Stabilization of pH gradients in buffer electrofocusing on polyacrylamide gel.

pH gradients in buffer electrofocusing on polyacrylamide gel (BEF) have been stabilized for at least 100 hr, 25°C, by replacing the strongly acidic and basic anolyte and catholyte with isoelectric buffers identical to the terminal constituents of the pH gradient and gel. Such stabilization leads to a constant pI position of an electrofocused protein. No stabilization of pH gradients is achieved under otherwise identical conditions when strongly acidic and basic anolyte and catholyte are used.     

Preparative separation of hemoglobins A and S by gel electrofocusing, using selective zone elution by gel transposition between suitable anolytes and catholytes.

Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection of the slice diffusates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem.75, 314–324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from a electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%, with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.     

The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of NISTmAb revealed by HDX‐MS,DSC, and nanoDSF

The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX‐MS), differential scanning calorimetry (DSC), and nano‐differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX‐MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the C_H2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry‐Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.     

Endogenous substrates for cyclic AMP-dependent and calcium-dependent protein phosphorylation in rabbit peritoneal neutrophils

As in other cells, cAMP-dependent (protein kinase A) and calcium-dependent protein kinases are present in the rabbit peritoneal neutrophil. The major substrates for protein kinase A in the cytosol of rabbit peritoneal neutrophil is a 43 kDa protein which appears to be actin (pI 5.7). The other substrates for protein kinase A in the cytosol are very acidic proteins with molecular weights of 135 000 (pI 4.6) and 130 000 (pI 4.8). Two classes of calcium-dependent protein kinases are present in the rabbit peritoneal neutrophil: one is calcium, calmodulin-dependent, the other is calcium, phosphatidylserine-dependent. Phosphatidylserine appears to be much more effective than calmodulin in stimulting calcium-dependent protein kinase activity. The phospolipid-sensitive, calcium-dependent protein kinase (protein kinase C), present only in the cytosol fraction, exhibits much higher activity than the cAMP-dependent protein kinase from the same source. At least four substrates (M_r 130 000 (pI 4.6) 43 000 (pI 4.8), 41 000 (pI 6.3) and 34 000) of the protein kinase C in the cytosol were identified. Trifluoperazine, a compound which inhibits the degranulation, aggregation and stimulated oxygen consumption of rabbit peritoneal neutrophils. (Alobaidi, T., Naccache, P.H. and Sha'afi, R.I. (1981) Biochim. Biophys. Acta 675, 316–321), also inhibits the activity of protein kinase C. The possible role of cAMP-dependent and calcium-dependent phosphorylation system in neutrophil function is discussed.     

Purification of the glial fibrillary acidic protein by anion-exchange chromatography

A procedure for the isolation of assembly-competent glial fibrillary acidic (GFA) protein from 2 m urea extracts of bovine spinal cord by anion-exchange chromatography is reported. The tissue was previously extracted with low-ionic-strength buffer. The procedure allowed the separation of nondegraded GFA protein from GFA protein comprising degraded species. As previously reported for neurofilament preparations obtained from porcine spinal cord (N. Geisler and K. Weber, J. Mol. Biol., 151, 565–571 (1981)), the procedure also allowed the simultaneous separation of the three neurofilament polypeptides (200,000; 150,000; and 70,000 daltons) contained in the 2 m urea extract. Brain filament proteins sequentially eluted at increasing salt concentration (25–200 mm NaCl) according to their isoelectric point. Proteins with higher pI eluted first. Tubulin eluted between the 200,000- and 150,000-dalton neurofilament polypeptides.     

Affinity and hydrophobic chromatography of Escherichia coli aspartate transcarbamoylase

Chromatography of aspartate transcarbamoylase from Escherichia coli on agarose-immobilized dyes and alkyl-agaroses of differing carbon length were investigated. The bacterial aspartate transcarbamoylase was bound by Procoin red HE3B-agarose and Cibacron blue F3GA-agarose nearly completely under the conditions chosen relative to other agarose-coupled dyes. The aspartate transcarbamoylase holoenzyme was eluted from the Procion red HE3B-agarose slightly later than from the Cibacron blue F3GA-agarose during salt gradient elution. The catalytic trimer of the enzyme as well as its regulatory dimer were eluted by a lower salt concentration from both dye-agarose gels than the concentration required to elute the haloenzyme. The interaction of the catalytic trimer with the Procion red HE3B-agarose and Cibacron blue F3GA-agarose gels may be a determinant in the holoenzyme being retained on these resins. Of those alkyl-agaroses tested, the ethyl-, propyl- and hexyl-agarose gels bound the majority of aspartate transcarbamoylase activity. Chromatography of aspartate transcarbamoylase on ethyl-agarose found it to be eluted by a low salt concentration. A purification scheme for relatively small amounts of aspartate transcarbamoylase utilizing Procion red HE3B-agarose and ethyl-agarose is presented. This purification scheme is particularly useful for mutant versions of aspartate transcarbamoylase which cannot be purified by literature procedures.     

Two-dimensional zymography in detection of proteolytic enzymes in wheat leaves

Proteolytic enzymes in wheat leaves were studied using zymographic detection of enzyme activities on one-way (1D) SDS-polyacrylamide gels and two-dimensional (2D) ones, on which protein samples were isoelectrofocused prior to PAGE separation. Gelatin of concentration 0.1 %, copolymerized into SDS-PAGE gels, digested by active proteinases enabled detection of those enzymes. On 1D gels, seven bands were seen and assigned to particular families through the use of specific inhibitors. Metalloproteinases inhibited by 20 mM EDTA were detected as 150 kDa band; aspartic proteinases were assigned to 115–118 kDa double band by using 25 mM pepstatin; 10 mM phenylmethylsulfonyl fluoride used for detection of serine proteinases pointed to band of 70 kDa and finally due to 10 μM E-64 inhibitor, cysteine proteinases of 37 and 40 kDa were detected. On 2D gels, additional separation according to protein isoelectric points enabled detection of proteinase isoforms. In the range of 4.5–6 pI, six metalloproteinases as well as ten aspartic proteinases were visible, ten serine- isoforms of pI 4.5–6.8 and four cysteine proteinases of 4.5–5.0 pI were found. Presented results were detected as reproducible results observed at least in four independent biological replications.     

COST-EFFECTIVE AND EFFICIENT APPARATUS FOR ELECTROELUTION OF MICRO- AND MACROGRAM QUANTITIES OF PROTEINS FROM POLYACRYLAMIDE GELS

Protein recovery from gel electrophoresis plays a significant role in functional genomics and proteomics. To assist in this, a simple, cost-effective, and efficient apparatus for electroelution of proteins has been designed. The performance of the apparatus was demonstrated using the proteins bovine serum albumin (BSA), phosphorylase, ovalbumin, pepsin, and trypsinogen. In all the cases the yield of elution was found to be consistently greater than 85% and the proteins could be eluted without degradation in less than 15 min. The utility of this method can be extended to protein elution from denatured and native polyacrylamide gels, DNA purification from agarose gels, and oligomeric primers purification from polyacrylamide gels. In addition to this, the method offers an effortless purification and characterization of microbial extracellular proteins. The eluted proteins can be directly used in N-terminal amino acid sequencing, and in amino acid and proteomics analyses.     

Purification of homogeneous forms of fungal peroxygenase

Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.     

An agarose derivative containing an arsenical for affinity chromatography of thiol compounds

An adsorbent for affinity chromatography of thiol compounds was prepared by covalently linking p-aminophenylarsine oxide to CNBr-activated Sepharose 6B. The adsorbent retained mono- and dithiols from acid and neutral solutions. Monothiols and 1,4-dithiols were eluted at pH 12, whereas more alkaline conditions were necessary for elution of 1,2- and 1,3-dithiols. The latter could also be eluted at pH 12 by 0.01 m phenylarsine oxide. Alternatively, thiol compounds may be eluted at neutral or acid conditions by other thiol compounds. Monothiols and 1,4-dithiols were thus eluted by cysteine, whereas 1,2- and 1,3-dithiols required dithiopropylamine for elution.     

Purification of murine thymocyte-stimulating factor from supernatants of mixed lymphocyte cultures

Thymocyte-stimulating factor (TSF) has been purified 360-fold with a yield of about 18% by the sequential use of gel filtration on Sephadex G-100, ultrafiltration, hydrophobic interaction chromatography on phenyl Sepharose and affinity chromatography on concanavalin A (Con A)-Sepharose. Most of the TSF activity was eluted from Con A-Sepharose columns with α-methyl-d-mannoside, thus suggesting the presence of glucose and/or mannose residues. At the same time, however, the eluted material showed a considerable degree of heterogeneity in the number of these carbohydrate residues. Column electrofocusing of the purest preparations showed two peaks of TSF activity with pI's of 4.7 and 5.1. Proteins were found to electrofocus at these pI's, while no detectable amounts of proteins were found at other pI's.     

Inhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation

The aim of this study was to investigate the mechanism of inhibition of mitochondrial aldehyde dehydrogenase (ALDH2) by carbon tetrachloride (CCl₄). CCl₄ administration caused marked hepatocyte ballooning and necrosis in the pericentral region. CCl₄ also inhibited hepatic ALDH2 activity in a time-dependent manner without altering the protein level, suggesting ALDH2 inhibition through covalent modifications such as phosphorylation by JNK. To demonstrate phosphorylation, the isoelectric point (pI) of ALDH2 in CCl₄-exposed rats was compared to that of untreated controls. Immunoblot analysis revealed that immunoreactive ALDH2 bands in CCl₄-exposed rats were shifted to acidic pI ranges on two-dimensional electrophoresis (2-DE) gels. Incubation with alkaline phosphatase significantly restored the suppressed ALDH2 activity with a concurrent alkaline pI shift of the ALDH2 spots. Both JNK and activated JNK were translocated to mitochondria after CCl₄ exposure. In addition, incubation with catalytically active JNK led to significant inhibition of ALDH2 activity, with an acidic pI shift on 2-DE gels. Furthermore, immunoprecipitation followed by immunoblot analysis with anti-phospho-Ser–Pro antibody revealed phosphorylation of a Ser residue(s) of ALDH2. These results collectively indicate a novel underlying mechanism by which CCl₄ exposure activates JNK, which translocates to mitochondria and phosphorylates ALDH2, contributing to inhibition of ALDH2 activity accompanied by decreased cellular defense capacity and increased lipid peroxidation.     

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.     

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.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

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.     

Isolation of a plant glycoprotein involved with control of intercellular recognition

A recognition molecule was isolated from stigmas of S-allele genotype S₂S₂ of Brassica oleracea var. capitata L. After Sephadex chromatography, it eluted as a single symmetrical peak during diethylaminoethane-cellulose chromatography. A high degree of purity was affirmed by: sedimentation as a single peak during ultracentrifugation through 5 to 20% sucrose gradients; elution as a single peak from Sephadex G-100; visualization as a single band which stains with Coomassie blue and periodic acid Schiff reagent after electrophoresis on polyacrylamide gels. Other criteria supporting the conclusion that it is a glycoprotein are: (a) the highly purified preparation is anthrone-positive and has a Lowry protein to anthrone-positive carbohydrate ratio of 1.3; (b) the preparation contains arabinose, galactose, glucose, and mannose, although it is not precipitated by concanavalin A; (c) the immunological properties of the molecule are lost following protease treatment, and it has a molecular weight of 90,000 by Sephadex gel-filtration analysis and 54,500 by velocity sedimentation analysis.     

Prevention of depurination during elution facilitates the reamplification of DNA from differential display gels.

DNA fragments that show a pattern of differential expression on differential display gels must be eluted from the gel matrix and reamplified to enable further analysis. Elution is usually achieved by heating excised gel slices in a small volume of either water or TE. Here we show that this elution step can adversely affect the ability of the eluted DNA to act as a template for PCR reamplification, probably via the process of depurination. Simply switching to an elution solvent designed to minimise depurination (PCR buffer) facilitates the elution of intact DNA fragments. This improvement is likely to be most beneficial when eluting higher molecular weight fragments (e.g. those >500 bp), in situations where the amount of DNA in an excised gel slice is limited or when contaminating differential display products co-migrate with the differentially expressed product.     

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.     

Schistosoma mansoni: One- and two-dimensional electrophoresis of proteins synthesized in vitro by males,females, and juveniles

Polyacrylamide gel electrophoresis coupled with fluorography is a sensitive method for visualizing individual gene products synthesized in vitro by Schistosoma mansoni (K. Atkinson and B. G. Atkinson 1980, Nature (London)283, 478–479). In vitro labelling with radioactive amino acids ensures that the proteins are of parasite origin and fluorography permits detection of minute amounts of newly synthesized, electrophoretically separated gene products. One-dimensional electrophoretic separation in polyacrylamide gels with sodium dodecyl sulphate and fluorography of juvenile and adult proteins reveal that juveniles produce most adult proteins. Although similar studies with proteins from sexed adults imply that analogous gene products are elaborated by both sexes, a number of sex-specific gene products are resolvable by more rigorous two-dimensional electrophoretic separations. The homogametic male produces 5 polypeptides not produced by the heterogametic female. Three outstanding male-specific gene products include a polypeptide with a molecular weight (MW) of 88 kilodaltons (kd) and an isoelectric point (pI) of 5.65, one with an MW of 66 kd and a pI of 5.25, and one with an MW of 58 kd and a pI of 5.25. Other, readily detectable male-specific polypeptides include one which coelectrophoreses with β-actin and one which coelectrophoreses with β-tropomyosin. The female synthesizes 4 specific polypeptides which have isoelectric points between 4.3 and 4.7, are of low molecular weight, and are resolvable only with 12% acrylamide gels. Two-dimensional electrophoresis resolves 74 major polypeptides synthesized by adult worms, and a code is presented which identifies each polypeptide by sex specificity, isoelectric point, and molecular weight.