Liquid Isoelectric Focusing Research Articles

Polygalacturonase produced by Botrytis fabae as elicitor of two furanoacetylenic phytoalexins in Vicia faba pods

Polygalacturonase, produced by Botrytis fabae in infected faba bean pods, was separated by isoelectric focusing and tested as possible elicitor of the phytoalexins wyerone acid and wyerone. The isoenzyme profile was characterised by multiple forms within a restricted range of isoelectric points (pI). These were indicated as very acidic, acidic and basic. Using liquid isoelectric focusing, polygalacturonase isoenzymes were separated into four different isoenzyme pools. These pools, tested at a range of concentrations, acted as an elicitor of wyerone acid and wyerone in endocarp pod tissues, the former being more prevalent. The level of phytoalexin accumulation varied depending on the enzymatic pool tested. Time course analysis revealed that phytoalexins accumulated at higher levels when lower enzyme doses were used. Each pool of isoenzymes was found to produce dark-brownish lesions similar to those observed from tissues inoculated with the pathogen. The possible dual role of polygalacturonase isoenzymes from B. fabae as putative pathogenicity determinants or defence responses elicitors during broad bean colonisation is discussed.

Size-exclusion chromatography in multidimensional separation schemes for proteome analysis

Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), well-established techniques relying upon similar criteria for separation. Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed.

Proteomic analysis using 2-D liquid separations of intact proteins from whole-cell lysates.

This unit describes procedures for 2-D liquid separations of proteins from whole-cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI-based fractionation using a batch-phase electrophoretic method, while chromatofocusing uses a column-based chromatographic method to generate the pH gradient. Using either method, a second-dimension fractionation is provided in the liquid phase using nonporous silica-based reversed-phase HPLC (NPS-RP-HPLC) to generate a 2-D liquid map of the protein content of the cell. The eluate of the 2-D liquid fractionation is directly coupled to a mass spectrometer for on-line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique.

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.

Comparison of the capabilities of liquid isoelectric focusing–one-dimensional nonporous silica reversed-phase liquid chromatography–electrospray ionization time-of-flight mass spectrometry and liquid isoelectric focusing–one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis mass mapping for the analysis of intact protein molecular masses

Nonporous silica reversed-phase HPLC coupled to electrospray ionization with on-line time-of-flight mass spectrometric detection (NPS-RP-HPLC–ESI-TOF-MS) is shown to be an effective liquid phase method for obtaining the molecular masses of proteins from pH fractionated cellular lysates where the method is capable of generating the same banding patterns typically observed using gel phase one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The liquid-phase mass spectrometry-based method provides a mass accuracy of at least 150 ppm, with 4000 mass resolution and provides improved sensitivity as the protein molecular mass (MW) decreases. The liquid and gel phase methods are shown to be complementary in terms of their mass range but the liquid phase method has the advantage over the gel method in that the analysis times are 50 times shorter, the mass accuracy is 70 times better and the resolution is 130 times higher. The liquid phase method is shown to be more effective for detection of proteins below 40 kDa, while the gel phase separation can access many more proteins, including more hydrophobic proteins, at increasing MW.

Isoelectric focusing of biotinidase and lipoamidase with the addition of non-ionic detergent

Isoelectric focusing (IEF) of purified biotinidase and lipoamidase (sialylated glycoprotein enzymes) was found to be reproducible and reliable when non-ionic detergent, Nonidet P-40 (NP-40), was added at 1% (v/v). Applications of the IEF containing 1% NP-40 on biotinidase and lipoamidase purifications were also successful; i.e. human serum biotinidase was purified by IEF after Affi-Gel Blue chromatography in the final step, and microsomal biotinidase and lipoamidase from guinea pig liver and kidney were purified by the preparative liquid IEF of Rotofor cell with 1% NP-40 followed by the final gel-permeation chromatography with Sephadex G-200. Thus, this high-recovery IEF with non-ionic detergent of NP-40 at 1% is shown to be a practical and reproducible method applicable to analysis and purification for several biotinidases and lipoamidases, and also expected to be generally applicable to the other relatively hydrophobic proteins.

Identification and purification of specific Penicillium marneffei antigens and their recognition by human immune sera.

Disseminated infection with the dimorphic pathogenic fungus Penicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.

Purification ofStaphylococcus aureusβ-Toxin: Comparison of Three Isoelectric Focusing Methods

β-Toxin (β-hemolysin) is one of several extracellular proteins produced byStaphylococcus aureus.It is a sphingomyelinase which disrupts the membranes of erythrocytes and other mammalian cells. Despite its characterized mechanism of action, the role of β-toxin in human and animal disease remains unclear. In this report, we compare three gentle, rapid methods to purify enzymatically active β-toxin. Extracellular proteins inS. aureusstrain RN4220 cell supernatants, containing a high concentration of the toxin, were precipitated by ethanol, dialyzed, and separated by preparative isoelectric focusing (IEF). We compared the efficiency of three preparative IEF methods: a Sephadex flat-bed IEF using pH 3.5–10.0 Ampholine, a liquid IEF using pH 7.8–8.9 Rotolyte buffers, and a liquid IEF with two consecutive steps using pH 3.0–10.0 Bio-Lytes in the first separation followed by a second step using pH 6.0–8.0 Bio-Lytes. All three IEF methods purified milligram amounts of enzymatically and biologically active β-toxin. Typically, 2–5 mg of purified β-toxin was obtained from 1.2 liters of culture medium. The total enzymatic activity recovered and overall yield were similar for all three methods. However, the single-step liquid IEF separation using Rotolyte buffers was the most preferred method because it purified β-toxin to >95% purity, did not require dialysis to remove ampholytes, and was the most rapid of the three methods.

Production of pectin-degrading enzymes by ericoid mycorrhizal fungi.

Production of enzymes which degrade plant cell wall macromoleculea has been studied in relatively few ericoid fungal isolates, although these polymers arc a major component of the organic litter and an important source of nutrients for these fungi. Our aims were to investigate whether the ability to degrade the wall pectic component, only reported for one isolate, is a general feature of ericoid fungi. Of about 35 isolates from different geographic regions, all were capable of growing on pectin as the sole carbon source. Polygalacturonase (PG) activity was detected to a different degree in the culture filtrates and independently of the fungal growth rate. Solid and liquid isoelectric focusing allowed separation and identification of several polygalacturonase isoforms. Among the fungal isolates investigated, those from the northern hemisphere produced mostly acidic isoforms, whereas isolates from South Africa secreted more abundantly basic isoforms. However, purification and biochemical characterization of several PG isoforms from the different isolates revealed an optimal activity in the acidic pH range for all the PG enzymes tested. Polygalacturonase enzymes seem to be an important component of the enzymatic arsenal secreted by ericoid fungi during their saprotrophic life. In addition, they could also play a role during root colonization, since penetration across the plant cell wall is a prerequisite for the establishment of endomycorrhizal symbiosis.

Isolation and partial characterization of a Paracoccidioides brasiliensis 58 kDa extracellular glycoprotein which is recognized by human immune sera

A novel 58 kDa antigenic determinant of the fungus Paracoccidioides brasiliensis was identified by enzymelinked immunosorbent assay using a panel of species-specific murine monoclonal antibodies (MAbs). Western immunoblot analysis, deglycosylation studies and isoelectric focusing indicated that this 58 kDa antigen is a glycoprotein, with a pI of approximately 5·2. The molecule was purified from P. brasiliensis culture filtrate and yeast cytoplasmic antigens by membrane ultrafiltration, liquid isoelectric focusing and gel filtration; N-terminal amino acid sequence data revealed no substantial homology with known proteins. The presence of the antigen in the cytoplasm of both yeast and mycelial forms of the fungus was demonstrated when these MAbs were used as markers in immunofluorescence, immunoperoxidase and immunoalkaline phosphatase techniques to label P. brasiliensis in cryostat sections. These MAbs also recognized the cytoplasm of P. brasiliensis yeast forms in paraffin-embedded pathological specimens from human cases. A preparation of the 58 kDa component from yeast cytoplasmic antigen was reacted by Western immunoblotting with 26 different serum samples from paracoccidioidomycosis patients, and 81% of them recognized it.

A 64-kDa protein is a substrate for phosphorylation by a distinct thylakoid protein kinase

Solubilization of spinach thylakoids with the nonionic detergent n-octyl-β-D-glucopyranoside (OG) releases active protein kinase from the membrane. Further purification was reported to demonstrate that a 64-kDa protein is the origin of this kinase activity (Coughlan S J and Hind G (1986) J Biol Chem 261: 11378-11385). The N-terminal sequence of this protein was subsequently determined (Gal A, Herrmann R, Lottspiech F and Ohad I (1992) FEBS Lett 298: 33-35). Liquid phase isoelectric focusing of the OG extract and an hydroxylapatite-purified fraction, derived from the OG preparation, reveals that the 64-kDa protein with this documented N-terminal sequence can be separated from the protein kinase activity. Experimental conditions were optimised by manipulation of ampholyte and detergent concentrations to maximise protein solubility and enzyme activity. The kinase-containing fraction was able to catalyze the phosphorylation of several proteins including the 64-kDa which was identified using antibodies raised against a synthetic peptide corresponding to the N-terminal sequence. The results described indicate that this 64-kDa protein is not the protein kinase responsible for the phosphorylation of the light-harvesting complex associated with Photosystem II.

The isolation and characterisation of jacalin [ Artocarpus heterophyllus (jackfruit) lectin] based on its charge properties

Jackfruit extracts contain a protein termed jacalin which possesses diverse biological properties. A detailed analysis of its charge properties has been lacking. The present investigation was initiated to study isoelectric properties of jacalin in detail and to isolate a single isoform of jacalin. Jacalin was isolated from jackfruit extracts by affinity chromatography on immunoglobulin-A immobilised to Sepharose 4B. Various techniques such as ion-exchange chromatography, isoelectric focusing (IEF) on polyacrylamide gels and preparative liquid IEF with the Rotofor cell were used. When analysed by IEF on thin layer polyacrylamide gels, jacalin was resolved into 35 bands over a pH range of 5.0–8.5. Upon SDS-PAGE in the second dimension all these charge species gave rise to only two-bands at 12 and 15.4 kDa. The lectin was mostly eluted with 50 and 100 mM sodium chloride when jackfruit extracts were fractionated on an anion-exchange column of DEAE-cellulose. In a single 6 hour run by preparative IEF with the Rotofor cell in the pH range of 3–9.5, it has been possible to isolate pure jacalin fractions containing fewer number of charged isomers. A single jacalin isoform was isolated by subjecting a Rotofor fraction containing fewer charged species to preparative IEF on thin layer polyacrylamide gel and eluting the band of interest from the gel. The isolated jacalin isoform was biologically active as it agglutinated erythrocytes. The study reveals the complexity of jacalin as it exists as multiple charge isomers over a broad pH range. By performing preparative IEF in solution as well as in thin layer polyacrylamide gels, it was possible to isolate a single jacalin isoform with the retention of biological activity.

Effect of culture conditions on the production of an extracellular proteinase byThermus sp. Rt41A

Thermus sp. Rt41A produced a single extracellular proteinase, as determined by fast protein liquid chromatography and isoelectric focusing. Proteinase activity was expressed from very early in the log phase, and halted when the growth substrate was exhausted. There was no continued proteinase production in the stationary phase. Proteinase production was not stimulated by O2 limitation, not repressed by amino acid growth substrates, and its production could not be correlated to the type or oxidation state of the carbon and energy source or the growth rate on different carbon and energy sources. Growth on certain substrates, e.g. glutamate and glucose, resulted in production of high levels of proteinase, whereas others, such as acetate, resulted in low proteinase levels. Acetate repressed proteinase production in cultures growing on L-glutamate. In continuous culture on L-glutamate, acetate or pyruvate, proteinase production was highest at higher growth (dilution) rates. The kinetics of proteinase production in continuous culture on L-glutamate can be interpreted as evidence for the constitutive nature of proteinase expression byThermus sp. Rt41A. The data obtained show that the control of proteinase production is different to that postulated forThermus sp. Ok6.A1.

Analytical and micropreparative two-dimensional electrophoresis of proteins

We describe procedures for the general separation of complex protein mixtures by high-resolution two-dimensional electrophoresis (2DE). Several refinements of O'Farrell's original technique that lead to improved reproducibility, resolution, and detection sensitivity are discussed. The advantages and versatility of a mid-sized (16 by 20 cm) gel format are emphasized. Optional (e.g., preparative liquid isoelectric focusing) and highly recommended steps in sample preparation are detailed, including descriptions of some of the pitfalls commonly encountered. Optimal buffer systems, gel construction, gel transfer from first to second dimension, gel removal, and protein detection (gel staining or membrane staining after electroblotting) are given. Further, our procedure for N-terminal and internal protein sequencing of 2DE protein spots, which requires < 50 pmol for the former and 250–500 pmol for the latter, is detailed. The working protocol for all these procedures and important troubleshooting points are listed in the appendix.

Pancreatic digestive enzymes and ultrastructure after chronic alcohol intake in the rat

The effects of chronic ethanol intake on the digestive enzyme content of rat pancreas, pancreatic juice and serum were studied by means of reversed-phase high performance liquid chromatography (HPLC), gel electrophoresis, isoelectric focusing and traditional enzymatic methods. The biochemical changes were further correlated to ultrastructural lesions caused by chronic alcohol consumption. 55 animals were given ethanol in their drinking water as 15% (w/v) solution up to 16 months. 49 control animals received water. The protein composition of the pancreatic juice was qualitatively similar in the alcoholic and control animals. Alcohol had a biphasic effect on pancreatic enzyme secretion. The protein and especially the trypsinogen concentrations of the pancreatic juice were increased after 8 months alcohol intake and diminished after 16 months. The changes in the acinar cell ultrastructure were marked only during the 2nd year of alcohol consumption. The amylase, lipase and especially the trypsinogen concentrations of pancreatic tissue were increased in the alcoholic animals. The results suggest that chronic ethanol consumption profoundly interferes with the pancreatic protein metabolism, especially that of trypsinogen and that the decrease in the enzyme secretion after 16 months alcohol intake may be due to acinar cell injury.

Effects of surfactants on human serum biotinidase

Isoelectric focusing (IEF) of purified biotinidase and lipoamidase (sialylated glycoprotein enzymes) was found to be reproducible and reliable when non-ionic detergent, Nonidet P-40 (NP-40), was added at 1% (v/v). Applications of the IEF containing 1% NP-40 on biotinidase and lipoamidase purifications were also successful; i.e. human serum biotinidase was purified by IEF after Affi-Gel Blue chromatography in the final step, and microsomal biotinidase and lipoamidase from guinea pig liver and kidney were purified by the preparative liquid IEF of Rotofor cell with 1% NP-40 followed by the final gel-permeation chromatography with Sephadex G-200. Thus, this high-recovery IEF with non-ionic detergent of NP-40 at 1% is shown to be a practical and reproducible method applicable to analysis and purification for several biotinidases and lipoamidases, and also expected to be generally applicable to the other relatively hydrophobic proteins.

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.

Phylogeny of immunoglobulin structure and function—VIII: Intermolecular heterogeneity of shark 19S IgM antibodies to pneumococcal polysaccharide

Significant IgM antibody responses to the pneumococcal capsular polysaccharide were demonstrated in nurse sharks immunized with pneumococcal vaccines. The antibodies isolated from immune sera by affinity chromatography were exclusively of the 19S variety; no 7S antibodies were isolated from any of six animals studied for periods up to twelve months. Equilibrium dialysis studies of the isolated antibodies demonstrated several important points: (1) the IgM antibodies contained ten combining sites per 19S molecule, (2) in all cases, the isolated antibodies showed marked heterogeneity of combining sites, (3) the antibodies were all of low average affinity, and (4) there was no increase in the average affinity of the antibodies isolated from any single animal for periods of up to twelve months after the start of immunization. In order to determine if a single IgM molecule contains ten equivalent combining sites, the antibodies isolated from several animals were fractionated by liquid isoelectric focusing. Equilibrium dialysis experiments using focused fractions showed the presence of ten functionally identical combining sites per IgM molecule. As a proof of the structural homogeneity of focused fractions, antibodies were separated into H and L chains, § recombined and tested for the regeneration of the original combining site by equilibrium dialysis. The results indicated that recombinants of focused antibody fractions contained binding sites identical to those of the intact antibody, whereas heterogeneous (unfocused) recombinants and isolated H and L chains failed to show any binding activity. The conclusion from this study is that the heterogeneity of ligand binding exhibited by nurse shark 19S antibodies to the capsular polysaccharide of the Type III pneumococcus can be attributed to intermolecular heterogeneity.

Separation of IgG Fab and Fc fragments by isoelectric focusing

Preparative liquid isoelectric focusing in a sucrose stabilized gradient was employed to separate Fab and Fc fragments of human and chicken IgG. Immunoelectrophoresis was used to confirm separation of the fragments. The advantages of isoelectric focusing over the classical method of ion exchange chromatography are discussed.

Heterogeneity of Factor VIII Antibodies: Further Immunochemical and Biologic Studies

Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.