Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

Electrophoresis of colloidal biological particles

Microscope electrophoresis was used to measure the electrophoretic mobility of polystyrene latex particles and bacterial, and mammalian tissue cells. The submicroscopic hydrophilic colloids (gelatin, serum albumin, and staphylococcal enterotoxin B) were adsorbed on latex carrier particles to determine their electrophoretic mobility and the effect of concentration, pH, electrolyte addition, and buffer ionic strength. Mobility curves as a function of pH were established for latex particles at 1 ppm concentration indicating an isoelectric point (IEP) at pH 3.6. The IEP for Escherichia coli B cells was measured at pH 2.8, Serratia marcescens at pH 2.6, Bacillus subtilis var. niger at pH 2.9, and L strain mouse fibroblast cells at pH 4.4. Using an adsorption technique, isoelectric points were measured for proteins: gelatin (acid form) at pH 9.4, serum albumin at pH 4.9, and staphylococcal enterotoxin B at pH 6.3. Procefures for examining electrophoretic characteristics of microscopic and submicroscopic biological particles are described in order to standardize procedures and to generate results applicable to an understanding of parameters influencing concentration and purification of colloidal biological particles.     

Surface characterization and on-line activity measurements of microorganisms by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to the electrophoretic characterization for microorganisms. The electrophoretic peaks detected using light scattering phenomena were characteristic of the microorganisms used. The electrophoretic mobility (μ) evaluated by CZE was in good agreement with that obtained by classical electrophoresis of microorganisms. The migration time was reproducible and depended on the ionic strength (I). Analysis of the μ vs. I relationship provided information regarding the charge density and the hardness of the microbial cell surface. The redox enzymatic activity of microorganisms was also evaluated by CZE using a running buffer containing a corresponding substrate and an appropriate exogenous electron acceptor. A decrease in the concentration of the electron acceptor due to microbial activity can be simultaneously monitored during the electrophoretic process without significant modification of the CZE instrument. Effects of some chemical treatments of microbial cells were also studied using this technique.     

An evaluation of the outer membrane charge and softness of<Emphasis Type="Italic">Thiobacillus ferrooxidans</Emphasis> by the Ohshima's electrophoretic model of a “soft” particle

The surface charge of bacterial cells plays an important role in their interfacial physiology and adhesion to substrata mediated by the electrostatic double-layer interaction. The surface charge or potential of biological cells is generally calculated from the experimentally measurable electrophoretic velocity of these cells migrating in an external electric field, applying the well-known Smoluchowski equation which is valid for “hard” particles with a sharp interface. However, bacterial cells possessing a structured outer membrane of a finite thickness (dependent on the ionic strength and pH of the surrounding liquid medium) are expected to obey Ohshima's electrophoretic mobility equation derived recently for ‘soft” particles. The electrophoretic mobility ofThiobacillus ferrooxidans was measured here by the fully automated technique of electrophoretic light scattering, based on the proportionality between the mobility and the Doppler shift in the frequency of light scattered by electrophoresing cells. Agreement was obtained between the experimentally determined electrophoretic mobility expressed as a function of low ionic strength (60–6000 μmol/L) at different pH values and the best-fit theoretical predictions of the “soft” particle electrophoresis theory, which is better than in the case of applying the Smoluchowski formula. The best-fit surface-charge and softness parameters predict a rather rigid and low-charge outer membrane of the bacterium examined, as compared to the parameters obtained for other bacteria in media of high ionic strength.     

Translocation of hyaluronic acid in cell surface of cultured mammalian cells after X-irradiation and its recovery by added adenosine triphosphate

To investigate the mechanism of radiation-induced decrease in cell electrophoretic mobility and its recovery by added adenosine triphosphate, specific enzymes and buffer solutions of different ionic strength were utilized. Decrease in the mobility of irradiated cells was detected only with the buffer solution of ionic strengths higher than 0.100. In this range of ionic strengths, removal of hyaluronic acid from cell surface by hyaluronidase had no effect on the electrophoretic mobility of irradiated cells, while the enzyme treatment resulted in 27% mobility reduction in non-irradiated cells. The removal of sialic acid and chondroitin sulfate by their specific enzymes resulted in the similar decrease in mobility either in irradiated and non-irradiated cells. These results suggest that the X-ray induced translocation of hyaluronic acid from the peripheral zone of 0–7.5 Å into the deeper zone of about 10–17 Å, if we use the Debye-Hückel's thickness of ion atmosphere for an approximate estimate of effective depth of electrokinetic plane of shear. Hyaluronic acid reappeared to the peripheral zone by the subsequent incubation after small dose irradiation, or by the addition of 1 mM adenosine triphosphate with Ca²⁺.     

Probing the electrostatic shielding of DNA with capillary electrophoresis

The free solution mobility of a 20-bp double-stranded DNA oligomer has been measured in diethylmalonate (DM) and Tris-acetate buffers, with and without added NaCl or TrisCl. DM buffers have the advantage that the buffering ion is anionic, so the cation composition in the solution can be varied at will. The results indicate that the free solution mobility of DNA decreases linearly with the logarithm of ionic strength when the ionic strength is increased by increasing the buffer concentration. The mobility also decreases linearly with the logarithm of ionic strength when NaCl is added to NaDM buffer or TrisCl is added to TrisDM buffer. Nonlinear effects are observed if the counterion in the added salt differs from the counterion in the buffer. The dependence of the mobility on ionic strength cannot be predicted using the Henry, Debye-Hückel-Onsager, or Pitts equations for electrophoresis. However, the mobilities observed in all buffer and buffer/salt solutions can be predicted within approximately 20% by the Manning equation for electrophoresis, using no adjustable parameters. The results suggest that the electrostatic shielding of DNA is determined not only by the relative concentrations of the various ions in the solution, but also by their equivalent conductivities.     

Comparison of mammalian mitochondrial ribosomal ribonucleic acid from different species

Mitochondrial ribosomal RNA species from mouse L cells, rat liver, rat hepatoma, hamster BHK-21 cells and human KB cells were examined by electrophoresis on polyacrylamide-agarose gels and sedimentation in sucrose density gradients. The S(E) (electrophoretic mobility) and S values of mitochondrial rRNA of all species were highly dependent on temperature and ionic strength of the medium; the S(E) values increased and the S values decreased with an increase in temperature at a low ionic strength. At an ionic strength of 0.3 at 23-25 degrees C or an ionic strength of 0.01 at 3-4 degrees C the S and S(E) values were almost the same being about 16.2-18.0 and 12.3-13.6 for human and mouse mitochondrial rRNA. The molecular weights under these conditions were calculated to be 3.8x10(5)-4.3x10(5) and 5.9x10(5)-6.8x10(5), depending on the technique used. At 25 degrees C in buffers of low ionic strength mouse mitochondrial rRNA species had a lower electrophoretic mobility than those of human and hamster. Under these conditions the smaller mitochondrial rRNA species of hamster had a lower electrophoretic mobility than that of human but the larger component had an identical mobility. Mouse and rat mitochondrial rRNA species had identical electrophoretic mobilities. Complex differences between human and mouse mitochondrial rRNA species were observed on sedimentation in sucrose density gradients under various conditions of temperature and ionic strength. Mouse L-cell mitochondrial rRNA was eluted after cytoplasmic rRNA on a column of methylated albumin-kieselguhr.     

Determination of the degree of substitution and its distribution of carboxymethylcelluloses by capillary zone electrophoresis

A method based on capillary zone electrophoresis (CZE) has been developed to determine the degree of substitution (DS) of carboxymethylcellulose (CMC). Separations were performed with borate buffer (pH 9, ionic strength 20 mM) as background electrolyte in capillaries of 75 microm ID, with an applied voltage of 10 kV, and for detection UV absorption at 196 nm was measured. The use of an internal standard (phthalic acid) to correct for mobility variations resulted in a strong improvement of the precision of the DS determination. Experiments with indirect UV detection indicated that the peak widths obtained actually reflect the variation in mobility, and with that of the DS value, of CMC samples. With the proposed method not only the average DS value but also its dispersity could be established for technical CMC samples. A small but definite effect of the polymeric size on the mobilities was observed. Therefore, DS calibration curves will have to be determined for a specific MM range. Since the size effect is small, a classification of CMCs as low-, middle-, or high MM will be sufficient to obtain accurate data on the DS distribution.     

Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system

Phosphorylated and nonphosphorylated forms of peptide substrates for protein kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were separated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillaries yielded asymmetric analyte peaks with substantial peak tailing. Some of the peptides also exhibited broad peaks with unstable migration times. To improve the electrophoretic separation of the peptides, several strategies were implemented: extensive washing of the capillary with a base, adding betaine to the electrophoretic buffer, and coating the capillaries with polydimethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base substantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both the migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvements in the resolving power of the separations. These modifications all utilized an electrophoretic buffer that was compatible with a living biologic cell. Consequently they should be adaptable for the new capillary electrophoresis-based methods to measure kinase activation in single cells.     

Electrophoresis of adsorbed DNA

The electrophoresis mobilities of native calf thymus DNA adsorbed on the charged solid particles were measured by a micro-electrophoretic method as functions of pII, ionic strength, and DNA concentration. The mobility data confirm the adsorption of DNA both on the positively charged alumina and negatively charged resin particles at wide range of pH and ionic strength. The mobility data also indicate significant DNA adsorption by negatively charged glass in the acidic range of pH. The electrophoretic mobilities of DNA adsorbed on different substrate particles under identical conditions do not differ widely, indicating the major role of the adsorbed DNA rather than the covered substrate in controlling the charge behavior of the particle. The mobilities of the adsorbed DNA at salt pH are of a comparable order of magnitude to those for the dissolved DNA in solution. The mobility of the adsorbed heat-denatured and alkali-denatured DNA is lower than that of the native adsorbed DNA under identical conditions of pH and ionic strength.     

Do zwitterions contribute to the ionic strength of a solution?

Capillary electrophoresis has been used to determine whether zwitterions contribute to the ionic strength of a solution, by measuring the mobility of a double-stranded DNA oligomer in cacodylate-buffered solutions containing various concentrations of the ionic salt tetraethylammonium chloride (TEA(+)Cl(-)) or the zwitterion tricine(+/-). The mobility of the DNA decreased as the square root of ionic strength, as expected from the Debye-Hückel-Onsager theory of electrophoresis, when TEA(+)Cl(-) was added to the buffer. However, the mobility was independent of the concentration of added tricine(+/-). Hence, zwitterions do not contribute to the ionic strength of a solution.     

Forced-flow electrophoresis of proteins and viruses

This article describes a model for forced-flow electrophoresis (FFE), considering the desired species fraction removal, other fraction removals, and outlet concentrations of all species in the system. The model predicts the necessary inlet flow rate of the retentate chamber and the rate of filtration and the voltage gradient and also provides an appropriate heat balance permitting consideration of possible heat denaturation of the species. The equipment consists of two membranes and a filter, the electric field being imposed by means of external electrodes, and two fractions are obtainable. The main discriminating factor is not the pore sizes of the filter but the relative solute ionization, which depends on the pH and the ionic strength of the buffer solution. Serum proteins (albumin, gamma-globulin) and bacteriophages (M13, MS2, phiX174) have been used to characterize the separation process.     

Heterogeneity of leghemoglobin from yellow lupine nodules

A possibility is demonstrated to separate summary lupine leghemoglobins (which are salted out within 55--90% of ammonium sulphate saturation) into Lb I and Lb II components by means of ionic exchange chromatography on DEAE-cellulose. Lb I is eluted at lower ionic strength buffer than LbII, and differs from the latter in the form and the size of crystals. Both components have the same electrophoretic mobility and contain N-terminal glycine. LbII and LbI precipitate under gradual salting out within 55--75% and 78--90% of saturation respectively.     

Separation of large circular DNA by electrophoresis in agarose gels

The electrophoresis of circular DNA, ranging in size from 4.4 kilobase pairs (kbp) to 220 kbp, was studied in agarose gels. Bacterial artificial chromosome (BAC) DNA was used as a source of large supercoiled and open circular (relaxed) forms. The open circles above approximately 50 kbp were trapped at the sample wells of 1% agarose gels during electrophoresis at 3 V/cm. Field inversion gel electrophoresis (FIGE) was used to relieve the trapping of the open circles in the gels. Using FIGE (30 s forward pulse time), open circles with sizes of 115 and 220 kbp required reverse pulse times of 3 and 6 s, respectively, to free the circles from open-ended gel fibers. A minimum in the gel velocity of the open circles was measured at approximately 20 kbp. Open circles below approximately 20 kbp migrated slower than the supercoiled forms, and above 20 kbp the order was reversed. These results indicate that when the size of the open circles exceeded the average pore size of a gel and it was forced to span multiple pores, the open circles gained a mobility advantage. Decreasing the ionic strength of the electrophoresis buffer significantly decreased the mobility of the smaller circles and slightly increased the mobility of the larger circles.     

Free solution mobility of oligomeric DNA

The free solution electrophoretic mobility of a charged oligomer in an ionic solvent that approximately takes into account relaxation field effects, screening of the velocity field, and the hydrodynamic interactions resulting from motions of the charges due to an electric field is described. For double‐stranded DNA, the free solution electrophoretic mobility under ionic strengths determined by the buffer and pH conditions relevant to capillary electrophoresis increases with increasing molecular weight up to few hundred base pairs. © 1999 John Wiley & Sons, Inc. Biopoly 49: 209–214, 1999     

Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides

Excellent correlation was observed for the electrophoretic mobilities measured by capillary zone electrophoresis versus q/MW2/3, where q is the calculated charge and MW is the molecular weight. Mobilities of a set of 33 diverse peptides from enzymatic digests and 10 intact proteins were measured for separations at pH 2.35, 8.0, and 8.15 with constant ionic strength, temperature, and viscosity. The correlation suggests that the frictional drag is proportional to the surface area of a sphere that has a volume proportional to the MW. The correlation of electrophoretic mobility with physicochemical properties will facilitate the elucidation of optimum separation strategies for protein and peptide mixtures.     

ELECTROPHORETIC STUDIES ON HUMAN RED BLOOD CELLS

1. The electrophoretic mobility of unhemolyzed human red cells has been determined as a function of ionic strength at approximately constant pH in isotonic mixtures of glucose solution and saline-phosphate buffer solution. 2. Above an ionic strength of about 0.02 the cells behave as particles with a smooth surface of large radius of curvature. Below an ionic strength of about 0.02, changes of the surface occur, probably involving a decrease of charge density and perhaps connected with injury of the surface. 3. The mobility as a function of pH at an ionic strength of 0.172 has been determined for human red cells, for the lipid extract of the cells, and for the stroma protein of the cells. The isoelectric points of cells, lipid, and protein have been found to be about 1.7, 2.6, and 4.7 respectively. 4. The pH-mobility data lead to the conclusion that a red cell surface is composed largely of lipid and dominated by strong acid groups, possibly the phosphoric acid groups of cephalin molecules.     

Analysis of the proteoglycans synthesized by human bone cells in vitro

Proteoglycans were isolated by ion-exchange chromatography from the extracted cell layer and culture medium of human bone cell cultures following incubation in the presence of [35S]sulfate and [3H]leucine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the synthesized proteoglycans consisted of at least five polydisperse species having median apparent Mr = 600,000, 400,000, 270,000, 135,000 and 40,000. When chromatographed further on octyl-Sepharose CL-4B, the proteoglycans of the cell layer resolved into three peaks. The unbound fraction (peak cell layer-I) contained a 40,000 species consisting of a single glycosaminoglycan chain with or without peptide. Peak cell layer-II contained three sulfated species on electrophoresis: a 600,000 species uniformly distributed across the peak, a 135,000 species enriched in the ascending limb (similar to bone PG-I as described previously), and a 270,000 species (similar to bone PG-I) enriched in the descending limb. Peak cell layer-III, eluting at 0.2% Triton X-100, was highly enriched in a 400,000 proteoglycan component. When media proteoglycans were chromatographed on octyl-Sepharose, two labeled peaks were found. Peak medium-I (unbound) contained a species exhibiting electrophoretic mobility similar to that of the 400,000 species present in peak cell layer-III. Peak II of the culture medium (medium-II) was apparently identical to that of peak cell layer-II, containing the 600,000, 270,000 and 135,000 species. No appreciable 40,000 species was observed in the medium. Treatment of the 600,000 species with either chondroitinase ABC or ACII generated a core protein preparation with bands of 390,000 and 340,000 on SDS gels. Neither the intact nor the chondroitinase ABC-treated 600,000 species was immunoprecipitated by a purified, polyclonal antiserum raised against the core protein of the large chondroitin sulfate proteoglycan of human articular cartilage. Treatment of the 270,000 and 135,000 proteoglycans with chondroitinase ABC, but not ACII, generated a core protein preparation with bands of 52,000 and 49,000 on SDS gels, indicating that they were dermatan sulfate-containing species. The 400,000 species contained both heparan sulfate and chondroitin sulfate, in approximately a 3:1 labeling ratio. This species changed in electrophoretic mobility following treatment with chondroitinase ABC, heparatinase, or both enzymes in combination, which suggested that it may be a hybrid proteoglycan (i.e. both types of glycosaminoglycan chain on the same core protein).(ABSTRACT TRUNCATED AT 400 WORDS)