SANS studies of interacting hemoglobin in intact erythrocytes.

Small angle neutron scattering (SANS) was used to investigate interaction forces between hemoglobin (Hb) molecules contained within human red cells. The scattering separately attributable to cell membranes and intracellular Hb was identified. A series of D2O-H2O contrast variation measurements were made in order to establish conditions for which scattering from the cell membrane is minimized (approximately 15% D2O). Measurements then were performed to examine changes in intermolecular Hb interactions occurring when the cells are contracted or swollen by varying the ionic strength of the suspension buffer. The scattering cross-sections were fitted to structure factors computed by a mean spherical approximation, and molecular parameters thereby extracted. Oxygenation studies on normal cells were performed, and results contrasted with those of similar studies of erythrocytes obtained from sickle cell disease patients.     

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.     

Deformation and fragmentation of human red blood cells in turbulent shear flow.

By means of glutaraldehyde fixation, human erythrocytes are "frozen" while suspended in turbulent shear flow. As the shearing is increased in steps from 100 to 2,500 dyn/cm2, the deformed cells evolve gradually toward a smooth ellipsoidal shape. At stresses above 2,500 dyn/cm2, approximately, fragmentation of the cells occurs with a concomitant increase in free hemoglobin content of the suspending medium. The photographic evidence suggests that the cells rupture in tension in the bulk flow.     

Small angle neutron scattering on an absolute intensity scale and the internal surface of diatom frustules from three species of differing morphologies

The internal nanostructure of the diatoms Cyclotella meneghiniana, Seminavis robusta and Achnanthes subsessilis was investigated using small angle neutron scattering (SANS) to examine thin biosilica samples, consisting of isotropic (powder) from their isolated cell walls. The interpretation of SANS data was assisted by several other measurements. The N₂ adsorption, interpreted within the Branuer–Emmet–Teller isotherm, yielded the specific surface area of the material. Fourier transform infrared (FTIR) and Raman spectroscopy indicates that the isolated material is amorphous silica with small amounts of organic cell wall materials acting as a filling material between the silica particles. A two-phase (air and amorphous silica) model was used to interpret small angle neutron scattering data. After correction for instrumental resolution, the measurements on two SANS instruments covered an extended range of scattering vectors 0.0011 nm^?1 < q < 5.6 nm^?1, giving an almost continuous SANS curve over a range of scattering vectors, q, on an absolute scale of intensity for each sample. Each of the samples gave a characteristic scattering curve where log (intensity) versus log (q) has a ?4 dependence, with other features superimposed. In the high-q regime, departure from this behaviour was observed at a length-scales equivalent to the proposed unitary silica particle. The limiting Porod scattering law was used to determine the specific area per unit of volume of each sample illuminated by the neutron beam. The Porod behaviour, and divergence from this behaviour, is discussed in terms of various structural features and the proposed mechanisms for the bio-assembly of unitary silica particles in frustules.     

Structure of RecA-DNA complexes studied by combination of linear dichroism and small-angle neutron scattering measurements on flow-oriented samples.

By combining anisotropy of small-angle neutron scattering (SANS) and optical anisotropy (linear dichroism, l.d.) on flow-oriented RecA-DNA complexes, the average DNA-base orientation has been determined in RecA complexes with double-stranded (ds) as well as single-stranded (ss) DNA. From the anisotropy of the two-dimensional SANS intensity representation, the second moment orientation function S is obtained. Knowledge of S is crucial for the interpretation of l.d. spectra in terms of orientation of the DNA bases and the aromatic amino acid residues. The DNA-base planes are essentially perpendicular to the fibre axis of the complex between RecA and dsDNA in the presence of cofactor ATP gamma S. A somewhat tilted base geometry is found for the RecA-ATP gamma S complexes with single-stranded poly(dT) and poly(d epsilon A). This behaviour contrasts the RecA-ssDNA complex formed without cofactor which displays a poor orientation of the bases. Well-ordered bases in the ssDNA-RecA complex is possibly reflecting the role of RecA in preparing a nucleotide strand for base-pairing in the search-for-homology process. While the central SANS intensity is essentially independent of the pitch of the helical complex, a secondary intensity maximum, which becomes focused upon flow orientation, is found to be a sensitive measure of the pitch. The pitch values for the complexes compare well with cryo-electron microscopy results but are slightly larger than those seen for uranyl-stained samples.     

Application of diffractometry and a linear image sensor to measurement of erythrocyte deformability.

We applied laser diffractometry and a linear image sensor to measurement of erythrocyte deformability to detect the light intensity pattern of the diffraction image. Deformability was evaluated as the deformability index (DI), calculated from the width and length of the diffraction pattern ellipse, estimated by the linear image sensor. With the erythrocytes under various shear stresses, the DI was linearly related to results by the geometric method (r = 0.996, p < 0.01). The coefficient of variance of DI at a shear stress of 236 dynes/cm2 was 0.2% (seven human blood samples), which was satisfactory for practical use. The DI was independent of the erythrocyte concentration in the range of 1.5 x 10(7)-5.0 x 10(7) cells/ml of suspension. Correlation between the DI and the logarithm of shear stress was linear in the range of 5 to 350 dynes/cm2 of shear stress in suspension media of different viscosities. Heat-treatment, which decreased membrane flexibility, caused parallel reduction of the DI plotted against the logarithm of shear stress. The method was sensitive and gave reproducible results. It may be useful for clinical applications.     

Measurements of viscosity of synthetic erythrocyte suspensions

Measurements were made of the viscosity of suspensions of synthetic erythrocytes composed of hemoglobin solutions encapsulated in liposomes, as a function of shear rate, temperature, suspension concentration, lipid membrane composition, and the viscosity of the suspending medium. It was found that the viscous behavior of the synthetic erythrocyte suspensions was non-Newtonian and nearly the same as that of suspensions of natural erythrocytes prepared similarly, with the major difference being that synthetic erythrocyte suspensions are somewhat more viscous. Suspensions of Fluosol FC-43 prepared similarly were found to be essentially Newtonian fluids, and substantially different and more viscous than either erythrocyte suspension. The higher viscosity of synthetic erythrocyte suspensions probably accounts for the ability of these suspensions to maintain normal systemic vascular resistance in transfusion experiments, in spite of the fact that synthetic erythrocytes are smaller than natural erythrocytes.     

Measurement of the erythrocyte orientation in a flow by spin labeling

When a suspension of erythrocytes labeled in their membrane with a fatty acid paramagnetic molecule is allowed to flow in a flat quartz sample cell, the recorded electron paramagnetic spectra change as a function of the orientation of the cell in the magnetic field. This indicates that the red cells are themselves oriented in the flow. Such spectral variations have been reproduced by a numerical simulation procedure, which allowed us to quantify the proportion of oriented red blood cells by measuring the amplitude of some characteristic lines on the experimental spectra. Orientation rates were then measured as a function of various rheological parameters, such as shear rate, hematocrit and viscosity of the suspending medium. The kinetics of the disorientation process was determined by stopping the flow.     

Viscoelasticity of packed erythrocyte suspensions subjected to low amplitude oscillatory deformation.

Concentrated adult erythrocyte suspensions were subjected to low amplitude oscillatory shear in a Weissenberg rheogoniometer equipped with a cone-and-plate assembly. The dynamic viscoelastic properties of the suspension were measured over a broad range of frequency by a numerical solution that accounted for fluid inertia. Variation of shear amplitude and cell volume percent, and comparison of buffered saline, plasma, and dextran as suspending media showed that the cellular elements had undergone small bending and shearing deformations. Studies of normal adult erythrocytes, hypotonically swollen cells, temperature-altered cells, and erythrocyte ghosts suggested that the method was evaluating membrane material properties. The normal membrane was found to exhibit a shear rate dependent elastic modulus that increased by more than a factor of 20 over a frequency range from 0.0076 Hz to 60 Hz. The membrane viscosity showed a substantial drop with frequency indicative of a frequency thinning phenomenon. At high frequency of deformation the viscous response of normal erythrocytes was no longer indicative of a membrane property due to the dominant influence of the internal hemoglobin solution. The studies generally supported the ability of the method to quantify relative membrane material properties and detect changes in membrane structure.     

Metabolic changes leading to oxidative lysis of erythrocytes maintained in a normal state in vitro

It was shown that in vitro oxidative hemolysis of human erythrocytes occurs as a result of a great increase in membrane permeability to cations leading to osmotic damage of the cells. Infusion at a steady rate with a solution of tert-butylhydroperoxide in an erythrocyte suspension resulted in a rapid fall of the reduced glutathione level down to 0, when the rate of infusion exceeded the maximal rate of pentose phosphate pathway. Under these conditions the potassium ions liberation from the erythrocytes began with the drop of the reduced glutathione level down to zero, and the hemoglobin liberation - at the moment when more than 60% of potassium ions were liberated from the erythrocytes. The kinetics of potassium ion liberation remained unchanged in anisotonic media, but hemoglobin liberation from the erythrocytes greatly increased in hypotonic media as compared with isotonic ones. The kinetics of K+ and hemoglobin liberation were correlated only with lipid peroxidation but not with the oxidation of protein SH-groups.     

Reduced erythrocyte deformability in Krushinsky-Molodkina rats with acute hemorrhagic stroke

Acute hemorrhagic stroke in Krushinsky-Molodkina rats was used to assess the ability of erythrocytes to change their shape in a shear flow. Membrane rigidity and internal viscosity of erythrocytes were measured by laser diffractometry (i.e., obtaining a diffraction pattern from a thin layer of an erythrocyte suspension in a shear flow followed by computer processing of the image). The results testify to reduced deformability of erythrocytes under hemorrhagic stroke.     

Double Superhelix Model of High Density Lipoprotein

High density lipoprotein (HDL), the carrier of so-called “good” cholesterol, serves as the major athero-protective lipoprotein and has emerged as a key therapeutic target for cardiovascular disease. We applied small angle neutron scattering (SANS) with contrast variation and selective isotopic deuteration to the study of nascent HDL to obtain the low resolution structure in solution of the overall time-averaged conformation of apolipoprotein AI (apoA-I) versus the lipid (acyl chain) core of the particle. Remarkably, apoA-I is observed to possess an open helical shape that wraps around a central ellipsoidal lipid phase. Using the low resolution SANS shapes of the protein and lipid core as scaffolding, an all-atom computational model for the protein and lipid components of nascent HDL was developed by integrating complementary structural data from hydrogen/deuterium exchange mass spectrometry and previously published constraints from multiple biophysical techniques. Both SANS data and the new computational model, the double superhelix model, suggest an unexpected structural arrangement of protein and lipids of nascent HDL, an anti-parallel double superhelix wrapped around an ellipsoidal lipid phase. The protein and lipid organization in nascent HDL envisages a potential generalized mechanism for lipoprotein biogenesis and remodeling, biological processes critical to sterol and lipid transport, organismal energy metabolism, and innate immunity.     

Shape changes in goose erythrocytes

Goose erythrocytes were subjected to agents and treatments that produce echinocytosis in human erythrocytes. In the presence of the ionophore A23187 and calcium at greater than micromolar concentrations, goose red cells retained their normal ellipsoidal symmetry, but developed extensive semiregular membrane wrinkles or corrugations. Metabolic NTP (nucleoside triphosphate) depletion, induced either by iodoacetamide or by incubating the cells without a substrate, initially produced a similar cell corrugation, but after prolonged incubation most cells became spherical with the nucleus displaced to the cell periphery. The echinocytic agents indomethacin and dimyristoylphosphatidylcholine had no effect on the gross morphology of goose erythrocytes.     

Intercomparison of small biomolecules by gel filtration and small angle scattering

This paper describes how small angle neutron scattering (SANS) can be used to confirm that gel filtration results are free of dimerization effects. After characterization by analytical gel filtration, concentrated solutions (in heavy water, D2O) of a cobra neurotoxin, a cytotoxin, and a cytotoxin analog are studied by SANS. Small differences in shape are shown to be discernible by means of least-square fits to ellipsoidal models. The parallel axis theorem is then invoked to assess dimerization levels statistically. The results are briefly discussed on the basis of function in relation to structure.     

Neutron-induced gamma dose from a reactor beam filter for boron neutron capture therapy

For the boron neutron capture therapy (NCT) of deep-seated metastatic melanoma, an epithermal (up to a few keV energy) neutron beam from a reactor horizontal facility could be useful if the inherent contamination from fast neutrons and gamma rays could be minimised. Calculations for ANSTO's 10 MW research reactor HIFAR have shown that, even though a filter material such as AlF3 attenuates the fast neutron dose, the beam quality improvement is counteracted by a relative increase in the gamma dose because of the gammas arising from neutron captures in the filter material, particularly the aluminium. The aluminium gammas, most of which arise from thermal neutron capture, are hard and cannot be attenuated by lead or bismuth without comparable attenuation of the epithermal neutron flux. Addition of an absorber such as 6Li to the AlF3 filter was investigated as a means of reducing the hard gamma dose, but the improvement in beam quality was small and at considerable cost to dose intensity. Dose characteristics calculations confirmed the superiority of a tangential beam over a radial beam with better results from an unfiltered tangential beam than from an AlF3 filter in a radial beam. This study showed conclusively that assessments of filter assemblies based on the effect of individual components on either the neutron or gamma dose in isolation are inadequate. In assessing any epithermal neutron filter, thermal neutron shield, and gamma shield combination, the total effect of each on the neutron, gamma, and boron-10 dose must be considered.     

Cell age-dependent changes in deformability and calcium accumulation of human erythrocytes

The deformability of human erythrocytes was measured in a rheoscope, as a function of intracellular calcium content (varied with ionophore (A23187) and CaCl2) without complete ATP depletion and echinocytic transformation. Loading calcium into intact erythrocytes (calcium content: 16.8 mumol/1 packed cells = 1.48 amol per cell), the cell volume and energy charge gradually decreased. Further, the membrane fluidity of the lipid portion decreased without crosslinking of membrane proteins. A distinct transition from deformable to undeformable cells was observed by the rheoscope technique: i.e., 50% transition occurred at 40-50 mumol calcium/1 packed cells (= 3.5-4.0 amol per cell) and more than 90% above 100 mumol/1 packed cells (= 6.5 amol per cell) at a shear stress of 140 dyn/cm2. The deformable cells maintained their deformability to ellipsoidal disks independent of the average calcium content. The underformable cells, separated as high-density cells by density gradient centrifugation after calcium-loading, showed lower glucose-6-phosphate dehydrogenase activity than low-density-deformable cells; thus, the calcium-loaded, undeformable cells were presumably in vivo aged cells. The younger cells, fractionated as low-density cells from intact erythrocytes, were more deformable than aged cells. Upon calcium-loading, the younger cells restored their cell volume and deformability, while the aged cells, containing originally more calcium and less ATP, decreased their volume and became undeformable. Therefore, calcium accumulation by ionophore-CaCl2 takes place in preference to aged cells of lower energy metabolism, and leads to cellular dehydration and loss of deformability, due to condensed hemoglobin and altered membrane organization.     

Characterization of conformational deformation-coupled interaction between immunoglobulin G1 Fc glycoprotein and a low-affinity Fcγ receptor by deuteration-assisted small-angle neutron scattering

A recently developed integrative approach combining varied types of experimental data has been successfully applied to three-dimensional modelling of larger biomacromolecular complexes. Deuteration-assisted small-angle neutron scattering (SANS) plays a unique role in this approach by making it possible to observe selected components in the complex. It enables integrative modelling of biomolecular complexes based on building-block structures typically provided by X-ray crystallography. In this integrative approach, it is important to be aware of the flexible properties of the individual building blocks. Here we examine the ability of SANS to detect a subtle conformational change of a multidomain protein using the Fc portion of human immunoglobulin G (IgG) interacting with a soluble form of the low-affinity Fcγ receptor IIIb (sFcγRIIIb) as a model system. The IgG-Fc glycoprotein was subjected to SANS in the absence and presence of 75%-deuterated sFcγRIIIb, which was matched out in D₂O solution. This inverse contrast-matching technique enabled selective observation of SANS from IgG-Fc, thereby detecting its subtle structural deformation induced by the receptor binding. The SANS data were successfully interpreted by considering previously reported crystallographic data and an equilibrium between free and sFcγRIIIb-bound forms. Our SANS data thus demonstrate the applicability of SANS in the integrative approach dealing with biomacromolecular complexes composed of weakly associated building blocks with conformational plasticity.     

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.     

A Computational Model of a Microfluidic Device to Measure the Dynamics of Oxygen-Dependent ATP Release from Erythrocytes

Erythrocytes are proposed to be involved in blood flow regulation through both shear- and oxygen-dependent mechanisms for the release of adenosine triphosphate (ATP), a potent vasodilator. In a recent study, the dynamics of shear-dependent ATP release from erythrocytes was measured using a microfluidic device with a constriction in the channel to increase shear stress. The brief period of increased shear stress resulted in ATP release within 25 to 75 milliseconds downstream of the constriction. The long-term goal of our research is to apply a similar approach to determine the dynamics of oxygen-dependent ATP release. In the place of the constriction, an oxygen permeable membrane would be used to decrease the hemoglobin oxygen saturation of erythrocytes flowing through the channel. This paper describes the first stage in achieving that goal, the development of a computational model of the proposed experimental system to determine the feasibility of altering oxygen saturation rapidly enough to measure ATP release dynamics. The computational model was constructed based on hemodynamics, molecular transport of oxygen and ATP, kinetics of luciferin/luciferase reaction for reporting ATP concentrations, light absorption by hemoglobin, and sensor characteristics. A linear model of oxygen saturation-dependent ATP release with variable time delay was used in this study. The computational results demonstrate that a microfluidic device with a 100 µm deep channel will cause a rapid decrease in oxygen saturation over the oxygen permeable membrane that yields a measurable light intensity profile for a change in rate of ATP release from erythrocytes on a timescale as short as 25 milliseconds. The simulation also demonstrates that the complex dynamics of ATP release from erythrocytes combined with the consumption by luciferin/luciferase in a flowing system results in light intensity values that do not simply correlate with ATP concentrations. A computational model is required for proper interpretation of experimental data.