Gelling properties of apohemoglobin S alone and in mixtures with hemoglobin S

Apohemoglobin S formed a gel in the cold (5 degrees C) with a protein concentration in the supernatants after centrifugation of the gels (Csat) near 27 g/dl, in 0.02 M phosphate buffer at pH 7.2. Under the same experimental conditions in mixtures of apohemoglobin S and deoxyhemoglobin S the solubility of hemoglobin S in the cold was decreased from Csat greater than 40 g/dl in the absence to about 18 g/dl in the presence of apohemoglobin S. Conversely, in the same mixture, Csat of apohemoglobin S was decreased to about 5 g/dl. Also, gelling occurred in mixtures of oxyhemoglobin S and its apoderivative. Apohemoglobin A alone did not form gels; however, it induced fiber formation in deoxyhemoglobin S in the cold; unlike apohemoglobin S, it was not included in the precipitate. Gels of apohemoglobin S were not birefringent, and inspection at the electron microscope failed to show the presence of organized structures. Excluded volume effects were probably at the origin of the decreased solubility of hemoglobin S and apohemoglobin S in the presence of each other.     

Elastic properties of deoxy hemoglobin S (deoxy-HbS) gels

Equilibrium shear moduli for deoxyhemoglobin S(HbS) gels were measured as a function of HbS concentration. The modulus was found to depend approximately on the 18th power of the HbS concentration. Deoxy-HbS gels were found at HbS concentration as low as 10 g/dl.     

Chemical potential measurements of deoxyhemoglobin S polymerization. Determination of the phase diagram of an assembling protein

We have used the "osmotic stress" method to determine the phase diagram of deoxyhemoglobin S polymerization. This method involves equilibration, through a semipermeable membrane, of the protein with solutions of inert polymers of known osmotic pressure. With deoxyhemoglobin A and S solutions, in which we have demonstrated achievement of equilibrium, plots of osmotic pressure versus concentration initially agree closely with the results of other methods of measurement of colligative properties. However, once the known solubility value is exceeded for the deoxyhemoglobin S solutions at various temperatures, there is a rapid rise in hemoglobin concentration over a narrow osmotic pressure range and then a more gradual increase in concentration. We believe that these two regions correspond, respectively, to the onset of the polymerization process, and of subsequent continuing growth and compression or alignment of polymer. We derive the thermodynamic values for these processes and show that the behavior of the deoxyhemoglobin S system is analogous to the phase transition for a simple chemical system. These results are relevant to understanding the intracellular polymerization of deoxyhemoglobin S in sickle cell disease, and these concepts are applicable to other protein assembly systems.     

Rheology of enzyme liquefied corn stover slurries: The effect of solids concentration on yielding and flow behavior

The measurement of yield stress and shear thinning flow behavior of slurries formed from unpretreated corn stover at solids loadings of 100–300 g/L provides a key metric for the ability to move, pump, and mix this lignocellulosic slurry, particularly since corn stover slurries represent a major potential feedstock for biorefineries. This study compared static yield stress values and flow hysteresis of corn stover slurries of 100, 150, 200, 250, and 300 g/L, after these slurries were formed by adding pellets to a cellulase enzyme solution (Celluclast 1.5 L) in a fed-batch manner. A rotational rheometer was used to quantitate relative yield stress and its dependence on processing history at insoluble solids concentrations of 4%–21% (wt/vol). Key findings confirmed previous observations that yield stress increases with solids loadings and reaches ~3000 Pa at 25% (wt/vol) solids concentration compared to ~200 Pa after enzyme liquefaction. While optimization of slurry forming (i.e., liquefaction) conditions remains to be done, metrics for quantifying liquefaction extent are needed. The method for obtaining comparative metrics is demonstrated here and shows that the yield stress, shear thinning and shear thickening flow behaviors of enzyme liquefied corn stover slurries can be analyzed using a wide-gap rheometry setup with relative measuring geometries to mimic the conditions that may exist in a mixing vessel of a bioreactor while applying controlled and precise levels of strain.     

Mechanical perturbation elicits a phenotypic difference between Dictyostelium wild-type cells and cytoskeletal mutants.

To determine the specific contribution of cytoskeletal proteins to cellular viscoelasticity we performed rheological experiments with Dictyostelium discoideum wild-type cells (AX2) and mutant cells altered by homologous recombination to lack alpha-actinin (AHR), the ABP120 gelation factor (GHR), or both of these F-actin cross-linking proteins (AGHR). Oscillatory and steady flow measurements of Dictyostelium wild-type cells in a torsion pendulum showed that there is a large elastic component to the viscoelasticity of the cell pellet. Quantitative rheological measurements were performed with an electronic plate-and-cone rheometer, which allowed determination of G', the storage shear modulus, and G", the viscous loss modulus, as a function of time, frequency, and strain, respectively. Whole cell viscoelasticity depends strongly on all three parameters, and comparison of wild-type and mutant strains under identical conditions generally produced significant differences. Especially stress relaxation experiments consistently revealed a clear difference between cells that lacked alpha-actinin as compared with wild-type cells or transformants without ABP120 gelation factor, indicating that alpha-actinin plays an important role in cell elasticity. Direct observation of cells undergoing shear deformation was done by incorporating a small number of AX2 cells expressing the green fluorescent protein of Aequorea victoria and visualizing the strained cell pellet by fluorescence and phase contrast microscopy. These observations confirmed that the shear strain imposed by the rheometer does not injure the cells and that the viscoelastic response of the cell pellet is due to deformation of individual cells.     

Quasi-elastic laser light scattering from solutions and gels of hemoglobin S.

Quasi-elastic light scattering has been used to examine solutions and gels of deoxyhemoglobin S. The autocorrelation function is found to decay with a characteristic exponential relaxation which can be ascribed to the diffusion of monomer (64,000 molecular weight) hemoglobin S molecules. In the absence of polymers, the relaxation time is in good agreement with previous measurements of the diffusion coefficient for solutions of normal human hemoglobin. In the presence of the polymer phase, a large (greater than 200-fold) increase in the scattered intensity is observed but no contribution to the decay of the autocorrelation function from the motion of the aligned polymer phase can be detected. Heterodyning between the time-independent scattering amplitude from the polymers and the time-dependent scattering of the diffusing monomers results in a twofold increase in the relaxation time arising from monomer diffusion.     

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.     

The Microrheology of Sickle Hemoglobin Gels

Sickle cell disease is a rheological disease, yet no quantitative rheological data exist on microscopic samples at physiological concentrations. We have developed a novel method for measuring the microrheology of sickle hemoglobin gels, based on magnetically driven compression of 5- to 8-μm-thick emulsions containing hemoglobin droplets ∼80 μm in diameter. Using our method, by observing the expansion of the droplet area as the emulsion is compressed, we were able to resolve changes in thickness of a few nanometers with temporal resolution of milliseconds. Gels were formed at various initial concentrations and temperatures and with different internal domain structure. All behaved as Hookean springs with Young's modulus from 300 to 1500 kPa for gels with polymerized hemoglobin concentration from 6 g/dl to 12 g/dl. For uniform, multidomain gels, Young's modulus mainly depended on the terminal concentration of the gel rather than the conditions of formation. A simple model reproduced the quadratic dependence of the Young's modulus on the concentration of polymerized hemoglobin. Partially desaturated samples also displayed quadratic concentration dependence but with a smaller proportionality coefficient, as did samples that were desaturated in steps; such samples were significantly less rigid than gels formed all at once. The magnitude of the Young's modulus provides quantitative support for the dominant models of sickle pathophysiology.     

Glycidol-modified gels for molecular-sieve chromatography. Surface hydrophilization and pore size reduction

Divinyl sulfone-crosslinked agarose gels were made hydrophilic by coupling glycidol to the agarose chains. The concentration of glycidol in the reaction mixture determines the pore size of the gels (the glycidol molecules probably form polymers, the degree of polymerization increasing with the glycidol concentration). Gels prepared with moderate glycidol concentrations are still porous enough to be used for separation of proteins and peptides. Gels with a high degree of glycidol polymerization are suited for desalting of low-molecular-weight compounds, for instance peptides.     

Cryosolvents effects on low temperature gel structure of denatured collagen

Water flux and crystallization are major problems for cryopreservation. The gel approach relies on the concept that a biological gel network might sufficiently entrap fluid within its pores, so as to maintain its osmotically inactive under the stresses usually encountered in the course of cryopreservation and limit the extent of crystallization. It could even induce vitrification. The effects of some cryosolvents on the structure of denatured collagen gels were studied as a function of temperature by hydraulic conductivity measurements and electron microscopy observations, so as to explore the porosity and fluid behavior of the gels. Gels formed in the presence of methanol exhibit a large increase in pore size as gelation temperature drops, whereas almost no variation is detected with ethylene glycol, where the porosity is finer. Gels in the presence of ethylene glycol display a larger fluid content, smaller flow rates, and better pressure resistance than those in the presence of methanol. Electron micrographs confirm the variations in gel structures depending on the cryosolvent and the temperature. Rheological measurements also support these observations. Upon rapid cooling, vitrification occurs only in gels with ethylene glycol. Ethylene glycol seems to have a specific interaction with denatured collagen gels which induces a finer network better adapted for trapping fluid osmotically inactive in the course of cryopreservation.     

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.     

Thermodynamic study of the crystallization of sickle-cell deoxyhemoglobin (hemoglobin S solubility/saturation concentration/enthalpy of crystallization/entropy of crystallization).

The solubility of crystalline deoxygenated sickle-cell hemoglobin has been determined by a new turbidometric technique. In the absence of the allosteric effector, inositol hexaphosphate, the solubility of sickle deoxyhemoglobin crystals is about 30% less than that of gels. The dependence of the solubility of crystals on temperature at pH 7.1 is very much the same as that of gels at pH 6.48. The change in Van't Hoff enthalpy with crystallization is positive but small (3.11 kcal mol^?1) and essentially independent of temperature below 30 °C. The presence of inositol hexaphosphate cuts the solubility almost in half. The entropy change when crystals form, just under 40 cal K^?1 mol^?1, is larger than the values of ΔS ° found for gels. None of the measurements reported required the estimation of pellet volumes and compositions.     

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.     

Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation

The effects of temperature, agitation rate and medium composition, including concentrations of glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of Rhizopus delemar (Rhizopus oryzae) NRRL1526 during pre-culture were studied. Inorganic ions and soybean peptone had negative and positive effects on pellet formation, respectively. The initial glucose and soybean peptone concentrations directly affected pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations; however, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for producing small pellets of R. delemar. Furthermore, dry cell mass and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% when fermented in a flask and 31.82% in stirred bioreactor tank fermentation.     

Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity: Changes in viscosity and volume on aggregation

The kinetics of gelation of deoxyhemoglobin S were investigated as a function of temperature, concentration of hemoglobin, and solvent composition. Measurements were made by continuously monitoring the changes in viscosity with time, after polymerization had been induced by rapidly raising the temperature. A specially constructed low-shear viscometer was used. The solution density was also measured continuously to determine whether a volume change accompanied aggregation.The results confirm earlier work in showing that the time-dependence of the viscosity is composed of a variable latent period (several minutes to tens of hours) during which there is only a slight and very gradual increase in viscosity, followed by a stage in which the viscosity rises very sharply within a very short time. The length of the initial latent period is highly dependent upon the HbS³ concentration (33rd ± 6 power) and temperature. If the duration is interpreted as the inverse of a reaction rate, the activation energy is 96 ± 10 kcal/mol for solutions containing inosital hexaphosphate. Unlike measurements reported by others, no dependence of the latent period on shear rate was observed at the low shear rate employed. When IHP is omitted from the hemoglobin solutions, qualitatively similar results are obtained; however, the latent period depends on the 26th ± 6 power of the deoxyhemoglobin S concentration and yields an average activation energy of 125 ± 10 kcal/mol. The length of the latent period is increased 40-fold. Tris is known to prevent gelation but the inhibition can be partly reversed by adding IHP. When this is done, highly concentration-dependent latent periods are again observed. The results may be interpreted in terms of nucleation kinetic theories: a critical nucleus composed of approximately 30 hemoglobin molecules is required for gelation; and the energy barrier (which is larger in the absence of IHP) to the formation of this critical aggregate is approximately 100 kcal/mol.Gelation is not accompanied by a detectable volume change (limits 5 × 10^?5 g/ml). This indicates that the volume change of the reaction must be less than + 60 cm³/mol when the aggregates represent one half of the HbS available for polymerization.     

Effect of heat treatment in vacuum on photoluminescence of anodic alumina

The results are presented of a study of the photoluminescent (PL) properties of an undoped porous anodic alumina (PAA) and PAA doped with manganese ions. The PAA samples were prepared by anodization of aluminum. The effect of annealing conditions in vacuum on the PL spectra was studied for the first time and a comparative analysis was made with the spectra of the PAA annealed in air. Vacuum annealing was used to obtain oxygen‐deficient alumina. A strong dependence of the PAA PL intensity on the annealing temperature in vacuum has been found: for the samples annealed at 600°С, the PL intensity is 15 times higher than that measured on the initial samples, whereas for the samples annealed in air it increases only 4.5‐fold with excitation at the wavelengths of 275 nm. This is the result of the formation of a high concentration of oxygen vacancies during annealing in vacuum under conditions of oxygen deficiency as compared with the samples annealed in air, where diffusion of oxygen from air leads to a decrease in vacancies. A significant increase in the PL intensity permits consideration of the vacuum‐annealed PAA as a promising material for dosimetry.     

Flow behaviour of a POSS biopolymer solution

A non-biodegradable polyhedral oligomeric silsesquioxane (POSS) nanocomposite biopolymer has been developed for fabrication of medical devices and for tissue engineering human organs. The polymer in solution, containing 2 wt% of POSS, has been synthesized, characterized and investigated to determine its key rheological properties. Thus, the variation of shear stress and viscosity as a function of shear rate has been determined at ambient temperature to estimate yield stress and the index of pseudoplasticity, respectively. The temperature dependence of viscosity and the effect of ageing on the viscosity of the polymer have also been investigated. Results are compared with those of a conventional polycarbonate urethane (PCU) polymer solution. The POSS-PCU polymer solution shows near-Newtonian behaviour in the shear rate range to 1000 s(-1), having an apparent viscosity of approximately 3000 mPa s and a pseudoplasticity index of 0.90, decreasing slightly as the polymer solution is aged over 9 months. The temperature dependence of viscosity of the POSS polymer is extremely low and does not change with ageing but the yield strength increases from 2.7 Pa to 8.3 Pa.     

Keratin filament suspensions show unique micromechanical properties.

All epithelial cells feature a prominent keratin intermediate filament (IF) network in their cytoplasm. Studies in transgenic mice and in patients with inherited epithelial fragility syndromes showed that a major function of keratin IFs is to provide mechanical support to epithelial cell sheets. Yet the micromechanical properties of keratin IFs themselves remain unknown. We used rheological methods to assess the properties of suspensions of epidermal type I and type II keratin IFs and of vimentin, a type III IF polymer. We find that both types of IFs form gels with properties akin to visco-elastic solids. With increasing deformation they display strain hardening and yield relatively rapidly. Remarkably, both types of gels recover their preshear properties upon cessation of the deformation. Repeated imposition of small deformations gives rise to a progressively stiffer gel for keratin but not vimentin IFs. The visco-elastic moduli of both gels show a weak dependence upon the frequency of the input shear stress and the concentration of the polymer, suggesting that both steric and nonsteric interactions between individual polymers contribute to the observed mechanical properties. In support of this, the length of individual polymers contributes only modestly to the properties of IF gels. Collectively these properties render IFs unique among cytoskeletal polymers and have strong implications for their function in vivo.     

Adhesion of the marine fouling diatom amphora coffeaeformis to non‐solid gel surfaces

Diatom adhesion to different gel surfaces was tested under different shear conditions, using the fouling marine diatom Amphora coffeaeformis as test organism. Four polymers were selected to obtain a test matrix containing gels with different surface charge as well as different surface energies, viz. agarose, alginate, chitosan and chemically modified polyvinylalcohol (PVA‐SbQ). Three experimental systems were applied to obtain different shear rates. Experimental system 1 consisted of gels cast in a cell culturing well plate for comparing initial adhesion as well as long term biofilm development in the absence of shear. In experimental system 2, microscope slide based test surfaces were tested in aquaria under low shear conditions. A rotating annular biofilm reactor was used to obtain high and controlled shear rates. At high shear rates A. coffeaeformis cells adhered better to the charged polymer gels (alginate and chitosan) than to the low charged polymer gels (agarose and PVA‐SbQ). In the system where shear was absent A. coffeaeformis cells developed a biofilm on agarose equivalent to the charged polymer gels, while adhesion to PVA‐SbQ remained low at all shear rates. It is concluded that non‐solid surfaces did not represent an obstacle to settling and growth of this organism. As observed for solid surfaces, low charge density led to reduced attachment, particularly at high shear.     

Shear creep of gelatin gels from mammalian and piscine collagens.

This paper describes new measurements on the creep rheological behaviour of gelatin gels from both traditional mammalian and piscine sources. Measurements on a series of concentrations of gels were obtained using a high-precision controlled stress rheometer. Results for the concentration dependence of compliance are close to those expected from dynamic oscillatory measurements of gel modulus, assuming ideal elasticity. The concentration dependence of viscosity approximates power law behaviour, with eta~C( approximately 2-3), lower than the exponent expected for semi-dilute solutions. The apparent contradiction implied by this is discussed and a novel gel viscosity versus concentration state diagram presented.