Robert S. Snyder

Control of electroosmosis in coated quartz capillaries

Abstract Analytical particle microelectrophoresis was used to evaluate the effectiveness of various coatings for control of the electroosmotic fluid flow that hinders electrophoretic processes. Mobilities of 2-μm-diameter glass and polystyrene latex spheres, exhibiting both negative and zero effective surface charge, were measured in 2-mm-diameter quartz capillaries filled with NaCl solutions within the pH range of 3.5 to 7.8. Covalent coating of capillary inner surfaces with poly(ethylene glycol) caused a reduction in the degree of electroosmotic flow which was related to the poly(ethylene glycol) molecular weight. Poly(ethylene glycol) coatings of 5000 molecular weight, or higher, greatly reduced electroosmosis within the selected pH range, were stable for long periods of time, and appeared to be more effective than dextran, methylcellulose, or silane coatings. Because of these characteristics, poly(ethylene glycol) coatings should be of considerable use for improving various electrophoretic processes as well as in the production of standard particles exhibiting controlled electrophoretic mobilities.

Titration curves of proteins by combined isoelectric focusing-electrophoresis in highly porous polyacrylamide matrices

Abstract Highly cross-linked (highly porous) polyacrylamide gels as described in the literature were found to be poorly characterized support media. Diallyltartardiamide (DATD) cross-linked gels contain up to 80–90% unpolymerized DATD, which reacts with proteins and produces gluey and highly stretchable matrices. The conversion into polymer could not be affected by time or higher temperatures, since DATD is an inhibitor of polymerization, but only by adding very high amounts of persulphate. Increased levels of riboflavin actually decreased the polymerization efficiency. Highly cross-linked N,N′-methylenebisacrylamide (BIS) gels, at 40–50% C, are too hydrophobic and produce a collapsed matrix which keeps exuding water. A happy compromise are 30% C BIS gels, which are stable and allow unhindered migration of globular proteins up to 5·10 5 daltons. BIS and DATD gels can be stabilized by covalent binding to a glass surface precoated with Silane A-174. The order of reactivity for monomers appears to be: acrylamide > BIS ⪢ DATD. Polymerization conditions are described which allow better than 96% conversion of monomers into the polymer.

Dispersion effects in capillary zone electrophoresis

Abstract We qauntitatively analyzed possible causes of the observed spreading of sample components which degrades separation compared with theoretical limits, and identified four potentially significant causes, electrokinetic dispersion, wall adsorption, enhanced diffusion due to Poiseuille flows driven by pressure gradients, and enhanced diffusion due to mobility variations associated with transverse temperature differences. Electrokinetic dispersion is caused by changes in the conductivity and pH distributions, proportional to the concentration of the sample relative to the buffer components, and independent of the tube diameter. One-dimensional numerical results for the separation of seven species in a sodium acetate buffer are presented to illustrate the effect. A detailed discussion of methods to control it is also presented. It is suggested that both wall adsorption of sample species and most coating methods used to control it or to reduce electroosmosis can be understood as aspects fo the Debye double-layer theory. Large molecules, with a high degree of ionization with the opposite sign to the wall charge, are preferentially attracted to the layer, excluding smaller molecules, and decreasing the wall potential and mobility. We demonstrate the importance of choosing a combination of pH and tube material such that the wall and the large proteins in the sample have the same charge sign and repel each other. The diffusion enhancement analysis is based on the analytic approximation of balancing the flow and mobility distrubance terms in the concentration equation for a sample species with the transverse diffusion term. This determines the fluctuating part of the concentration, with zero transverse average. The interaction of this concentration distribution with the fluctuations modifies the equation for the transverse average of the concentration, by adding diffusivity a2Y2/48Di·. Here a is the radius, Di is the diffusivity of the species, and Y is a disturbance speed and is a sum of contributions from the pressure-gradient-driven Poiseuille flow, the transverse mobility variation due to the heating in the tube, and other effects which we believe are smaller. The numerical factor of 48 is exact for the Poiseuille flow and the transverse mobility variations effects, and presumably it improves the estimate for the other effects. The Poiseuille flow profile is driven by variations in the electroosmotic slip velocity, which are mostly caused by conductivity variations along the tube and by changes in the Debye layer structure associated with composition changes along the tube. Contributions from temperature or radius variations along the tube are estimated, and are apparently smaller. Relatively insignificant causes of dispersion include sample dispersion by the difference between the electroosmotic flow and the slower flow in the Debye layer (the layer is to thin), variations in the tube radius, and convection and electrohydrodynamics flows. Molecular diffusion is important (and theoretical plate numbers are achieved) if other dispersion effects are small; it is larger for sample species with small molecules.

Electrohydrodynamic distortion of sample streams in continuous flow electrophoresis

Abstract Prior theories involving electroosmotic, electrokinetics, and other effects have proved inadequate to explain the observed sample spreading and performance degradation in continuous flow electrophoresis (CFE). We suggest electrohydrodynamic flows as the main cause. These findings should contribute to efforts to improve the performance of CFE. It is shown theoretically that an electric field (AC or DC), perpendicular to a circular filament of conducting fluid surrounded by fluid of a different conductivity, produces an electrohydrodynamic flow, which distorts the filament into an ellipse. This distortion is similar to that described by G. I. Taylor for spherical drops. The major axis of the ellipse is either parallel to or normal to the field, depending on the conductivity and dielectric constant of the filament fluid relative to those of the surrounding fluid. For equal dielectric constants, the major axis is parallel to the field if the filament conductivity is greater than that of the surrounding fluid, and normal to the field otherwise. The flow and distortion rate is proportional to the square of the applied field. It is further shown theoretically that the flow associated with an elliptic cross section maintains the elliptic shape while it continues the distortion, provided the deviation from a circular cross section is small. As the ellipse stretches, small deviations from an elliptic cross section appear. It is shown, using an energy argument based on the assumption of an elliptic cross section, that the circular filament continues to flatten indefinitely, forming a ribbon, either parallel to the field or normal to it. The nature of the analysis, the physics of the flow, and the related experiments appear to confirm this behavior. For our experiments, we used an aqueous electrolyte (barbital buffer) and a sample of the same material with polystyrene latex added for visibility, in a CFE-type apparatus. The flow rate and configuration were typical of CFE. Electrokinetic and electrophoretic effects, and electroosmosis, were eliminated by using an AC field. Distinctive ribbons were formed, in both directions, at small fields of order 50 V/cm or less. All observations were consistent with the theories described above.

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.

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.

Effects of zero van der Waals and zero electrostatic forces on droplet sedimentation

Abstract The stability of concentrated suspensions of glutaraldehyde-fixed human erythrocytes layered on a D2O cushion was studied by taking as a criterion the maximum cell concentration that could be sustained without giving rise to droplet sedimentation. Greatest stability of the cell suspensions prevailed under conditions of zero van der Waals attraction and high negative cellular surface potential. The van der Waals attraction between the cells could be reduced to zero by lowering the surface tension of the medium by the admixture of 12% (v/v) dimethyl sulfoxide (DMSO), corresponding to a surface tension of the liquid of ≈65 ergs/cm2. This conforms closely to the surface tension of ≈64.5 ergs/cm2 found for the fixed erythrocytes by means of a freezing front technique. The electrostatic repulsion between the cells could be reduced to zero by the admixture of 5 × 10−8M lanthanum nitrate. In the region of zero electric charge, maximum stability, although occurring at significantly lower cell concentrations, also was achieved at zero van der Waals attraction. Mechanisms other than electrostatic repulsion and van der Waals attraction, such as density differences, mass diffusion, etc., clearly also play a role in droplet sedimentation.

Protein crystal growth results for shuttle flights STS-26 and STS-29

Abstract Recent advances in protein crystallography have significantly shortened the time and labor required to determine the three-dimensional structures of macromolecules once good crystals are available. Crystal growth has become a major bottleneck in further development of protein crystallography. Proteins and other biological macromolecules are notoriously difficult to crystallize. Even when usable crystals are obtained, the crystals of essentially all proteins and other biological macromolecules are poorly ordered, and diffract to resolutions considerably lower than that available for most crystals of simple organic and inorganic compounds. One promising area of research which is receiving widespread attention is protein crystal growth in the microgravity environment of space. A series of protein crystal growth experiments were performed on US shuttle flight STS-26 in September 1988 and STS-29 in March 1989. These proteins had been studied extensively in crystal growth experiments on earth prior to the microgravity experiments. For those proteins which produced crystals of adequate size, three-dimensional intensity data sets with electronic area detector systems were collected. Comparisons of the microgravity-grown crystals with the best earth-grown crystals obtained in numerous experiments demostrate that the microgravity-grown crystals of these proteins are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions. Analyses of the three-dimensional data sets by relative-Wilson plots indicate that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts.

Protein crystallization facilities for microgravity experiments

Abstract The first experiments demonstrating the enhancement of protein crystallization in microgravity were performed on the European Space Agencys first Spacelab mission in 1983 using the liquid/liquid interface diffusion technique and this experiment method will be continued on the European Retrievable Carrier. The current facility fromthe National Aeronautics and Space Administration employs a modified version of the “hanging drop” or the vapor diffusion technique. Protein crystal growth programs of NASA, ESA. and other national space programs now utilize a variety of instrumentation, and new methods are being developed to grow and study crystals of biological macromolecules in microgravity. This paper summarizes the present status of the NASA Vapor Diffusion Apparatus and ESA Protein Crystallization Facility.

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.