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.     

Rapid purification of extracted bacterial lipopolysaccharides by continuous free-flow electrophoresis

The use of continuous free-flow electrophoresis for the purification of extracted lipopolysaccharides ( LPSs ) was investigated. Commercial (nucleic acid contaminated) LPS preparations, isolated by the hot phenol-water method of Westphal from Salmonella typhimurium and Escherichia coli 0111: B4, were analyzed. Continuous free-flow electrophoresis for purification of crude LPSs proved to be a rapid and useful means for the continuous purification of large amounts of LPS (more than 45 mg crude LPS per hr) and it showed good reproducibility and pure LPS. The electrophoretic profile of both crude LPSs obtained by continuous free-flow electrophoresis showed two distinct, sharp peaks; one representing the nucleic acid fraction and the other the LPS fraction. Under the continuous free-flow electrophoresis conditions employed, nucleic acid in the crude LPSs possessed low electrophoretic mobility, whereas LPS migration was negligible. Thus for both preparations pure LPS (no detectable nucleic acid) was obtained. Electrophoretic profiles of these purified LPSs on sodium dodecylsulfate-polyacrylamide gel electrophoresis were similar in both cases to those of crude LPS and of LPS purified by repeated ultracentrifugation. By immunological analysis using double immunodiffusion and immunoelectrophoresis, it was found that two components of crude E. coli 0111: B4 LPS were eliminated by continuous free-flow electrophoresis, but each component of purified E. coli 0111: B4 LPS was immunologically identical to the corresponding component in its crude LPS. In S. typhimurium LPS, none of its components were influenced by continuous free-flow electrophoresis but not by ultracentrifugation. In spite of these results, both purified LPSs possessed stronger mitogenic activity than each crude LPS. These results indicated that continuous free-flow electrophoresis is a useful means of purifying extracted crude LPS.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

The preparation of an enzyme associated with aflatoxin biosynthesis by affinity chromatography

An affinity matrix for the purification of norsolorinic acid dehydrogenase, an enzyme involved in aflatoxin biosynthesis, was prepared by coupling norsolorinic acid to an agarose gel. This matrix was found to be ineffective in isolating active enzyme, and was therefore modified by methylation, using diazomethane. The methylated matrix produced a one-step purification of the enzyme from a crude homogenate, resulting in a 138-fold purification. The active isolate was found to contain one major and two minor bands upon nondenaturing electrophoresis, and all the norsolorinic acid dehydrogenase activity was associated with the major band. It was concluded that the matrix exhibited true affinity for the enzyme, and that affinity chromatography was a valuable approach to isolating other secondary metabolic enzymes involved in the biosynthesis of the aflatoxins.     

Involvement of cis and trans Golgi apparatus elements in the intracellular sorting and targeting of acid hydrolases to lysosomes

To delineate the traffic route through the Golgi apparatus followed by newly synthesized lysosomal enzymes, we subfractionated the Golgi apparatus of rat liver by preparative free-flow electrophoresis into cisternae fractions of increasing content of trans face markers and decreasing contents of markers for the cis face. NADPase was used to mark median cisternae. Beta-Hexosaminidase, the high mannose oligosaccharide processing enzyme, alpha-mannosidase II, the two enzymes involved in the biosynthesis of the phosphomannosyl recognition marker, and the phosphomannosyl receptor itself decreased in specific activity or amount from cis to trans. Additionally, these activities were observed in a fraction consisting predominantly of cisternae, vesicles and tubules derived from trans-most Golgi apparatus elements. These results, along with preliminary pulse-labeling kinetic data for the phosphomannosyl receptor, suggest that lysosomal enzymes enter the Golgi apparatus at the cis face, are phosphorylated, and appear in trans face vesicles by a route whereby the phosphomannosyl receptor bypasses at least some median and/or trans Golgi apparatus cisternae.     

NADP phosphatase as a marker in free-flow electrophoretic separations for cisternae of the Golgi apparatus midregion

Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH-cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6-phosphatase. The pH optimum was 5.0, the K_m for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific market to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations.     

Study of human placental estradiol-17β dehydrogenase/20α-hydroxysteroid dehydrogenase by preparative disc-gel electrophoresis

The soluble enzyme, estradiol-17β dehydrogenase from human term placenta, appears to co-purify with a second soluble enzyme, 20α-hydroxysteroid dehydrogenase. The enzyme, which had been partially purified by affinity chromatrography, fractionated on a preparative electrophoresis gel to a homogeneous preparation containing both estradiol-17β dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in a ratio of ～100:1. Analytical polyacrylamide disc-gels resolved this homogeneous preparation as a single band by both protein and activity staining techniques. Homogeneous enzyme inactivated and affinity-radioalkylated by 16α-[2′-su14C]bromoacetoxyprogesterone or 16α-[2′-su14C] bromoacetoxyestradiol 3-methyl ether, and when analyzed by SDS disc-gel electrophoresis, gave a single protein band which corresponded identically to the radioactivity peaks. These observations support the hypothesis that estradiol-17β dehydrogenase and 20α-hydroxysteroid dehydrogenase represent dual oxidoreductase activity in one enzyme.Preparative disc-gel electrophoresis, a technique which has not been previously adapted to purification of these human placental enzyme activities, was useful to rapidly (3 days) effect a 15-fold enrichment of the estradiol-17β dehydrogenase specific activity from “heat-treated cytosol”. Thus, laboratory-scale preparative disc-gel electrophoresis is useful for rapid, small-scale enrichment of this soluble enzyme.     

Isolation of plasma membrane from amphibian epidermis: evidence for a basal-to-apical charge and activity gradient

Aqueous two-phase partition and preparative free-flow electrophoresis were used in series to isolate the plasma membranes of amphibian epidermis. Fractions obtained by two-phase partition were 40-fold enriched in a K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase relative to the total homogenate and based on morphology were representative isolates of all epidermal cells together. Small mucosal granules and mucin aggregates were the primary contaminants. Based on activities of marker enzymes, contents of mitochondria, Golgi apparatus and endoplasmic reticulum were low (0.15 that of total homogenate) or absent. When plasma membranes isolated by aqueous two-phase partition were subjected to preparative free-flow electrophoresis, they were distributed toward the anode in a series of fractions of increasing net negative charge, sialic acid content and specific activity of the K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase reminiscent of the activity gradient from base to apex for frog epidermis observed from cytochemical investigations. The most electronegative fractions nearest the anode and to the left of the main protein peak were enriched in both sulfate groups and thick membranes of the stratum corneum. A fraction migrating less toward the anode and to the right of the main protein peak contained hemidesmosomes together with the lowest enrichments of sialic acid, sulfate and the phosphatase. The results suggest that the plasma membranes isolated from mixed cell populations, such as those encountered in epidermal homogenates, may be resolved by free-flow electrophoresis according to cell type of origin following activity gradients present in the original tissue. Additionally, the findings provide independent biochemical confirmation of a base-to-apex gradient of transport (ATPase) activity associated with the plasma membranes of cells of the different strata of the amphibian epidermis.     

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 mM ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.     

Separation of plant membranes by electromigration techniques

The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented.     

Purification and properties of L-3-glycerophosphate dehydrogenase from pig brain mitochondria.

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.     

On the nature of the activating enzyme of the inactive form of delta-aminolevulinate synthetase in Rhodopseudomonas spheroides.

The activating enzyme of the inactive form of Fraction I of delta-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas (R.) spheroides was purified about 1,000-fold from an extract of R. spheroides cells grown anaerobically in the light. The purification of the activating enzyme was achieved by fractionating the 100,000 X g supernatant fraction of the crude extract with ammonium sulfate and acetone, followed by Sephadex G-200 chromatography, pyridoxamine phosphate-Sepharose 4B chromatography, and preparative gel electrophoresis. The final preparation of the activating enzyme still contained a minor contaminant (less than 20%) as judged by disc gel electrophoresis. The activating enzyme exhibited cystathionase [EC 4.4.1.1] activity throughout the purification. These two enzyme activities were not separated at all during any step of the purification. An apparently homogeneous preparation of cystathionase [EC 4.4.1.8] purified from rat liver also exhibited activating activity in the presence of L-cystine. It was concluded that the activating enzyme is a cystathionase.     

Purification of creatine kinase from beef heart mitochondria]

53-fold purified creatine kinase is isolated from beef heart mitochondria by phosphate buffer extraction followed by chromatography on DEAE-cellulose and KM-cellulose and preparative electrophoresis in phosphate buffer density gradient. The purified enzyme was homogenous under electrophoresis in agarose gel and moved to cathode. The enzyme did not enter into separating gel under disc electrophoresis in conditions for the separation of neutral anc acid proteins, while under conditions for separating alkaline proteins it produced five fractions. The stability of creatine kinase under storage considerably decreased after the purification.     

On-line optical fiber detection in a preparative free-flow electrofocusing apparatus

Many analytical isoelectric focusing (IEF) instruments are equipped with on-line detection, e.g., conductivity or UV-vis absorbance. Most of their preparative counterparts have not integrated such detection systems and thus require labor-intensive off-line analysis to quantify separation results. This paper describes the incorporation of an optical fiber based, on-line detection system that allows one to follow the evolution of the protein bands in a preparative IEF apparatus. An array of four optical fibers was designed to deliver light to the annulus of a free-flow electrophoresis apparatus, to detect the transmitted light passing through the separation media and to determine the protein concentration at vertical positions along the annulus of a vortex-stabilized focusing chamber using a 1024 bit CCD line camera. The final concentration of the major myoglobin band was 21.0 mg/mL at electric field strengths as high as 333 V/cm. Spectrophotometric analysis indicated a final concentration of 18.9 mg/mL, 10% less than that reported by the optical fibers.     

Improved enzyme screening by automated fast protein liquid chromatography

The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with non-specific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.     

Evaluation of crude dextran as phase-forming polymer for the extraction of enzymes in aqueous two-phase systems in large scale

A detailed study of the influence of crude dextran on enzyme extractions in aqueous phase systems is presented in this article. The physical parameters of crude dextran, a purified T-500 fraction from Pharmacia, and a hydrolyzed crude dextran are compared and their influence on the phase system parameters investigated. Initially there is a drastic increase in the viscosity of the lower dextran-rich phase and a significant shift in the macroscopic structure of these phases, observed as the "gel-forming" properties of the dextran phases. The latter can be important for the partition of any enzyme by influencing the effect of phosphate concentration on the partition of proteins, although these experiments show that the partition coefficient of several enzymes is not much altered. The partition parameters allow the substitution of Dextran T-500 fractions by crude dextran or unfractionated, slightly hydrolyzed fractions. Using crude dextrans the performance and technical realization of enzyme extraction processes are demonstrated for pullulanase from Klebsiella pneumoniae and formate dehydrogenase from Candida boidinii.Both enzymes were recovered in comparable high yields. The equipment performance was quite good, as indicated by the high throughput values of the separators employed. Especially when using nozzle separators for phase separation there is a better performance in comparison to the Dextran T-500 fraction. No serious technical problems were encountered when replacing the expensive fractionated dextran with a crude dextran. In this way aqueous two-phase systems containing dextran become more feasible for enzyme purification from an economic point of view. The price of about 1.30 German marks (DM) per liter for a useful phase system already appears acceptable for the production of valuable intracellular enzymes.     

Purification and characterisation of a fructosyltransferase from Rhodotorula sp

The present work was devoted to investigations concerning the purification and characterisation of the fructooligosaccharide (FOS)-producing extracellular enzyme of Rhodotorula sp. LEB-V10. FOS are functional food ingredients showing prebiotic properties, meaning that it could stimulate selectively the growth and/or activity of probiotic bacteria in the gut. The purification of the enzyme was carried out according to the following sequential procedure: cell separation by centrifugation, recovering by ethanol precipitation and purification by anion exchange chromatography. The molecular weight was estimated to be 170 kDa by preparative gel filtration and 77 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, signifying that the native enzyme exists as a dimer. With sucrose as substrate, the data failed to fit the Michaelis-Menten behaviour, rather showing a sigmoid shape similar to that of the allosteric enzymes (cooperative behaviour), requiring high sucrose concentrations to obtain high reaction rates. The enzyme showed both fructofuranosidase (FA) and fructosyl-transferase (FTA) activities. The optimum pH and temperature for FA activity were found to be around 4.0 and 72-75 degrees C, respectively, while FTA showed optimum activity at pH 4.5 and 65-70 degrees C. Both activities were very stable at temperatures below 66 degrees C, while for FA, the enzyme was more stable at pH 4.0 and for FTA at pH 5.0.     

Free-flow electrophoretic separation of Plasmodium berghei sporozoites

Sporozoites of the rodent malaria, Plasmodium berghei, were obtained from infected Anopheles stephensi by grinding mosquitoes, prepurifying the material in a discontinuous Hypaque gradient and further purifying by means of continuous free-flow electrophoresis. Bacteria, debris, mitochondria, mitoplasts, and other contaminants were removed in the electric field. The isolated sporozoites were morphologically intact and were positive in indirect immunofluorescence assay. They were infective to mice prior to and following free-flow electrophoretic separation. The surface of the sporozoites exhibited a polysaccharide-rich layer. The predominant surface protein labelled after surface iodination had a molecular weight between 42,000 and 46,000 daltons.     

Purification and molecular weight determination of glucose-6-phosphate dehydrogenase and malic enzyme from mouse and Drosophila.

A facile two-step procedure was employed for simultaneous purification of glucose-6-phosphate dehydrogenase and malic enzyme from mouse (strain $\text{DBA}\text{2J}$) and Drosophila melanogaster. This involved the use of an 8-(6-aminohexyl)-amino-2′,5′-ADP-Sepharsoe affinity column chromatography followed by DEAE-Sephadex chromatography. The native and subunit molecular weights of these two homogeneous enzymes were determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. From this study, it was concluded that the two enzymes are tetrameric and have native molecular weights between 200,000 and 280,000 in both species.     

Purification of yeast isocitrate dehydrogenase

The NAD-linked isocitrate dehydrogenase from baker's yeast was purified to homogeneity (as judged by gel filtration and polyacrylamide-gel electrophoresis) with an overall yield of 50% by using dilute solutions of the allosteric effector (AMP) to elute the enzyme specifically from CM-cellulose. This method preserves the allosteric properties of the crude enzyme. Although the pure enzyme shows only a single band on electrophoresis in the presence of sodium dodecyl sulphate, two types of subunit are observed in 8m-urea. The isoelectric point of the enzyme rises during purification, and this may reflect the partial loss of an additional low-molecular-weight component. Values are included for the amino acid composition and extinction coefficients of the pure enzyme.