoglobin gelation.

We have used two techniques to characterize the gelation of deoxyhemoglobin S, a high sensitivity heat-flow calorimeter to measure the heat of gelation and a simple light-transmission method to measure the optical birefringence resulting from the alignment of deoxyhemoglobin S fibers in the gel. A theory for the interpretation of the birefringence measurements is presented. We combine the results of the calorimetric and optical measurements with those of sedimentation experiments to obtain enthalpy changes for gelation. The enthalpy change obtained from scanning and isothermal calorimetric measurements (0.25 m-potassium phosphate, 0.05 m-sodium dithionite, pH 6.9) varies from 4000 to 2200 cal mol⁻¹ hemoglobin between 16 and 25 °C. There is a large apparent heat capacity change of −130 to −190 cal deg.⁻¹ mol⁻¹. The apparent enthalpy change estimated from solubility measurements and birefringence melting experiments is 2200 ± 500 cal mol⁻¹ in qualitative agreement with the calorimetric results. Analysis of the time dependence of the calorimetric and optical progress curves at 20 °C leads to a rough estimate of 1800 to 4000 and −800 to 1500 cal mol⁻¹ hemoglobin for the enthalpies of polymerization and alignment of fibers, respectively. The small magnitude of the observed enthalpy change is in accord with the view that no large conformational change takes place in the deoxyhemoglobin S molecule upon gelation.     

Gelation of sickle cell hemoglobin in mixtures with normal adult and fetal hemoglobins.

We report the results of thermodynamic and kinetic studies on the gelation of mixtures of sickle cell (S) deoxyhemoglobin with normal human adult (A) and fetal (F) deoxyhemoglobins. The delay time of thermally induced gelation was monitored by the increase in turbidity. At the completion of gelation the solubility was determined by sedimenting the polymers and measuring the supernatant concentration spectrophotometrically. Addition of hemoglobins A or F, at mole fractions from 0 to 0.6, resulted in large increases in both the solubility and the delay time. For a 50:50 mixture of deoxyhemoglobin F with deoxyhemoglobin S, the solubility increased by a factor of 1.8 and the delay time by a factor of 10⁷ relative to pure deoxyhemoglobin S at the same total concentration, while for a 50:50 mixture of deoxyhemoglobins A and S the solubility increased by a factor of 1.4 and the delay time by a factor of 10⁴. The relative delay times were independent of both temperature and total hemoglobin concentration. The data have been analyzed according to theoretical models which treat the effects of temperature, concentration, non-ideality and solution composition on the thermodynamics and kinetics of gelation. The increased solubility in mixtures with deoxyhemoglobin F is fully explained by a model in which only deoxyhemoglobin S molecules polymerize. The effect of fetal hemoglobin (α₂γ₂) and hybrid α₂γβ^S molecules is to increase the solution non-ideality through the contribution of their excluded volume. The smaller increase in the solubility observed in comparable mixtures with deoxyhemoglobin A requires that the hybrid α₂β^Aβ^S molecules copolymerize with the deoxyhemoglobin S. The kinetic results for the mixtures can be quantitatively accounted for using a nucleation model in which the equilibrium properties of the polymer are used to describe the critical nucleus. The very large increases in delay time observed for the $\text{S}\text{F}$ mixtures can be explained by assuming that only α₂β₂^S molecules participate in the formation of a nucleus containing about 25 monomers. As in the thermodynamic analysis, the smaller effect of adding deoxyhemoglobin A can be attributed to the contribution of the hybrid molecules in forming the critical nucleus. Thus the difference between the polymerization properties of mixtures of deoxyhemoglobin S with deoxyhemoglobins A and F can be attributed solely to the copolymerization of the α₂β^Aβ^S hybrid molecule and the absence of any significant copolymerization of the α₂γβ^S hybrid.     

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.     

Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

Kinetics of the polymerization of hemoglobin S: studies below normal erythrocyte hemoglobin concentration.

The kinetics of polymerization of deoxyhemoglobin S have been studied by measuring transverse water proton relaxation times (T₂) in hemoglobin solutions. As seen by other techniques, the kinetic profile consists of a delay time followed by a decrease in T₂ during polymerization. The length of the delay time can be decreased and the rate of change of T₂ can be increased by increasing the concentration of hemoglobin S or non-gelling hemoglobin or ovalbumin. At a total protein concentration of about 210 mg/ml the kinetic profiles in all three cases are indistinguishable suggesting that a non-specific protein-protein interaction may be involved in the kinetics of polymerization. In addition, it is suggested that no polymer formation occurs during the delay period.     

NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

Hemoglobin-water interactions in normal and sickle erythrocytes by proton magnetic resonance T1ϱ measurements

The dependence of the water proton magnetic resonance spin-lattice relaxation rate (T_1?^?1) in the rotating frame on the strength of the spin-locking (H₁) field has been investigated for packed oxy and deoxy normal and sickle erythrocytes at temperatures from 9 to 40 °C. The T_1?^?1 of oxy or deoxy normal erythrocytes shows no dependence on H₁ up to ~7 G at any temperature studied. On the other hand, T_1?^?1 decreases from about 40 s^?1 to 15 s^?1 (H₁ from 0 to ~7 G) for deoxygenated packed sickle cells at 40 °C. The magnitude of this variation of T_1?^?1 with H₁ decreases with decreasing temperature. Oxy packed sickle cells also show a dependence of T_1?^?1 on H₁ but the magnitude is <10% of that of the deoxygenated samples. These results suggest that water proton T_1?^?1 measurements are a sensitive probe of hemoglobin S polymerization and provide a novel technique for the study of slow water motions in these systems. The T_1?^?1 results are compared with low frequency T₁^?1 results of other investigators on hemoglobin S solutions. Analysis of the data suggests that water proton motions with correlation times of the order of 10^?5 s are present in the deoxygenated sickle cell samples at temperatures above 10 °C.     

Observation of allosteric transition in hemoglobin

Two conclusions have been drawn from NMR studies of mixed state hemoglobins. First the α and β subunits in hemoglobin are not equivalent in their conformational properties. Second the mixed state hemoglobin (α^IIICN β^II)₂ can take two different quaternary structures without changing the degree of ligation. One of the two structures is similar to that of deoxyhemoglobin and the other to that of oxyhemoglobin.     

Photon correlation spectroscopy of hemoglobin: Diffusion of Oxy-HbA and Oxy-HbS

Translation diffusion coefficients have been measured for oxyhemoglobin A and oxyhemoglobin S over the concentration range 0.1–37 g/dl by means of photon correlation spectroscopy. The solutions were 0.1 M in KCl and in each case the pH was adjusted to the isoelectric point of the hemoglobin species present. No significant differences were found between the HbA and HbS results; and after correction to water at 20°C, the diffusion coefficients could be described by the equation where D_o = (6.93 ± 0.06) × 10^?7 cm²/sec and c ids the concentration in units of g/dl. No evidence was found for the aggregation of oxy-HbS at high concentrations which was reported by Lindstrom et. al. [(1976) Biophys. J. 16 , 679–689].     

Demonstration of lag phase in the sol-gel transformation of deoxygenated S hemoglobin without temperature alteration.

1). During the sol to gel transformation of deoxygenated sickle hemoglobin, a time-dependent process preceding gel formation (lag phase) was demonstrated that was inversely proportional to a function of the hemoglobin concentration and that occurred without alteration in temperature, pH, or oxygen tension. 2). As determined by the Schachman modification of the capillary viscometer, preparations of oxyhemoglobin S and A and deoxyhemoglobin A were indistinguishable when compared over a wide range of concentrations. Up to the concentration at which gelling occurred, deoxyhemoglobin S exhibited the same viscosity behavior. The viscosity of deoxygenated hemoglobin S within the lower gelling concentration range was normal during the lag phase and became abnormally high only at the time of gelation.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

Kinetics of polymerization of deoxyhemoglobin S and mixtures of hemoglobin A and hemoglobin S at high hemoglobin concentrations

Transverse water proton relaxation times (T₂) have been measured as a function of time after deoxygenation of solutions containing hemoglobin S. The shortened T₂ values observed upon deoxygenation of hemoglobin S result from an increase in the correlation time (τ_c) of the water fraction irrotationally bound to deoxyhemoglobin S as it polymerizes. Therefore, the change in τ_c as a function of time after deoxygenation can be used to measure the rate of polymer formation. The change in τ_c observed is reasonably fit by the first-order equation τ = τ₀ (1 ? e^?kt) + τ_oxy. At a total hemoglobin concentration of approximately 300 mg/ml, the pseudo-first-order rate constant in a heterozygous AS sample is 25 times slower than in a homozygous S sample, k = 0.019 and 0.47 s^?1, respectively. Since the transit time for an erythrocyte in vivo is approximately 15 s, these results suggest that the heterozygous A/S erythrocyte would traverse the circulation and become reoxygenated before extensive polymerization and, therefore, cell sickling could occur. For the homozygous S/S erythrocyte, there is ample time for polymerization and for cell sickling during circulation.     

Rheology and Confocal Reflectance Microscopy as Probes of Mechanical Properties and Structure during Collagen and Collagen/Hyaluronan Self-Assembly

In this work, the gelation of three-dimensional collagen and collagen/hyaluronan (HA) composites is studied by time sweep rheology and time lapse confocal reflectance microscopy (CRM). To investigate the complementary nature of these techniques, first collagen gel formation is investigated at concentrations of 0.5, 1.0, and 1.5 mg/mL at 37°C and 32°C. The following parameters are used to describe the self-assembly process in all gels: the crossover time (t_c), the slope of the growth phase (k_g), and the arrest time (t_a). The first two measures are determined by rheology, and the third by CRM. A frequency-independent rheological measure of gelation, t_g, is also measured at 37°C. However, this quantity cannot be straightforwardly determined for gels formed at 32°C, indicating that percolation theory does not fully capture the dynamics of collagen network formation. The effects of collagen concentration and gelation temperature on k_g, t_c, and t_a as well as on the mechanical properties and structure of these gels both during gelation and at equilibrium are elucidated. Composite collagen/HA gels are also prepared, and their properties are monitored at equilibrium and during gelation at 37°C and 32°C. We show that addition of HA subtly alters mechanical properties and structure of these systems both during the gelation process and at equilibrium. This occurs in a temperature-dependent manner, with the ratio of HA deposited on collagen fibers versus that distributed homogeneously between fibers increasing with decreasing gelation temperature. In addition to providing information on collagen and collagen/HA structure and mechanical properties during gelation, this work shows new ways in which rheology and microscopy can be used complementarily to reveal details of gelation processes.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.