Two-dimensional electrophoresis of membrane proteins. Factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI greater than 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112-120) gives good resolution of proteins of pI 5-8, including cytochromes P-450, but fails to resolve a major acidic protein of pI less than 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R . N+(CH3)2 . CH2CH2CH2SO3- are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C14 or C16) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents.

In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB₁₄, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7–11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0–4°C, at 200 V in 6-cm gels of 5% T/15% C_Bis with 0.1 n H₂SO₄ and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.     

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.     

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.     

Determination of isoelectric points in thin-layer isoelectric focusing: The importance of attaining the steady state and the role of CO2 interference

Isoelectric points differing by 1 to 2 pH units are measured for horseradish peroxidase and lactoperoxidase depending upon the technique of isoelectric focusing, namely, the density gradient technique or systems stabilized by either granulated (Sephadex, Bio-Gel) or compact polyacrylamide gels. Conditions standardized for the determination of pI values of selected pH marker proteins proved inadequate for the predominant isoenzyme of horseradish peroxidase which requires an excessively long focusing time to attain the steady state. Carbon dioxide interferes with the determination of pI values >8.2 to 8.3. Thin-layer isoelectric focusing in a CO₂-free atmosphere followed by pH measurements also in a CO₂-free atmosphere, yields for alkaline marker proteins and the predominant peroxidase isoenzyme, pI values in excellent agreement with these found by the density gradient technique. The isoionic point of the predominant peroxidase isoenzyme determined by ion exchange desalting is identical with the isoelectric point found by density gradient and thin-layer isoelectric focusing in a CO₂-free atmosphere.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

Highly purified neurophysin proteins: Method of isolation based on their acidic properties

The isolation of highly purified bovine neurophysins I and II from freshly frozen posterior pituitaries is reported. The method can also be used for the isolation of neurophysins from other species, and acetone-desiccated preparations may serve as starting material as well. Crude posterior pituitary extract was obtained as described by Hollenberg and Hope (1967, Biochem. J., 104, 122–127). Basic and neutral proteins were then separated from the acidic neurophysins by cation-exchange chromatography on Cellex-CM (carboxymethyl). Neurophysin I was separated from neurophysin II by anion-exchange chromatography on DEAE-(diethylaminoethyl)-Sephadex with a continuous sodium chloride gradient (0 to 0.4 m). Highly purified bovine neurophysin I was also secured with a stepwise sodium chloride gradient (0.22 m starting gradient followed by a steep gradient from 0.22 to 0.4 m). The current method yields neurophysin proteins in a higher overall yield than previous procedures, as determined by single radial immunodiffusion and concentration-dependent absorption after disc electrophoresis. The method also gives neurophysins of greater purity than standard procedures currently in use. The proteins are characterized by a single, sharp precipitation band on immunodiffusion and immunoelectrophoretic analysis against antiporcine neurophysin antibody, by single bands on analytical gel disc electrophoresis at a running pH of either 8.8, 5.9, or 4.0. Isoelectric focusing on polyacrylamide gel gave an apparent pI value of 4.31 ± 0.07 for neurophysin I and a value of 4.79 ± 0.11 for neurophysin II. Radioimmunoassay revealed barely detectable levels of adrenocorticotropin-like material in neurophysin I (12 pg/100 μg of neurophysin) and no detectable levels in neurophysin II. Both proteins were devoid of avian vasodepressor activity in the conscious chicken, melanotropic activity in vitro in frog skin, and did not effect electrolyte excretion in hydropenic rats.     

Two-dimensional electrophoresis of small-molecular-weight proteins

We have developed a procedure for two-dimensional separation of small-molecular-weight (9000–30,000), acidic (pI 4–6) proteins that allows the use of strips cut from horizontal isoelectric-focusing slab gels for the first dimension, and discontinuous gels containing sodium dodecyl sulfate and high concentrations of urea in the second dimension. This technique facilitates the screening of large numbers of samples and the evaluation of electrofocusing artifacts. We emphasize measures to prevent major problems encountered in the use of this technique, particularly those caused by diffusion and aggregation. We also describe an extension of the method which allows the two-dimensional comparison of many samples in a selected narrow pH zone of interest.     

Tissue inhibitor of metalloproteinases. Identification of precursor forms synthetized by human fibroblasts in culture

Precursor forms of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) synthesized by human fibroblasts in culture have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of specific immunoprecipitates. Translation of mRNA extracted from fibroblasts in the cell-free rabbit reticulocyte lysate system yielded a single immunoprecipitable precursor of tissue inhibitor of metalloproteinases, M_r 22 000. Intact fibroblasts cultured in the presence of tunicamycin synthesized an M_r 20 000 form of tissue inhibitor of metalloproteinases, detectable intracellularly and extracellularly. This is in contrast to the predominantly intracellular M_r 24 000.form synthetized during monensin treatment of cells and the normal secreted form of tissue inhibitor of metalloproteinases, M_r 29 000. Isoelectric focusing of the various immunoprecipitable precursor forms showed a progressive increase in positive charge and microheterogeneity of the protein during cellular processing. The data suggest that the inhibitor protein core, of basic pI, is glycosylated initially by the addition of mostly neutral sugars and subsequently by acidic sugars, prior to secretion.     

A unified method for purification of basic proteins

Protein purification is still very empirical, and a unified method for purifying proteins without an affinity tag is not available yet. In the postgenomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied for purifying any recombinant basic protein from Escherichia coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI−1 ? 6.94) may be purified from E. coli in a single step using a cation-exchanger resin, SP-Sepharose, and a selected buffer pH, depending on the pI of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step.     

Resolution of fibronectin and other uncharacterized proteins by two-dimensional polyacrylamide electrophoresis with thiourea

Several proteins, which are recognized components of serum, are not resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) under standard conditions. One major example is fibronectin, which is detected in fairly high concentration (milligram range) by immunoassays, while undetectable in 2D-PAGE gels. Following several experiments with a combination of zwitterionic and chaotropic substances we obtained a good resolution of the protein in gels containing 0.5 M thiourea plus 8 M urea. By this technique, fibronectin was, for the first time, found to be microheterogeneous between pI values of 5.3 and 5.6 . Besides fibronectin we detected three other families of uncharacterized proteins with M_r of 130?000, 110?000 and 34?000 respectively, whose identity and function are currently under investigation.     

Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LM₂ were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LM₄ was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02–0.1 m hexafluoroacetone/NH₃, pH 7.2 for highly acidic peptides; and (2) 6 m formic acid/0.13 m trimethylamine, pH 1.5, vs 4 m formic acid/0.09 m trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterification of serine and threonine residues and appear to be reversible by aminoethanol treatment.     

Un cytochrome de type P450 dans le spadice d'arum

A cytochrome of the P₄₅₀ type has been shown to exist in the microsomal fraction derived from Arum spadices. It appears to be present in greater quantities than the cytochrome b₅. It is transformed, partially at least, into a pigment of type P₄₅₀ under the action of salts and detergents.     

High-resolution two-dimensional electrophoretic analysis of acidic,basic and surface proteins of mouse neutrophils

The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 200 proteins detected) and with fluorography or autoradiography after [³⁵S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of ¹²⁵I associated with actin) yielded more than 20 major ¹²⁵I-labelled proteins on high-resolution electrophoretic maps. The major ¹²⁵I-labelled protein (M_r ≈ 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [³⁵S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.     

Antitumor activity of some organomettalic bismuth(III)thiolates

The neutral organobismuth(III)bis(thiolates) CH₃Bi(SCH₃)₂ and CH₃Bi(p-SC₆H₄NH₂)₂ and the ionic mercaptoanilinium derivative [CH₃Bi(p-SC₆H₄NH₂CH₃)₂]²⁺2I⁻ were tested for antitumor properties in the fluid Ehrlich ascites tumor systems of mice. They all effected an optimum cure rate of 100% and were characterized by values of the therapeutic index ranging between 3.2 and 5.0.     

Azido auxins : photoaffinity labeling of auxin-binding proteins in maize coleoptile with tritiated 5-azidoindole-3-acetic Acid

Tritiated 5-azidoindole-3-acetic acid (5-N₃-[7-³H]IAA), a photoaffinity labeling agent, was used to photolabel proteins of a crude microsomal preparation from maize (Zea mays L., Bear Hybrid, WF9 × BR38) coleoptile. Approximately 50% of the bound radioactivity was solubilized in 5 molar urea containing Triton X-100, and the extract was fractionated using a variety of techniques. High performance liquid chromatography demonstrated that, although many membrane proteins incorporated tritiated label, only a few showed reduced incorporation in the presence of excess indole-3-acetic acid. By contrast, no detectable reduction in incorporation was observed in the presence of excess naphthalene-1-acetic acid. Results from isoelectric focusing gel electrophoresis indicate that the proteins that showed reduced incorporation of photolyzed 5-N₃-[7-³H]IAA in the presence of IAA fell into two main groups: one which focuses between pH 5.2 and 5.7 (pI 4.8-5.3) and another around pH 6.2 (pI 5.8). In sodium dodecylsulfate polyacrylamide gel electrophoresis, the proteins migrated as four bands with apparent molecular weights of 60, 49, 45, and 37 kilodaltons. The auxin-transport inhibitor, 2,3,5-triiodobenzoic acid, competes for the labeling by 5-N₃-[7-³H]IAA, suggesting that some of these proteins may be involved in auxin transport.     

Density functional theory study on the reaction mechanism of synthesizing 1,3-dimethyl-2-imidazolidinone by urea method

We report a first-principles density functional theory investigation on tailoring the fundamental reaction mechanism of synthesizing 1,3-dimethyl-2-imidazolidinone (DMI) through the urea method with water serving as both solvent and catalyst. The nucleophilic cyclization reaction is implemented by two ammonia removal steps. One –NH group of dimethylethylenediamine (DMEDA) first attacks the carbon atom of urea, eliminating one –NH₃ group and forming an intermediate state CH₃NHC₂H₄N(CH₃)CONH₂ (IM^I). IM^I subsequently undergoes the cyclization process through a secondary ammonia removal via similar manner. Without water, the two ammonia removal steps are both slightly exothermic with high activation barriers (~50 kcal mol^-1). As water participated in the reaction, the kinetics of the two steps can be significantly improved, respectively. The role that water plays, beside as solvent, more importantly, is to serve as a proton exchange bridge. Due to the spatial configuration, the direct proton migration from the N atoms of ethylenediamine to urea is difficult to occur. The water bridge facilitates the proton migration by shortening the migration distance. As a consequence, the activation barriers are considerably lowered down to ~30 kcal mol^-1, indicating a strong catalytic effect from water. In contrast, the three possible side reactions of IM^I, even catalyzed by water, have higher activation barriers due to strong steric inhibitive effect and consequently become difficult to occur at the same condition. The current computational understanding on the prototypical reaction to DMI can be extended to guide developing more efficient routes to synthesize imidazolidinone derivatives through the urea method.     

Rapid and efficient extraction of soluble proteins from Gram-negative microorganisms without disruption of cell walls

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably penetrate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.     

Rapid chromatofocusing of proteins

Chromatofocusing, a chromatographic technique whereby proteins are selectively eluted from an ion-exchange support according to their pI values, has been adapted to high-performance liquid chromatography. It was found that chromatofocusing can detect heterogeneity in protein preparations not demonstrated by reverse-phase or size exclusion chromatography and that the resolution of chromatofocusing is comparable to ion-exchange chromatography. Although chromatofocusing may not resolve proteins as well as isoelectric focusing, it has advantages over this technique in both speed and capacity. The usefulness of chromatofocusing as an additional technique in the analysis and preparation of proteins is discussed. The rapid separation technique described here is able to resolve protein mixtures in the chromatofocusing mode in approximately 30 min.     

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.