Studies on the analytical performance of a non-covalent coating with N,N-didodecyl-N,N-dimethylammonium bromide for separation of basic proteins by capillary electrophoresis in acidic buffers in 25- and 50-microm capillaries.

Capillaries (25- and 50-microm inner diameter) coated with a double-alkyl-chain cationic surfactant N,N-didodecyl-N,N-dimethylammonium bromide (DDAB) were used for the separation of four basic standard proteins in buffers of pH 4 at various ionic strengths. The choice of buffer is critical for the analytical performance. Ammonium ions must be avoided in the buffer used in the non-covalent coating procedure owing to competition with the surfactant. Phosphate buffer gave a better separation performance than some volatile buffers; the reason seems to be a complex formation between the proteins and dihydrogenphosphate ions, which decreases tendencies for adsorption to the capillary surface. The DDAB coating was easy to produce and stable enough to permit, without recoating, consecutive separations of the proteins for up to 100 min with good precision in migration times and peak areas. A strong electroosmotic flow gives rapid separations, which is of special importance when commercial instruments are used, since the choice of the length of the capillary is restricted.

Partial purification of a human liver sulphotransferase active towards bile salts.

An enzyme that catalyzes the transfer of sulphate from 3'-phosphoadenosine 5'-phosphosulphate to bile salts was purified from human liver cytosol by chromatography on DEAE-Sephadex and Sephadex G-200, by agarose suspension electrophoresis and by isoelectric focusing in free solution. The purified enzyme was also active towards oestrone, dehydroepiandrosterone and phenol. No other liver steroid sulphotransferases could be detected during this purification procedure. Km values of 1.8 . 10(-6) M and 3.3 . 10(-6) M for glycolithocholate and 3'-phosphoadenosine 5'-phosphosulphate respectively were found. The sulphotransferase has an isoelectric point of 5.5. The enzyme was markedly activated by Mg2+, Mn2+ and Co2+ and inhibited by Cu2+, Fe2+ and Zn2+. Chenodeoxycholate and deoxycholate were sulphated at the 7-OH and 12-OH position, respectively. No bile salt disulphate formation was detected. A 30-fold increase in specific activity was obtained, although the purification based on ultraviolet light measurements was considerably higher.

Purification and characterization of two forms of a low-affinity Ca2+-ATPase from erythrocyte membranes.

A low-affinity Ca2+-ATPase from erythrocyte membranes has been purified by agarose suspension electrophoresis and polyacrylamide gel electrophoresis in the absence of detergents. For maximal activity a calcium concentration above 10 mM is required. The activity is independent of magnesium. The Km value for ATP is about 60 microM. The enzyme appears in two forms (A and B) with similar amino acid composition. The specific activity of A is higher than that of B. Gel electrophoresis in SDS of A gives a pattern consisting of two bands. B gives the same pattern; the only difference between the patterns is the ratio of the amounts of protein in the bands. The apparent molecular weight of the proteins in the two SDS bands has been estimated at 23000 and 21000, respectively. The results obtained can be explained by assuming that the two proteins corresponding to the two bands obtained in SDS electrophoresis have a similar structure and can associate to complexes A and B. We have also shown that electrophoretic and chromatographic supporting media can induce aggregation of (membrane) proteins. Artificial complexes can thus be formed and cause misinterpretation of the data obtained. This may be the reason why some authors have speculated that Ca2+-ATPase is active only in complex with other proteins such as spectrin and actin.

Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis.

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.

Chromatographic purification of a mammalian histidine decarboxylase on charged and non-charged alkyl derivatives of agarose.

Histidine decarboxylase (EC 4.1.1.22) from a mouse mastocytoma has been purified by chromatography on charged and non-charged n-alkyl derivatives of agarose. The former was represented by the coupling product of CNBr-activated agarose and alkylmonoamines (alkylamino-agarose), the latter by the coupling of agarose and alkylglycidyl ehters (alkyl agarose). The choice of fractionation medium was restricted by the enzyme stability; excessively high ionic strength media could not be used. Under the conditions investigated, the best result was obtained with the non-charged ocytl agarose. The enzyme was adsorbed to this gel at a relatively high ionic strength, and on stepwise decrease in ionic stength of the eluting buffer it was desorbed with a total recovery of 80%. There was an approx. 10-fold increase in specific activity. The histidine decarboxylase, thus purified, retained 90-100% of its activity for 10 days or more at 6-8 degrees C. Some general comments on protein fractionation on charged and non-charged alkyl derivatives of agarose are given. The complexity of protein interaction with the charged alkyl derivatives is illustrated by experiments with a colored protein, phycoerythrin.

The major sialoglycoprotein of the human erythrocyte membrane. Release with a non-ionic detergent and purification.

The major sialoglycoprotein of the human erythrocyte membrane has been selectively released by the non-ionic detergent Tween 20 and further purified in detergent-free buffers by hydroxyapatite chromatography and, finally, by hydrophobic interaction chromatography on pentyl-Sepharose. The purified glycoprotein shows one main zone, PAS-1, and up to three minor zones after staining both for protein and carbohydrate in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The relative staining intensities are concentration dependent. When the purified glycoprotein has been heated to 100 degrees C in dodecyl sulfate, more stain appears in the most rapid zone, PAS-2, and less in the slower zones, indicating a disaggregation of oligomeric forms of this glycoprotein, including a dimer, PAS-1.

Purification of membrane proteins in SDS and subsequent renaturation.

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.

The glucose transport activity of human erythrocyte membranes. Reconstitution in phospholipid liposomes and fractionation by molecular sieve and ion exchange chromatography.

Human erythrocyte membranes, at a protein concentration of 1-2 g/l, were solubilized with 0.12 M cholate in the presence of 0.06 M phospholipid (egg yolk phospholipids or phosphatidylcholine). More than 40% of the protein was solubilized. Cholate was removed by molecular sieve chromatography, whereby liposomes formed. These liposomes exchanged D-glucose faster than L-glucose. The recovery of glucose transport activity in the reconstituted system was estimated to be higher than 16%. The liposomes were heterogenous in size, as shown by molecular sieve chromatography on Sepharose 4B, and small liposomes predominated. In liposomes formed with phosphatidylcholine, the distribution of glucose transport activity did not parallel the distribution of protein or phospholipid, and the activity was found mainly in the smallest liposomes. The proteins were incorporated mainly in the liposomes that eluted at the lowest ionic strength upon ion exchange chromatography. The glucose transport activity separated into three main peaks upon ion exchange chromatography of egg yolk phospholipid liposomes. The activity eluted at low ionic strength. The liposomes contained proteins mainly from the 3- and 4.5-regions (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1-19). The activity peaks were highest in the first part of the chromatogram. The protein distribution did not coincide with the variation in activity over each peak. Therefore, it cannot be excluded that a minor component not seen in the electrophoretic analyses might be responsible for the glucose transport activity.

Hydrophobic interaction chromatography on non-charged Sepharose derivatives. Binding of a model protein, related to ionic strength, hydrophobicity of the substituent, and degree of substitution (determined by NMR).

A series of agarose gels, substituted with hydrophobic groups, has been synthesized and used for binding studies with the coloured model protein, phycoerythrin. The degrees of substitution for the derivatives can easily be estimated with proton magnetic resonance (NMR). It has been found that the capacity of the derivatives for phycoerythrin increases with increasing hydrophobicity of the substituent, degree of substitution and increasing ionic strength. For column experiments the degree of substitution should lie in the range 40-100 mmol substituent/mol galactose. When it is excessively high, the flow characteristics of the columns are unsatisfactory and difficulties to achieve complete desorption may arise.

Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane.

The membrane proteins from Spiroplasma citri have been resolved into 16 components by SDS-polyacrylamide gel electrophoresis. By this technique it was also shown that the molecular weights of these proteins ranged from 13000 to 160 000. One of the proteins, which had an apparent molecular weight of 26 000 was the most abundant and represented more than 22% of total membrane protein. We have designated this protein spiralin. None of the proteins contained carbohydrate. Spiralin has been isolated by a procedure which involves removal of some membrane proteins with the neutral detergent Tween 20, selective solubilization of the Tween residue in DOC and fractionation of the DOC-soluble material by agarose-suspension electrophoresis. The homogeneity of spiralin was demonstrated by analytical polyacrylamide gel electrophoresis under different conditions and by crossed immunoelectrophoresis. Spiralin appeared to bind less DOC than the other membrane proteins of S. citri. This observation does not imply, however, that the binding of DOC to spiralin is weak. Spiralin was neither soluble in detergent-free buffers nor in Tween 20, which indicated that it is an intrinsic membrane protein. The amino-acid composition of spiralin was quite different from that of the membrane. Spiralin lacked methionine, histidine and tryptophan, and had a low content of glycine, leucine, tyrosine and phenylalanine, but a high content of threonine, alanine and valine.

A new test based on 'salting out' to measure relative surface hydrophobicity of bacterial cells.

A simple method for quantification of the hydrophobic surface properties of bacteria is described. The method is based on precipitation of cells by salts, for instance (NH4)2SO4. The order in which cells are precipitated is a measure of their surface hydrophobicities, the most hydrophobic cells being first precipitated at low salt concentration. Temperature, pH, time and the bacterial cell concentration were shown to affect the results. When these variables were kept constant the method was highly reproducible. This 'salting out' method was applied to enterotoxigenic Escherichia coli strains with different surface protein antigens (fimbriae, fibrillae and colonization factor antigen, CFA). These enterotoxigenic E. coli strains were found to have surface hydrophobicity in the following order: CFA/I greater than CFA/II greater than K88 similar to K99 greater than type 1.

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.

Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis).

The use of displacement electrophoresis (synonymous to isotachophoresis, steady-state stacking, and moving boundary electrophoresis) for recovery of DNA fragments from agarose and polyacrylamide gels is described. Complete recovery of DNA molecules ranging from oligonucleotides to 20 000-basepairs-long fragments was achieved. The DNA is recovered in a small volume (0.1-0.3 ml) and can be used directly in enzyme-mediated cleavage and ligation reactions. The recovered DNA contained no inhibitory contaminants as revealed by ligation or restriction enzyme cleavage.

Protein immunoassay based on agglutination of antibody-coated ferric oxide particles.

A novel immunoassay is described based on the agglutination by antigens of antibody-coated ferric oxide (Fe2O3) particles. The procedure for attachment of antibodies to the ferric oxide particles is simple and fast. The assay was used for identification and quantification of gamma-globulin in human serum and in chromatographic fractions as well as of gp70 (an envelope protein from feline leukemia virus) in the culture medium and for identification of the thyroid-stimulating hormone (TSH).

Surface hydrophobicity and electrophoretic mobilities of staphylococcal exotoxins with special reference to toxic shock syndrome toxin-1.

The surface hydrophobicities of eleven staphylococcal toxins were estimated and compared with those of standard proteins on an octyl agarose column by high-performance hydrophobic-interaction chromatography (HP-HIC). Staphylococcal enterotoxins (SE) D, C3, C2, C1 and B showed a low surface hydrophobicity whereas alpha-toxin and gamma-toxin had a moderate surface hydrophobicity. SEA, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal epidermolytic toxin (SET) showed high surface hydrophobicity and delta-toxin was the most hydrophobic protein. The electrophoretic mobility of the toxins was determined by free zone electrophoresis (FZE). All toxins except SEC1 and one of the two SEA species showed negative charge at pH 8.6. Charge heterogeneity was observed in SEA, SEC1, SEC3 and TSST-1: SEA and SEC1 had two overlapping components, whereas SEC3 and TSST-1 were resolved into two distinct components. The mobilities of the two TSST-1 components were estimated at -2.12 x 10(-5) and -3.60 x 10(-5) cm2v-1s-1, respectively, at 10 degrees C, and both fractions were immunologically indistinguishable as tested by specific TSST-1 antibodies with ELISA. An asymmetric peak was obtained in hydrophobic-interaction chromatography of TSST-1 indicating heterogeneity.

Mitogenic properties of two distinct forms of toxic shock syndrome toxin-1 separated on hydroxyapatite by high-performance liquid chromatography.

The homogeneity of a purified staphylococcal toxic shock syndrome toxin-1 (TSST-1) was tested by high-performance methods. This preparation was homogenous in ion-exchange chromatography and isoelectric focusing (pI = 7.4), but was resolved into two distinct peaks by high-performance hydroxyapatite chromatography. Both components, TSST-1hA and TSST-1hB had similar molecular weights (22 kD) and amino acid compositions. TSST-1 did not dimerize or polymerize upon heating at 60 degrees C for 30 min or in solutions with pH varying from 4.0 to 8.5. TSST-1hA and TSST-1hB showed similar immunological reactivity to native TSST-1 goat polyclonal antibodies. TSST-1hA and TSST-1hB as well as staphylococcal enterotoxin A and staphylococcal exfoliative toxin were potent mitogens in lymphocyte proliferation assays. The lymphocyte proliferative response to 10 pg of TSST-1hB was comparable to a response elicited by 10 ng of TSST-1hA, suggesting that the former component is a more potent mitogen. Rabbit or goat polyclonal antibodies to native TSST-1 efficiently neutralized both TSST-1 components. Heat treatment at 80 degrees C for 15 min had minimal or no effect on the mitogenic properties of TSST-1hA and TSST-1hB.

Neuron-specific enolase in relation to differentiation in human neuroblastoma.

The presence of the two forms of enolase, neuron-specific enolase (NSE) and non-neuronal enolase (NNE), have been examined in biopsy material of human neuroblastoma, ganglioneuroblastoma, ganglioneuroma and cultured neuroblastoma cells, after separation with ion exchange chromatography. The enolase activities were inhibited in the presence of NaCl but remained active in KCl, which were used in the chromatographic step. The relative NSE levels in the neuroblastoma tissues were found to be lower than in the histopathologically more differentiated forms of the tumour, i.e. ganglioneuroblastoma and ganglioneuroma. The human neuroblastoma in vitro cell lines SK-N-SH, SH-SY5Y, SK-N-MC and IMR-32 contained considerably lower relative levels of NSE compared to the levels in the neuroblastoma biopsies. After treatment of the cultured cells with nerve growth factor or dibutyryl-cAMP some cells showed morphological differentiation and concomitantly an increase in the NSE levels. The results indicate that NSE might be useful as a marker for differentiation in human neuroblastoma.

Capillary and rotating-tube isoelectric focusing of a transmembrane protein, the human red cell glucose transporter.

The human red cell glucose transporter (Glut1) is a transmembrane protein. Monomeric Glut1 was purified by ion-exchange chromatography in the presence of the non-ionic detergent n-dodecyl octaoxyethylene (C12E8). For focusing, the ionic strength of the solution of C12E8-Glut1 complexes with co-purified lipids was lowered by dialysis, the detergent concentration was increased and carrier ampholytes were added. Focusing was done for 5 min at 3000 V in a methyl cellulose-coated glass capillary (50 microns I.D.). The anolyte H3PO4 was then replaced by NaOH for mobilization towards the anode. Absorbance monitoring at 280 nm showed two groups of zones at pH 6 and 8. Similarly, isoelectric focusing in a rotating quartz tube (3 mm I.D.) gave Glut1 zones at pH 5.5 and 8.0. Phosphorus analysis revealed that the Glut1 zone at pH 8 contained more phospholipids than did the other one. The above results together with previously determined and calculated isoelectric points (pI) of Glut1 indicate that the Glut1 at pH 8 is monomeric and that the zone at pH 5.5-6 represents oligomeric materials. The pI 8.0 at 22 degrees C applies for monomeric Glut1 in the absence of urea. The results exemplify that capillary isoelectric focusing of hydrophobic membrane proteins is possible.

Immobilized liposome chromatography of drugs on capillary continuous beds for model analysis of drug-membrane interactions.

Liposomes were immobilized in capillary continuous beds with covalently linked C4 or C8 alkyl ligands for chromatographic analysis of drug interaction with phospholipid bilayers, as reflected by drug retention volumes and calculated differences in interaction free energies. This procedure is a high-resolution micro-scale version of immobilized liposome chromatography for prediction of diffusion of drugs across biological membranes. The logarithm of the specific capacity factors of several structurally unrelated drugs showed a linear correlation with the logarithm of known apparent drug permeabilities through Caco-2 epithelial cell monolayers. The latter values are used for prediction of absorption of orally administered drug doses.

A simple and inexpensive chromatographic method for the purification of gamma-globulin from human serum.

Inexpensive cation- and anion-exchangers based on continuous polymer beds were prepared directly in a plastic syringe. These beds are unique in the sense that they are synthesized by a very simple and cost-effective procedure; for instance, no preparation of beads is required as in conventional methods. Highly purified gamma-globulin could be prepared easily by step-wise elution accomplished by forcing buffers of increasing salt concentration through the column with the aid of the plunger.