Ionic interactions between proteins in nonequilibrium pH gradient electrophoresis: histones affect the migration of high mobility group nonhistone chromatin proteins

In two-dimensional gel electrophoresis of the high mobility group (HMG) proteins, it has proved necessary to use nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension rather than isoelectric focusing, because of the basic character of most of the HMG proteins [D. Tyrell, P. J. Isackson, and G. R. Reeck (1982) Anal. Biochem. 119, 433-439]. In this paper it is reported that in samples that contain histones, the mobilities of HMG proteins (particularly HMG-1, HMG-2, and HMG-E) are severely distorted in NEPHGE. This presumably results from formation of complexes between histones and HMG proteins through ionic interactions. Analysis of HMG proteins by NEPHGE/sodium dodecyl sulfate-gel electrophoresis is thus precluded in samples containing histones. Our results raise the possibility of similar artifacts occurring in NEPHGE (or isoelectric focusing) analysis of other proteins with regions of high charge density.     

Gradiflow as a prefractionation tool for two-dimensional electrophoresis

The Gradiflow trade mark, a preparative electrophoresis instrument capable of separating proteins on the basis of their size or charge, was used to separate whole cell lysates, prepared from bakers yeast (Saccharomyces cerevisiae) and Chinese snow pea seeds (Pisum sativum macrocarpon), into protein fractions of different pH regions. Both broad and narrow range (with a difference of approximately 1 pH unit) pH fractions were obtained. Analysis of the protein fractions by isoelectric focusing gels and two-dimensional (2-D) polyacrylamide gel electrophoresis indicated minimal overlap between the pH fractions. Further, when the prefractionated acidic samples were analyzed on pH 4-7 immobilized pH gradient 2-D gels, improved resolution of the proteins within the chosen pH region was achieved compared to the unfractionated samples. This study demonstrates that the Gradiflow could be used as a preparative electrophoresis tool for the isolation of proteins into distinct pH fractions.     

Technical improvement to 2D-PAGE of rice organelle membrane proteins

Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.     

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.     

Effect of alkylation on the physical properties of simian virus 40 T-antigen species.

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.     

Scanning isoelectric focusing of cholera enterotoxin in polyacrylamide gels

Scanning isoelectric focusing has been employed for continuous monitoring of the isoelectric spectrum of highly purified cholera enterotoxin in 4% polyacrylamide gels containing 2% ampholytes pH 3–10. The resolution obtained by this technique is of high order because at no instance during focusing interruption of current occurs and thus diffusion of the isolated protein moieties is suppressed. An added aspect of scanning isoelectric focusing was that it allowed estimation of the minimal focusing time of cholera enterotoxin. Thus under the standard assay procedure, the main basic component of cholera enterotoxin was focused in 5800 sec, while the other at least 3 minor acidic and anodic components were focused in approximately 19000 sec. Focusing of cholera enterotoxin in the presence of 6m urea allowed the visualization of 5 well defined and about equal components. The proteinaceous nature of the observed peaks was verified by scanning at wavelengths other than 280 nm, staining of gels for protein, and varying the concentration of the enterotoxin. The design of scanning isoelectric focusing equipment is presented. Reproducibility, economy of sample, and ampholytes and simplicity of experimental technique were some of the features of this apparatus. The resolution of scanning isoelectric focusing was found to be superior to that of ordinary disc and SDS gel electrophoresis.     

Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

Analysis of ultra acidic proteins by the use of anodic acidic gels

Ultra acidic proteins, generated by posttranslational modifications, are becoming increasingly important due to recent evidence showing their function as regulatory elements or as intermediates in degradation pathways in bacteria. Such proteins are important in neurodegenerative diseases and embryonic development, and they include the Alzheimer-related tau (τ) protein (resulting from posttranslational modifications) and the phosphor-storage embryonic proteins. The ultra acidic proteins are difficult to study because standard two-dimensional gel electrophoresis is inadequate for their analysis. Here we describe a novel electrophoresis system of anodic acidic gels that can replace isoelectric focusing as the first dimension of separation in two-dimensional electrophoresis. The system is based on a sodium acetate buffer (pH 4.6), is compatible with traditional stains (e.g., Coomassie blue) as well as novel fluorescent dyes (e.g., Pro-Q Diamond), and is quantitative for the analysis of ultra acidic proteins. The anodic acidic gels were used for the functional classification of the ultra acidic part of the Bacillus subtilis proteome, showing significant improvement over traditional two-dimensional electrophoresis.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Charge-dependent staining of proteins after electrophoretic separation in polyacrylamide gels

A staining procedure is described which allows for the identification of basic and acidic proteins after gel electrophoresis. This includes electrophoresis of sodium dodecylsulfate (SDS) membrane proteins not accessible to isoelectric focusing as a means of charge-dependent separation. Nonfixative charge-dependent staining can be used for detection of proteins after separation in gels, when further investigation of the intact protein is desired.     

Evidence against electrophoresis as the principal mode of protein transport in vitellogenic ovarian follicles of Drosophila

Charged cell constituents in polytrophic insect follicles are thought to be transported in the nurse cell-oocyte syncytium by way of electrophoresis. This concept, proposed by Woodruff & Telfer (1980) was based on electrophysiological data and microinjection of heterologous proteins using Hyalophora follicles. By microinjecting fluorescently labelled acidic and basic proteins into the nurse cells or oocyte of vitellogenic Drosophila follicles, we failed to obtain evidence for charge-dependent migration of these molecules. We have also analyzed the proteins of nurse cells and oocyte on isoelectric focusing gels, by means of two-dimensional gel electrophoresis, and by ion exchange chromatography to see if basic or acidic proteins accumulate in vivo in nurse cells and oocyte, respectively. For the bulk of the follicular proteins we found no accumulation. Further evidence against an electrophoretic transport system in Drosophila was obtained by estimating the intracellular pH from the colour of indicator dyes microinjected into the follicles; the results indicate that the pH in the nurse cell cytoplasm is lower than that in the ooplasm. According to the model developed for Hyalophora, electrophoretic transport would be favoured by high pH in the nurse cell cytoplasm.     

Isoelectric points and molecular weights of salt-extractable ribosomal proteins.

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCL were characterized by acid urea-polyacrylamide gel electrophoresis, sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

An isoelectric focusing procedure for erythrocyte membrane proteins and its use for two-dimensional electrophoresis

Procedures are described and evaluated for one-dimensional isoelectric focusing of erythrocyte membrane dissolved in lysine, urea, and Triton X-100 without using sodium dodecyl sulfate (SDS) and for two-dimensional electrophoresis with SDS in the second dimension. The membrane was completely dissolved, most of the proteins including the anion porter(s) entered the focusing gel, and complex, well-resolved patterns were seen. Ampholines, 2-mercaptoethanol, or SDS in the applied sample each seriously reduced focusing resolution and phenylmethylsulfonyl fluoride blurred the patterns. The two-dimensional patterns showed more and sharper spots than did patterns obtained from membrane initially dissolved with SDS. Anion porter spots were seen with both procedures. However, major cytoskeletal proteins were much less well recovered with the former procedure than with the latter.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose. Effect of protein charge on immobilization

An uncharged N-hydroxysuccinimide ester derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5--11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3--5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl2, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new N-hydroxysuccinimide ester derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9--4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.