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.     

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.     

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.     

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.     

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.     

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.     

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.