Dipetalonema viteae: isoelectric focusing and immunochemical studies on somatic extracts of adult worms and microfilariae.

Isoelectric focusing (IEF) of a somatic extract of adult worms (SEAW) yielded nine fractions. Most of the applied protein antigen was recovered in five fractions with pI values of 5.2, 4.4, 4.3, 4.0, and 3.3, respectively. The nine IEF fractions of SEAW gave a total of 37 bands following electrophoresis on polyacrylamide gel (PAGE). IEF fractionation of a somatic extract of microfilariae (SEM) yielded nine fractions. Two fractions with pI values of 4.4 and 3.2, respectively, contained most of the applied protein. The nine IEF fractions of SEM gave a total of 32 bands following PAGE. Crossed immunoelectrophoresis (CI) of SEM and SEAW against serum from hyperinfected hamsters yielded five and eight precipitation peaks, respectively. CI of SEM and SEAW against their homologous rabbit antisera gave 7 and 10 major precipitation peaks, respectively. Immunodiffusion of the nine IEF fractions from both SEM and SEAW against both their homologous and heterologous rabbit antisera indicated three and four precipitin bands peculiar only to SEM and SEAW, respectively. The remaining 23 bands from both preparations showed lines of identity.     

Protein composition of human submandibular secretions

The protein composition of human submandibular saliva obtained from a single donor has been investigated, and 21 proteins have been resolved. On Sephadex G-100, submandibular secretions (unstimulated) separated into four fractions, I, II, III, and IV. Each fraction was analyzed further by isoelectric focusing and disc gel electrophoresis. The major components detected in each fraction along with their isoelectric point (pI) are as follows: I, blood group specific substance (2.3), immunoglobulin A (5.0–6.0), and immunoglobulin G (4.5–6.5); II, albumin (4.9), two glycoproteins (5.0), and acid phosphatase (5.2); III, three phosphoproteins (4.3–4.4), isoamylase 1A (5.9), isoamylase 1B (6.4), unidentified protein (7.1), lysozyme (>10), and a basic protein (>10); and IV, isoamylase 2A (5.9) and isoamylase 2B (6.4). Isoamylase 1A and IB are glycoproteins. Stimulated submandibular secretions were also resolved into four protein fractions by gel filtration. Fraction III, compared with unstimulated secretions, showed the greatest percent increase in protein. Analysis of this fraction by disc gel electrophoresis demonstrated the presence of four protein bands which were not detected in the unstimulated secretion. One of these proteins is tentatively identified as a phosphoprotein and two as basic proteins (pI > 10). The protein composition of submandibular, parotid, and sublingual secretions is compared.     

Isoelectric focusing in immobilized pH gradients: Generation of extended pH intervals

A new technique for generatiing extended pH gradients (3–4 pH units) in Immobiline gels for isoelectric separations is described. A five-chamber gradient mixer has been built, based on the ‘Varigard’-type mixers of Peterson and Sober (Anal. Chem. 31, 1959, 857–862). Each chamber contains one of the following Immobilines, in this order: pK values 4.4, 4.6, 6.2, 7.0 and 8.5, titrated in the pH 4–8 interval with non-buffering Immobilines pK 9.3 (in the case of the two acidic Immobilines) and pK 3.6 (in the case of the three basic Immobilines). In this way it is possible to cast, in a highly reproducible way, an immobilized pH gradient in thepH range 4.0 to 7.5, which should be ideal for isoelectric separations in the first dimension of two-dimensional techniques. A computer program is also described which, given the molarities and pK values of the different Immobilines in the chambers of the Varigrad mixer, can generate the theoretical pH profile, together with the buffering capacity (β) and ionic strength (I) courses.     

Properties of albumins of milled rice

Extraction studies on IR36 milled rice showed that albumins solubilized by 0.1–0.15 M (NH₄)₂SO₄ consisted of about 20% high(～5%) lysine, fast-migrating proteins on electrophoresis at pH 8.3 and about 80% lower ～2%) lysine proteins of slower mobility. The 2%-lysine albumins were insoluble in 1.8 M (NH₄)₂SO₄ while the higher lysine albumins required 4 M (NH₄)₂SO₄ to precipitate. The 2%-lysine albumins were not fractionated by gel filtration and gave only one major fraction with MW 19 000. SDS-polyacrylamide gel electrophoresis confirmed the major subunit to be of MW 17 000. These albumins were separated by DEAE-Sephacel chromatography at pH 8.5 into three fractions of similar aminograms but differing in analytical get electrophoretic and isoelectric focusing patterns.     

Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea–urea mixtures

Capillary isoelectric focusing (cIEF) is normally run under denaturing conditions using urea to expose any buried protein residues that may contribute to the overall charge. However, urea does not completely denature some proteins, such as the tetrameric enzyme Erwinia chrysanthemil-asparaginase (ErA), in which case electrophoresis-compatible alternative denaturants are required. Here, we show that alkylureas such as N-ethylurea provide increased denaturation during cIEF. The cIEF analysis of ErA in 8 M urea alone resulted in a cluster of ill-resolved peaks with isoelectric points (pI values) in the range 7.4 to 8.5. A combination of 2.0 to 2.2 M N-ethylurea and 8 M urea provided sufficient denaturation of ErA, resulting in a main peak with a pI of 7.35 and an acidic species minor peak at 7.0, both comparing well with predicted pI values based on the sum of protein residue pK_a values. Recombinant deamidated ErA mutants were also demonstrated to migrate to pI values consistent with predictions (pI 7.0 for one deamidation). The quantitation of ErA acidic species in samples from full-scale manufacturing (1.0–3.5% of total peak area) was found to be reproducible and linear. Use of alkylureas as denaturing agents in capillary electrophoresis and cIEF should be considered during biopharmaceutical assay development.     

Trichinella spiralis: Immunization of rats with an antigen fraction enriched for allergenicity

Trichinella spiralis whole muscle larval extract was fractionated by gel filtration and anion-exchange chromatography, and the protein fractions were assayed for allergenicity by a footpad-swelling test in mice; IgE antibody levels in rats immunized with the fractions were determined by passive cutaneous anaphylaxis test in rats. By these methods, an allergenic fraction from T. spiralis was isolated. The fraction, F1-b, was shown to be monodisperse by analysis with SDS-PAGE, IEP, and isoelectric focusing, indicating that it is a single protein moiety with a molecular weight of approximately 45,000 and a pI of approximately 5.1. The Schiff-periodate test showed Fl-b to be a glycoprotein. Rats immunized with Fl-b had significantly fewer intestinal worms than did nonimmunized controls at 24 hr and 7 days after oral challenge with T. spiralis larvae.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Purification and hydrophobic properties of the δ-endotoxin in the parasporal crystal produced by Bacillus thuringiensis var. entomocidus

Parasporal crystals of Bacillus thuringiensis var. entomocidus were separated from spores and other cell debris by the water-chloroform biphase procedure. The solubilization and fractionation were carried out under mild conditions at 4°C. Crystals were solubilized in 0.01 M dithiothreitol and 0.2 M glycine NaOH buffer at pH 10.0. The solution was treated overnight with 0.01 M Tris-HCl buffer, pH 5.5, containing 0.1% Triton N-101 and 0.1% sodium cholate, and then placed on a Sepharose 6B column, equilibrated, and later developed with the same buffer. Under these conditions, four fractions were obtained, one of which had a molecular weight ranging from 60,000 to 70,000, and demonstrated a high insecticidal activity on second instar larvae of Spodoptera litioralis. The LC₅₀ value of this fraction was a half of that of the solubilized crystals. The other three fractions had a lower activity. The active fraction was further fractionated on an octyl-Sepharose 4B resin. Elution of this column with the same buffer separated the proteins into two fractions. The first eluted fraction was highly active, while the second demonstrated a very low activity. The active fraction was further purified by loading on a short column of octyl-Sepharose 4B and eluted with a linear gradient of the same detergents. Under these conditions, the highly active fraction gave a sharp and symmetrical peak that revealed five close bands at the pH range of 6.1–6.5 on isoelectric focusing gel electrophoresis.     

Electrophoretic variation of seed proteins among U.S. populations of Tripsacum dactyloides Var. dactyloides

Accessions from across the range of Tripsacum dectyloides in the US. were assayed electrophoretically for interpopulation variation in seed proteins. Clustering of isoelectric focusing patterns for Tris-HCl and water-soluble proteins revealed high levels of homogeneity. Preliminary resultswith alcohol-soluble proteins, in contrast, showed this fraction to be intrinsically much more variable. Divergent functional roles of the protein fractions themselves could account for this observed difference in variation and are discussed in the paper.     

Immunological relationships and a genetic interpretation of major and minor acidic proteins in human parotid saliva

Isoelectric focusing was performed on parotid salivas selected for their electrophoretic phenotypes of proline-rich acidic salivary proteins. Fractions encompassing narrow pH regions were pooled and examined by polyacrylamide gel electrophoresis. Isoelectric focusing yielded partial purification of major and minor acidic proline-rich proteins which were subsequently compared by immunoelectrophoresis and double immunodiffusion against goat anti-human parotid saliva. Cross-reactivity without spurring between all fractions containing major Pr proteins in both immunoelectrophoresis and double immunodiffusion suggests that these proteins are immunologically very similar or identical.This study was supported in part by an award from the American Cancer Society Institutional Grant IN-88F to Fels Research Institute.     

Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M_r and pI of the 14gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.     

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Purification and heterogeneity

The enzyme fructose- 1,6-diphosphatase (FDPase), involved in the reductive cycle of the pentose phosphate pathway, has been purified from spinach leaves by heating (30 min at 60°), “salting out” with ammonium sulphate (between 30–70% of saturation), filtration through Sephadex G-100 and G-200, fractionation on DEAE-52 cellulose and preparative electrophoresis on polyacrylamide gel. Filtration through DEAE-cellulose led to the isolation of two active fractions (fractions I and II) with very close MWs and isoelectric points. By electrophoresis on acrylamide gel, both fractions gave two active fractions (fractions I_a-I_b and II_a-II_b). The fractions with low electrophoretic migration rate—I_b and II_b—are stable in acid and neutral pH, have a MW between 90 000 and 110 000 and constitute the native form of the photosynthetic enzyme. The fractions of faster migration rate—I_a and II_a-originate from the corresponding fractions I_b and II_b under alkaline conditions, show half the MW of the respective fractions, and behave as subunits of the original dimer form. Measured by electrofocusing, the four active fractions have isoclectric points in the range 4·10–4.30.     

Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)₃-p-nitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts.     

Schistosoma mansoni: Characterization of antigens in excretions and secretions

Excretory and secretory antigens of Schistosoma mansoni were obtained by in vitro cultivation of worms in Medium H-199, under sterile conditions at 37 C, in the dark, in an atmosphere of 92% air and 8% CO₂. This procedure yielded about 1 μg soluble excretion-secretion products per worm per 24 hr. The composition of the “excretory and secretory antigen” (ESA) preparation is complex. Analysis by isoelectric focusing revealed the presence of about 10 major and about 30 minor protein components. Immunological analysis of the ESA preparation was performed by immunoelectrophoresis. At least five precipitin arcs were seen with infected mouse serum, and seven with rabbit anti-ESA serum. Immunoelectrophoresis of molecular-weight fractions of ESA showed a total of 17 different antigens. One of these antigens was excreted exclusively by female worms. The antibody response in rabbits to preparations obtained by homogenization of adult worms, or by extraction of the tegument, was very different from the response to excretory and secretory antigens. Considerable cross-reactivity between these preparations did, however, occur.     

Isoenzymes of ferredoxin-NADBΔ oxidoreductase from the cyanobacterium Nostoc strain MAC

Ferredoxin-NADP⁺ oxidoreductase from the cyanobacterium Nostoc strain MAC was separated into two fractions by ion-exchange chromatography. Both were purified to electrophoretic homogeneity and exhibited diaphorase and ferredoxin-dependent cytochrome c reductase activity. The activities with three different electron carriers in this latter assay were similar for the two fractions, as were the pH optima in both assays. Each fraction, however, could be resolved into several active components by isoelectric focusing, both after initial separation and following apparent purification by gel filtration on Sephadex G-150, further chromatography on DEAE-cellulose, and use of hydroxylapatite columns.Abbreviation DCIP = phenolindo-3,6-dichlorophenol>     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Purification and some properties of an Mg2+-, Ca2+- and calmodulin-stimulated ATPase from spinach chloroplast envelope membranes

Spinach chloroplasts display an ATPase activity which is associated with the envelope. This envelope-bound activity is stimulated by Ca²⁺, Mg²⁺ and calmodulin (Nguyen, T.D. and Siegenthaler, P.A. (1983) FEBS Lett. 164, 67–70). The Triton X-100-solubilized enzyme was retained specifically on a calmodulin-Sepharose affinity column in the presence of calcium. The fractions eluted by EGTA contained two proteins characterized by pI values of 7.3 and 6.0 (isoelectric focusing). Both proteins, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), were resolved into a single polypeptide having and identical apparent M_rmr of 65 000. This suggests that the two initial proteins might be isoelectric variants. However, the amount of the enzyme fraction obtained by the calmodulin-Sepharose column was small and the ATPase activity was very labile. A linear glycerol gradient allowed the recovery of a greater amount of the enzyme which was, however, only partially purified, but the activity of which was much more stable. Electrophoresis of the ATPase-containing fractions in a native polyacrylamide gradient gel permitted the separation of a 260 kDa protein which was resolved by SDS-polyacrylamide gel electrophoresis into a single polypeptide of 65 kDa. Thus, the chloroplast envelope-bound ATPase might be a tetramer (260 kDa) consisting of 4 identical monomers (65 kDa). The purified ATPase had properties similar to that of the envelope-bound enzyme. TheK_m value for ATP was 0.45 mM. The activity was stimulated by Ca²⁺ and Mg²⁺, and further enhanced by calmodulin. The physiological significance of the chloroplast envelope-bound ATPase is discussed.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing

When hydrophobic samples, or membrane proteins, are disaggregated in buffers containing detergents (e.g. Nonidet P-40), urea and 2-mercaptoethanol, and applied at the cathodic end of a gel cylinder or slab for isoelectric separation, as routinely performed for two-dimensional techniques, a severe disturbance of the alkaline region of the pH gradient ensues. This phenomenon has been attributed to high protein loads, which supposedly overcome the buffering power of isoelectric carrier ampholytes. On the contrary, in the present study it has been found that this suppression of the alkaline end of the pH gradient is due to 2-mercaptoethanol, which is a buffer with pK 9.5. This compound ionizes at the basic gel end and is driven electrophoretically along the pH gradient, sweeping away, along its path, and focused carrier ampholytes.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.