Dielectric Properties and Ion Mobility in Erythrocytes

The impedance of erythrocytes of man, cattle, sheep, dog, cat, rabbit, and chicken was measured in the range from 0.5 to 250 Mc. The dielectric constant of the red cell interior is 50 at 250 Mc, varies but little with species, and can readily be accounted for by the cells' hemoglobin content. The electrical conductivity of the red cell interior was determined between 70 and 100 Mc. The values differ from species to species within the rather limited range from 4.4 to 5.3 mmho/cm. Removal of the cell membranes does not affect the conductivity. Hence, the cell interior behaves, from an electrical point of view, like a highly concentrated hemoglobin solution. A theoretical value for the electrical conductivity of erythrocyte interiors, which is calculated on the basis of the salt content of the cell, ion mobility, and the volume concentration of the hemoglobin, is roughly twice as large as the measured value. This discrepancy is typical not only of the red blood cell. Pertinent measurements show that it is probably caused by hydrodynamic and possibly by electrostatic effects also, which lower the mobility of the ions. From the lower electrical mobility it appears that a lowered diffusion constant of the electrolytes and nonelectrolytes within the cell is indicated.     

Modifications in erythrocyte membrane zeta potential by Plasmodium falciparum infection

The zeta potential (ZP) is an electrochemical property of cell surfaces that is determined by the net electrical charge of molecules exposed at the surface of cell membranes. Membrane proteins contribute to the total net electrical charge of cell surfaces and can alter ZP through variation in their copy number and changes in their intermolecular interactions. Plasmodium falciparum extensively remodels its host red blood cell (RBC) membrane by placing 'knob'-like structures at the cell surface. Using an electrophoretic mobility assay, we found that the mean ZP of human RBCs was -15.7 mV. In RBCs infected with P. falciparum trophozoites ('iRBCs'), the mean ZP was significantly lower (-14.6 mV, p<0.001). Removal of sialic acid from the cell surface by neuraminidase treatment significantly decreased the ZP of both RBCs (-6.06 mV) and iRBCs (-4.64 mV). Parasite-induced changes in ZP varied by P. falciparum clone and the presence of knobs on the iRBC surface. Variations in ZP values were accompanied by altered binding of iRBCs to human microvascular endothelial cells (MVECs). These data suggest that parasite-derived knob proteins contribute to the ZP of iRBCs, and that electrostatic and hydrophobic interactions between iRBC and MVEC membranes are involved in cytoadherence.     

The Electrophoretic Velocity of Human Red Cells, of Their Ghosts and Mechanically Produced Fragments, and of Certain Lipid Complexes

Ghosts prepared in CO₂-saturated water from unwashed human red cells can be fragmented mechanically, but ghosts from thrice washed cells cannot. If the ghosts are prepared by freezing and thawing, this difference is not observed. The electrophoretic velocity varies also with the way in which the ghosts are prepared. The pH-mobility dependence of washed red cells flatten off to a plateau at pH 9, and the electrophoretic velocity is zero at about pH 2. Ghosts prepared by freezing and thawing have almost the same pH-mobility dependence, but if the ghosts are prepared in CO₂-saturated hyptonic saline, the mobility at pH 9.4 is 0.75 times that of washed cells. Fragments of ghosts of unwashed red cells have a smaller mobility than that of the red cells. Trypsin reduces the mobility of washed red cells and of ghosts. Sols of lipid complexes (lecithin, cephalin, and lipositol), at varying pH's, have a mobility 1.2 times that of the washed red cell. The pH-mobility relation is otherwise similar. These complexes can be coated with dextran and trypsin.     

Different red cell populations in newborn, genetically low potassium sheep: relation to hematopoietic, immunologic and physiologic differentiation

Three red cell populations have been distinguished in genotypically low potassium (LK) newborn sheep by an improved electrical sizing method and were best approximated by a logarithmic normal distribution. Labeling studies with ⁵¹Cr and ⁵⁹Fe exclude transformation of the three red cell populations into each other. Population I, consisting of large red cells (mean volume 36 μm³), with a comparatively slow electrophoretic mobility is present at birth and disappears within three to four weeks from circulation. These cells possess a high potassium (HK) steady state concentration, a K⁺ pump influx activity at least 5-fold greater than observed in adult LK red cells, very low amounts of the L antigens generally associated with the LK property, and do not respond to the stimulatory action of the L antibody. The first population is gradually replaced by population II comprising small red cells (mean volume 28 μm³) of intermediate electrophoretic mobility and with a peak production around day 20 after birth. The potassium concentration, [K⁺]_c, in these cells appears to be lower than in the cells of population I but the L antigen content is increased. Formation of population III (mean volume 30 μm³ and comparatively fast electrophoretic mobility) follows closely that of population II and is preceded by a sharp increase in reticulocytosis. The red cells of population III exhibit parameters characteristic for adult LK cells: low [K⁺]_c and K⁺ pump activity, fully developed L antigen content, and an almost maximal response to the K⁺ pump stimulating effect of anti-L. In L and M antigen positive LK red cells of newborn sheep, the development of the M antigen parallels that of the L antigen. The data are consistent with the hypothesis that cellular replacement and not maturation is the major factor in controlling the HK-LK transition in newborn sheep.     

Lateral mobility of proteins and lipids in the red cell membrane and the activation of adenylate cyclase by beta-adrenergic receptors

Models of beta-adrenergic signal transduction in red blood cell membranes frequently assume that at least one of the membrane-bound components is laterally mobile and distributes the hormonal signal in the membrane plane. However, direct measurements reveal that protein lateral mobility in the red cell membrane is severely restricted. Furthermore, the spectrin-actin compartmentalizes the cytoplasmic face of the red cell membrane into a regular array of small elementary areas. These considerations support models in which the beta-adrenergic signal is spread in the membrane plane by a molecule which has binding sites on the membrane but diffuses in the aqueous compartment.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

Lateral mobility of integral proteins in red blood cell tethers.

The red blood cell membrane is a complex material that exhibits both solid- and liquidlike behavior. It is distinguished from a simple lipid bilayer capsule by its mechanical properties, particularly its shear viscoelastic behavior and by the long-range mobility of integral proteins on the membrane surface. Subject to sufficiently large extension, the membrane loses its shear rigidity and flows as a two-dimensional fluid. These experiments examine the change in integral protein mobility that accompanies the mechanical phenomenon of extensional failure and liquidlike flow. A flow channel apparatus is used to create red cell tethers, hollow cylinders of greatly deformed membrane, up to 36-microns long. The diffusion of proteins within the surface of the membrane is measured by the technique of fluorescence redistribution after photobleaching (FRAP). Integral membrane proteins are labeled directly with a fluorescein dye (DTAF). Mobility in normal membrane is measured by photobleaching half of the cell and measuring the rate of fluorescence recovery. Protein mobility in tether membrane is calculated from the fluorescence recovery rate after the entire tether has been bleached. Fluorescence recovery rates for normal membrane indicate that more than half the labeled proteins are mobile with a diffusion coefficient of approximately 4 x 10(-11) cm2/s, in agreement with results from other studies. The diffusion coefficient for proteins in tether membrane is greater than 1.5 x 10(-9) cm2/s. This dramatic increase in diffusion coefficient indicates that extensional failure involves the uncoupling of the lipid bilayer from the membrane skeleton.     

Electrokinetic behavior of inside-out vesicles from human red cell membranes

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (<5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.     

Hemolytic anemia due to pyruvate kinase deficiency: characterization of the enzymatic activity from eight patients.

We have studied the red cell pyruvate kinase (PK) variants from eight patients representing five families with pyruvate kinase deficiency-associated hemolytic anemia. The kinetic properties, electrophoretic mobilities, and immunological reactivity with anti-normal red cell pyruvate kinase were determined. The patients differ in the severity of their clinical condition and in the molecular properties of their red cell pyruvate kinase variants. The most seriously affected patient (PK Beaverton) has no electrophoretically demonstrable red cell isozymes. The activity present is due to the M2 isozyme, however red cell isozyme can be detected immunologically. PK Molalla and PK Lake Oswego are thermolabile variants with normal kinetic parameters. PK Molalla, in addition, has altered electrophoretic mobility. PK Multnomah and PK Milwaukie have decreased affinity for the substrate phosphoenolpyruvate, and PK Multnomah also has altered electrophoretic mobility. PK Coos Bay shows electrophoretic variation and a slightly decreased affinity for phosphoenolpyruvate consistent with an increased modulating effect of fructose-1,6-diphosphate.     

EFFECT OF PROTEINS ON ELECTROPHORETIC MOBILITY AND SEDIMENTATION VELOCITY OF RED CELLS

The isoelectric point of normal red cells cannot be measured but is certainly lower than that of any plasma protein. Red cells are easily damaged so that they will adsorb proteins from low concentrations. Normal red cells do not adsorb protein even from concentrated solutions, as is evidenced by the finding that the ratio of the mobility of the cells to that of the proteins themselves is at least as high in concentrated casein, albumin, gelatin, or fibrinogen solutions as in dilute. The finding that the observed mobility of red cells is unchanged or only slightly decreased when bulk viscosity is increased by added protein is interpreted as indicating that the red cell surfaces are hydrated. The aggregating effect of certain proteins has been determined and is assumed to be due to their dehydrating effect on the cells. Some types of cells, as beef, are not aggregated, presumably because they are resistant to this dehydrating effect. The difference in the behavior of different types of red cells demonstrates the importance of the nature of the cell as well as of the medium in determining the rate of aggregation and therefore of sedimentation.     

Reaction of colloidal silica with membranes of intact mammalian cells

Colloidal silical particles were produced at a size that permitted reaction with human erythrocytes and rat macrophages without affecting cell integrity. Binding of colloid was shown by increased electrophoretic mobility of red cells and also resulted in changes in the surface topography of red cells as seen with scanning electron microscopy. The degree to which colloid binds to red cells was determined by microprobe analysis of single intact cells. Furthermore, the capacity of red cells to bind silica was increased if sialic acid residues were removed enzymatically from the cell surface.     

Aging of erythrocytes results in altered red cell surface properties in the rat, but not in the human. Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

The absence of electrically demonstrable polarizing factors in Hydra

Various investigators have shown that in the marine hydroids, Tubularia, Obelia, Eudendrium, and Pennaria, regeneration and polarity is affected by an electrical field applied parallel to the regenerate. Using electrical currents up to the physiological limits for Hydra, no relation between electrical current and regeneration rate or polarity could be demonstrated. This is in spite of the fact that adult Hydra are normally electrically polarized with the distal end approximately–18 mV relative to the proximal end of the animal. When the electrophoretic mobility and isoelectric point of cells from distal, central and proximal thirds of Hydra were measured, a significant difference was found between cells of the two cell layers but not between cells of the three body thirds. These results are discussed in relation to Hydra growth factors described by various other authors.     

Label-free determination of the number of biomolecules attached to cells by measurement of the cell's electrophoretic mobility in a microchannel

We developed a label-free method for a determination of the number of biomolecules attached to individual cells by measuring the electrophoretic mobility of the cells in a microchannel. The surface of a biological cell, which is dispersed in aqueous solution, is normally electrically charged and the charge quantity at the cell's surface is slightly changed once antibody molecules are attached to the cell, based on which we detect the attachment of antibody molecules to the surface of individual red blood cells by electrophoretic mobility measurement. We also analyzed the number of antibody molecules attached to the cell's surface using a flow cytometer. We found that there is a clear correlation between the number of antibody molecules attached to the individual cells and the electrophoretic mobility of the cells. The present technique may well be utilized not only in the field of cell biology but also in the medical and pharmaceutical industries.     

Intracellular Localization and Dichroic Orientation of Phytochrome in Plasma Membrane and/or Ectoplasm of a Centrifuged Protonema of Fern Adiantum capillus-veneris L.

Protonemata of the fern Adiantum capillus-veneris grown undercontinuous red light for 6 days were kept in darkness for 15h and subsequently centrifuged 3 times in different directions,so that oil droplets and other cytoplasm were removed from theapical region of the protonemata. Electron micrographs clearlydemonstrated that cell wall, plasma membrane, ectoplasm andmicrotubules remained in the apical and subapical regions afterthe centrifugal treatments. A brief local exposure of the flankof the subapical region of the centrifuged protonemata to amicrobeam of red light effectively induced a phototropic responsetoward the irradiated side, suggesting that phytochrome is locatedin the ectoplasm and/or plasma membrane. When the flank of thecentrifuged protonema was irradiated with linearly polarizedred or far-red light, red light with an electrical vector parallelto the cell surface was more effective than that perpendicularto the cell surface. The direction of the electrical vectorof far-red light for reversion of the preirradiated red lighteffect, however, was opposite. These results suggest that differentdichroic orientations of P_R and P_FR exist in the plasma membraneor ectoplasm. (Received May 26, 1983; Accepted September 1, 1983)     

STUDIES OF CELL DEFORMABILITY : IV. A Possible Role of Calcium in Cell Contact Phenomena

Cells grown in suspension culture were incubated with EDTA-disodium salt and shown to have more easily deformable surfaces and raised electrophoretic mobility than controls, following this treatment. The reversibility of these observations by the addition of calcium ions, and other parallel experiments, support the conclusion that, in these cells, calcium is bound to anionic sites at the cell periphery, some of which are located at the cellular electrokinetic surface. These cells should, therefore, exhibit demonstrable calcium-sensitive aggregation, if current theories on the role of calcium in the physiological situation are correct. The fact that no calcium-sensitive aggregation was observed suggests that calcium does not form "bridges" between the adjacent anionic sites on different cells, and does not act directly by its effects on the diffuse electrical double-layer in this situation. An alternative hypothesis is advanced for the role played by calcium in cell adhesion and separation processes.     

Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane

Intracellular Ca2+ at concentrations ranging from 0 to 10 mumol/l increases the shear modulus of surface elasticity (mu) and the surface viscosity (eta) of human red blood cells by 20% and 70%, respectively. K+ selective channels in the red cell membrane become activated by Ca2+. The activation still occurs to the same extent when the membrane skeleton is degraded by incorporation of trypsin into resealed red cell ghosts, suggesting that the channel activation is not controlled by the proteins of the membrane skeleton and is independent of mu and eta. Incorporation of trypsin at concentrations ranging from 0 to 100 ng/ml into red cell ghosts leads to a graded digestion of spectrin, a cleavage of the band 3 protein and a release of the binding proteins ankyrin and band 4.1. These alterations are accompanied by an increase of the lateral mobility of the band 3 protein which, at 40 ng/ml trypsin, reaches a plateau value where the rate of lateral diffusion is enhanced by about two orders of magnitude above the rate measured in controls without trypsin. Proteolytic digestion by 10-20 ng/ml trypsin leads to a degradation of more than 40% of the spectrin and increases the rate of lateral diffusion to about 20-70% of the value observed at the plateau. Nevertheless, mu and eta remain virtually unaltered. However, the stability of the membrane is decreased to the point where a slight mechanical extension, or the shear produced by centrifugation results in disintegration and vesiculation, precluding measurements of eta and mu in ghosts treated with higher concentrations of trypsin. These findings indicate that alterations of the structural integrity of the membrane skeleton exert drastically different effects on mu and eta on the one hand and on the stability of the membrane on the other.     

Various topochemical arrangements of sialic acids on human erythrocytes as detected by partition in aqueous two-polymer phase systems

Human and rabbit red blood cells were subjected to partition in an aqueous, buffered Ficoll-Dextran two-phase system. The effect of neuraminidase treatment on the cell partition behaviour was examined and compared with the amount of sialic acids released from the cell surface and with the change in the electrophoretic mobility of the cells. The data obtained in the study indicate that there are two main groups of sialic acids differing in their topochemical arrangement on the human erythrocyte surface, and their relative hydrophobicity was evaluated. The results obtained in the case of rabbit red cells seem to indicate that sialic acids present on the cell surface are not the only major ionogenic surface components as is the case for human red cells. The data obtained support the assumption that the membrane surface charge is the determinant of cell partition only as a factor affecting the relative hydrophobicity of the cell surface.     

Red cell membrane protein lateral mobility in diabetes mellitus.

In a group of 24 diabetics subdivided for type, we evaluated the red cell membrane protein lateral mobility marking intact red cells with pyrene-3-maleimide (3-PM) and calculating the dimer to monomer fluorescence intensity ratio (Iex/Im). The same fluorescent parameter was determined in a group of 13 normal controls. From the obtained data, it is evident that the red cell membrane protein lateral mobility clearly discriminates normals from diabetics of type 1 and 2. In normals and in diabetics of type 1 and 2 no relationship is present between this fluorescent determinant and the glycometabolic parameters (FBGL and HbA1c); considering all the diabetics, a negative relationship is evident between Iex/Im ratio and HbA1c only.