Electrophoresis in space. Foreword II.
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Biotechnology↗
Biomedical subjects
R S Snyder
Publications and source records attributed to R S Snyder.
Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.
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Biotechnology↗
Electrohydrodynamic effects in continuous flow electrophoresis.
We demonstrate experimentally and theoretically the importance of electrohydrodynamic (EHD) flows in continuous-flow electrophoresis (CFE) separations. These flows are associated with variations in the conductivity or dielectric constant, and are quadratic in the field strength. They appear to be the main cause of extraneous and undesired flows in CFE which have degraded separation performance and have until now not been explained. We discuss the importance of EHD flows relative to other effects. We also describe possible techniques for reducing the associated degradation of CFE separations.
Buffers↗
Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I.
A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable plate-like crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.
Antibodies, Monoclonal↗
Protein crystal growth in microgravity.
The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. space shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on Earth.
Animals↗
Application of some droplet sedimentation theories to layered erythrocyte suspensions.
The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehyde-fixed erythrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.
Animals↗
Cell separation by immunoaffinity partitioning with polyethylene glycol-modified protein A in aqueous polymer two-phase systems.
Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG-dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work we show that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, we provide a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.
Animals↗
Thermally reversible gels in electrophoresis. I: Matrix characterization.
Two series of thermally reversible, hydrogen-bonded gels have been characterized: 5% PVA-4% PEG and 5% PVA-0.04% borate gels. They both have extremely low melting points (16-17 degrees C) and could be of potential interest for recovery of proteins after preparative electrophoresis. The PVA-borate gels can be exploited in the pH range 7-11 by progressively increasing the borate content in the pH interval 8 to 7 and concomitantly decreasing the borate levels in the pH zone 8 to 11. It is hypothesized that the low melting point of these gels is due to the fact that they are sparingly and sparsely hydrogen bonded along the PVA chain: on the average, 1 -OH group out of 3 or 4 -OH's in the PVA polymer should be engaged in H-bond formation.
Electrophoresis↗
Phase partitioning in space and on earth.
In aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system consisting of a PEG-rich phase floating on top of a dextran-rich phase. Biological particles and macromolecules tend to partition differentially between the phases and the liquid-liquid phase interface in these systems. Bioparticle partitioning has been shown to be related to physiologically important surface properties such as membrane charge or lipid composition. Affinity partitioning into the PEG-rich phase can be accomplished by coupling PEG to a ligand having affinity for specific cells or macromolecules. Subpopulations can be identified or separated using multi-step countercurrent distribution (CCD). Incomplete understanding of the influence of gravity on the efficiency and quality of the impressive separations achievable by partitioning, and appreciation for the versatility of this efficient technique, have led to its study for low-gravity biomaterials processing. On Earth, two-phase systems rapidly demix because of density differences between the phases. In low-gravity, demixing has been shown to occur primarily by coalescence. Polymer surface coatings, developed to control localization of demixed phases in low-g, have been found to control electroosmosis which adversely affects electrophoretic separation processes on Earth and in space. In addition PEG-derivatized antibodies have been synthesized for use in immunoaffinity cell partitioning.
Dextrans↗
Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals.
A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.
Animals↗
Immuno-affinity partition of cells in aqueous polymer two-phase systems.
Poly(ethylene glycol) (PEG) was covalently coupled to IgG antibody preparations directed against human red blood cells. This modification reduces the tendency of the antibody to agglutinate cells and increases its affinity for the upper phase in dextran-PEG aqueous two-phase systems. These effects are related to the molecular weight of the PEG used for modification and to the number of PEG molecules attached to the antibody. Exposure of human red blood cells to PEG-modified antibody causes a substantial and specific increase in cell partition into the PEG-rich phase in a number of PEG-dextran aqueous two-phase systems. Pertinent phase-system parameters were examined. Following a single incubation with PEG-derivatized antibody, a mixture of sheep and human red blood cells was completely separated in 100 min by a 30-transfer countercurrent extraction using a two phase system which normally offers little resolution.
Animals↗
Vertical ascending electrophoresis of cells with a minimal stabilizing medium.
Vertical fractionation of a mixture of fixed horse and human red blood cells layered over a stabilizing support medium was done to give a valid comparison with proposed space experiments. In particular, the effects of sample thickness and concentration on zone migration rate were investigated. Electrophoretic mobilities of horse and human cells calculated from zone migration rates were compatible with those obtained by microelectrophoresis. Complete cell separation was observed when low power and effective cooling were employed.
Animals↗
Settling of fixed erythrocyte suspension droplets.
The fractionation of micron-size particles according to physical properties of size, density and surface characteristics by centrifugation and electrophoresis is hindered when the particles behave collectively rather than individually. The formation and sedimentation of droplets containing particles is an extreme example of collective behavior and a major problem for these separation methods when large quantities of particles need to be fractionated. In this paper, experiments that measured droplet sizes and settling rates for a variety of particles and droplets are described. Expressions are developed relating the particle concentration in a drop to measurable quantities of the fluids and particles. The number of particles in each droplet was then estimated along with the effective droplet density and certain trends are noted. Since a major application of this work is the purification of biological cells in the range of 10 microns, for which monodisperse inert particles are not available, red blood cells from different animals fixed in glutaraldehyde provided model particle groups with the necessary size range, visibility and stability for these fluid dynamical studies.
Animals↗
On the limiting pore size of hydrophilic gels for electrophoresis and isoelectric focusing.
The maximum pore diameter that can be obtained in hydrophilic gels, either highly diluted agarose (0.16%C) or highly cross-linked polyacrylamide (45%CBis or 60%C DHEBA) is around 500 nm. An empirical equation has been derived linking the mean pore diameter (mean p) to gel concentration (C) in dilute agarose gels: mean p = 140.7 x C(-0.7). It is suggested that other equations hold for concentrated gels and for highly cross-linked polyacrylamides, since the matrix structure is different. Most of the cross linkers for polymerizing polyacrylamide gels have been tabulated and their properties studied. A new gel matrix is described: a highly cross-linked N,N'-(1,2-dihydroxyethylene)bisacrylamide gel, which is hydrophilic, highly porous and can be conveniently used for electrophoresis in horizontal, ultrathin layers cast on silanized glass surfaces.
Electrophoresis↗
New experimental approaches to the isoelectric fractionation of cells.
Several parameters that could affect the survival rate and the resolution of cells in continuous-flow isoelectric focusing have been investigated. Cell survival at progressively lower pHs was found to be a function of the osmolarity of the medium: at pH 4 there was an absolute requirement for a 300 mOsm environment. A pH-dependent interaction of the carrier ampholytes with the cell surface has been demonstrated. The binding was very strong at pH 4, weak at pH 5 and totally abolished above pH 5. The same interaction was obtained with pentaethylenehexamine, the polyamino backbone of carrier ampholytes, stripped of carboxyl groups. A model was derived showing how at pH 4 the oligocationic carrier ampholytes, via their nitrogen groups, would bind to the polyanionic cell surface. The Hannig apparatus was adapted for cell separation in a continuously flowing curtain of isoelectric ampholytes. The medium osmolarity was maintained with glycine (delta pK 7.2), taurine (delta pK 8.5) and trimethylaminopropionyl sulphonate (TMAPS, delta pK 11). With increasing delta pK, the pH range that can be created in the presence of these compounds progressively widens, from a pH 4.3-7.5 range with glycine, to pH 3.5-7.5 with taurine up to pH 3.5-9.5 with TMAPS. The 48 fractions collected were routinely assayed for conductivity, osmolarity and pH.
Adult↗
Experimentation in space and biorheology.
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Cytological Techniques↗