Edwin Donath

Electrophoretical study of cell surface properties.The influence of the surface coat on the electric potential distribution and on general electrokinetic properties of animal cells

Summary The electric potential distribution near a cell surface coated witha layer of charged glycoproteins and glycolipids (GPL) was calculated. Some distributions of the fixed charge inside the GPL with respect to the resulting electric potentials were analyzed. It is shown that the cell surface potential is no measure for a cell property such as the total fixed charge. Furthermore, the electrophoretic mobility of a cell with a charged GPL was claculated taking into account the frictional interaction of the liquid flow with the GPL molecules. A fundamental result is that the electrophoretic mobility of the cell does not tend to become zero at high ionic strenghts in contrast to the classic case of a smooth particle. A comparison with experimental data allowed the estimation of the thickness of the GPL. The result is in good agreement with the usually assumed thickness of this layer. Thus it follows that cell electrophoretical measurements become very promising for the direct study of the surface properties of the cell.

Influence of surface charge and transmembrane potential on rubidium-86 efflux of human red blood cells

SummaryThe dependence of the rate constant of Rb+ efflux on extracellular cation concentration was measured. At low ionic strengths Rb+ efflux increased strongly. Permeability coefficients were calculated from the rate constants measured, using the Goldman flux equation, with and without making allowance for surface potentials. Only when allowance was made for surface potentials and the associated differences beween ion concentrations in the bulk solutions and at the membrane surface, the permeability coefficient remained constant. Best agreement between experimental data and theoretically calculated values was obtained when an interior surface potential of − 110 mV was assumed.When the surface charge of erythrocytes is reduced by neuraminidase, the rate constants for Rb+ efflux decreased, indicating a significant influence of surface potential.

Charge distribution within cell surface coats of single and interacting surfaces—a minimum free electrostatic energy approach. Conclusions for electrophoretic mobility measurements

The fixed charge distribution in a cell surface coat model was calculated using a variational approach based on the minimum of electrostatic free energy. The influence of ionic strength on distribution was analyzed. It is shown that interaction with other coats or smooth surfaces changes the fixed charge distribution. The special effect of a possible membrane charge was calculated in addition. In this case there is a fixed, charge-free space near the membrane, depending on coat thickness, ionic strength and membrane charge/surface coat charge ratio. For several cases energies, electric potentials and fixed charge distributions are given. The general result is that fixed charge distribution is always composed of a constant space charge density over a part of or the whole surface coat and, with some exceptions, of a surface charge density at the border of the surface coat. Conclusions for electrophoretic mobility measurement interpretations are drawn.

The influence of the antiviral drugs amantadine and rimantadine on erythrocyte and platelet membranes and its comparison with that of tetracaine

The influence of the antivirus drugs amantadine and rimantadine and of the anionic analogue 1-adamantane-carboxylic acid on a range of properties of human erythrocyte membrane and of thrombocytes has been compared with the effect of the local anaesthetic tetracaine. At low antiviral drug concentrations the abilities of the drugs to induce erythrocyte shape change and suppress osmotic haemolysis were quantitatively proportional to their clinical potency (rimantadine more effective than amantadine at the same concentration). Rimantadine was also more effective than amantadine in suppressing influenza virus-erythrocyte fusion and viral induced haemolysis. The antiviral drug effects were qualitatively similar to those induced by tetracaine. At the quantitative level, tetracaine was more efficient than the antiviral drugs in inhibiting osmotic haemolysis, virus membrane fusion and platelet aggregation. In the absence of any specificity of the antiviral drug effects we argue for a lysosomotropic mode of drug action, i.e. that the drugs modify virus-membrane interactions by changing the endosomal or lysosomal pH.

Spectroscopic characterization of vesicle formation on heated human erythrocytes and the influence of the antiviral agent amantadine

EPR investigations on the vesiculation process of heated human erythrocytes were performed, using different fatty acid spin labels. Spectrin denaturation and vesiculation do not influence the fluidity of the lipid phase of the remaining membrane of human erythrocytes: Vesicles released differ in chemical composition as well as in the lipid fluidity of their membrane from the intact human erythrocyte membrane. A reduced cholesterol-to-phospholipid ratio and a depletion of spectrin was found. By changing the ionic concentration of the suspension medium an effect on membrane spectra and on vesicle release was established. The adamantane derivative amantadine causes fluidization of the human erythrocyte membrane and inhibits vesicle release. Based on these results, a model for the mechanism by which adamantane-like molecules could interact with membranes is proposed.

Electrorotation — a new method for investigating membrane events during thrombocyte activation. Influence of drugs and osmotic pressure

The measurement of the spin of cells in rotating high-frequency electric fields (electrorotation) make possible the investigation of dielectric membrane properties of single cells. This method was applied to membrane permeability changes accompanying thrombocyte activation and compared with light-scattering data. Describing the dielectric behavior of platelets by a single-shell model and assuming a sufficiently low membrane conductivity of 1 X 10(-7) S/m we found for nonactivated platelets a membrane capacity of 5.5 mF/m2 and the conductivity of the internal medium was estimated to be 0.12 S/m. Upon activation, the electrorotation decreased continuously, with half-times in the range of few minutes. This could be explained assuming a 500-fold increase in membrane conductivity. The application of both local anesthetics and virostatics inhibited the decrease of electrorotation, as did hypertonic osmotic pressure. In all cases this was accompanied by inhibition of platelet aggregation. Hypotonic solutions induced self-aggregation and spontaneous loss of electrorotation. It was concluded that the increase in permeability of the granule membrane is a crucial step in the release reaction and that the electrorotation method is able to detect the incorporation of the granule membranes into the plasma membrane during activation. The advantage of this electrorotation method is that it enables measurements on a single-cell level, thus avoiding interactions between platelets.

Measurement of inherent particle properties by dynamic light scattering: introducing electrorotational light scattering.

Common dynamic light scattering (DLS) methods determine the size and zeta-potential of particles by analyzing the motion resulting from thermal noise or electrophoretic force. Dielectric particle spectroscopy by common microscopic electrorotation (ER) measures the frequency dependence of field-induced rotation of single particles to analyze their inherent dielectric structure. We propose a new technique, electrorotational light scattering (ERLS). It measures ER in a particle ensemble by a homodyne DLS setup. ER-induced particle rotation is extracted from the initial decorrelation of the intensity autocorrelation function (ACF) by a simple optical particle model. Human red blood cells were used as test particles, and changes of the characteristic frequency of membrane dispersion induced by the ionophore nystatin were monitored by ERLS. For untreated control cells, a rotation frequency of 2 s-1 was induced at the membrane peak frequency of 150 kHz and a field strength of 12 kV/m. This rotation led to a decorrelation of the ACF about 10 times steeper than that of the field free control. For deduction of ERLS frequency spectra, different criteria are discussed. Particle shape and additional field-induced motions like dielectrophoresis and particle-particle attraction do not significantly influence the criteria. For nystatin-treated cells, recalculation of dielectric cell properties revealed an ionophore-induced decrease in the internal conductivity. Although the absolute rotation speed and the rotation sense are not yet directly accessible, ERLS eliminates the tedious microscopic measurements. It offers computerized, statistically significant measurements of dielectric particle properties that are especially suitable for nonbiological applications, e.g., the study of colloidal particles.

Conformational alterations within the glycocalyx of erythrocyte membranes studied by spin labelling

The structure of the glycocalyx of the membrane of human erythrocytes and spectrin-depleted vesicles was studied under various conditions by two spin-labelling approaches: covalently labelling sialic acid residues of the glycocalyx and incorporation of a charged hydrophobic spin probe, CAT 16, being sensitive to alterations on the membrane surface into the lipid phase. Although cell electrophoretic measurements which were performed, additionally, indicated an erection of the glycocalyx upon decreasing the ionic strength of the suspension medium a more restricted mobility of spin-labelled sialic acid residues was found, in this case probably due to electrostatic interactions. The enhanced mobility of the spin probe CAT 16 at low ionic strength as well as in the case of neuraminidase-treated cells could be caused by reduced steric and electrostatic interaction with glycoproteins and glycolipids. La3+ adsorption and virus attachment on the human erythrocyte membrane were accompanied with a reduced mobility of sugar headgroups of the surface coat. No indication of cluster formation or lateral segregation of glycophorin molecules was found upon virus binding. After denaturation of the spectrin cytoskeleton of intact erythrocytes, increased mobility of spin-labelled sialic acid residues was observed.

Free energy of the electrostatic interaction of cells with adjacent charged glycoprotein layer: A theoretical approach

Abstract The distance-dependent free energy of the electrostatic interaction is calculated for thick dielectric membranes of low dielectric constant in electrolyte solutions with adjacent charge-carrying glycoprotein layer (glycocalix). The charges are assumed to be continuously distributed. For the description of the space dependence of the charge density on the space coordinate perpendicular to the membrane surfaces various functions are used. The free energy corresponding to the space charge arrangement is determined by a stepwise charging process where the mobile ions are in thermodynamic equilibrium. For various charge arrangements of the interacting glycocalices the electric potential profile between the cells and the electrostatic contribution to the free energy are calculated. The model can be applied within the range of validity of the linearized Poisson-Boltzmann equation.

Electrorotation of particles measured by dynamic light scattering : a new dielectric spectroscopy technique

Electrorotation provides information about the dielectric and conductive properties of particles over a large frequency range, which is not easily available from other techniques. Numerous applications of the dielectric particle spectroscopy technique have already been demonstrated by microscopic measurements in the field of biology. Aware of the serious limitations of microscopy for applications in colloid science, we demonstrate that dynamic light scattering can be used to measure electrorotation spectra. A suitable chamber was designed which fits into a commercial correlation spectrometer. Measurements on human red blood cells showed that the apparent diameter of the particles recorded as a function of the frequency of the rotating field matches the microscopically measured electrorotation spectrum. However, the applicability of conventional dynamic light scattering sizing-devices to the recording of electrorotation spectra is limited, since the autocorrelation function in the presence of particle rotation is a superposition of zero-order Bessel functions. Consequently, new mathematical techniques have to be developed. Another limitation is that the direction of rotation is not yet detectable. Nevertheless, since the new method overcomes the restrictions of microscopic observations it offers a potentially wide range of applications for colloidal particles, vesicles, microemulsions, subcellular particles, and cells.