Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.

The three-dimensional structures of the class II anticoagulant phospholipase A2 (PLA2) toxin RVV-VD from the venom of Russell's viper, Vipera russelli russelli, and the class I neurotoxic PLA2 Notechis II-5 from the Australian tiger snake, Notechis scutatus scutatus, were determined to 2.2Å and 3.0Å resolution, respectively. Both enzymes are monomeric and consist of 121 and 119 residues, respectively. A comparison of ten class I/II PLA2 structures showed, among other differences, that the β-sheet of these enzymes (residues 76–83) is about 90° less twisted in class I than in class II PLA2s. This, along with the insertion of some residues in the region 57–59 in class I enzymes (the elapid loop), could be the main reason for the significant difference in the anticoagulant and (presynaptic) neurotoxic properties between the two classes of PLA2. It seems apparent from sequence and structural comparisons that the toxic site of PLA2 responsible for the strong anticoagulancy of these toxins consists of a negatively charged part, Glu53, together with a positively charged ridge of lysine residues free for intermolecular interactions. These lysines differ between the two classes of PLA2.

In packed chromatographic beds, both Eddy diffusion and the relatively long time the analytes stay in the mobile phase until they collide and interact with the ligands attached to the beads (the residence time in the mobile phase) contribute considerably to zone spreading. One should not expect Eddy diffusion to occur in macroscopically homogeneous separation media, such as gels or polymer solutions, and residence time should be shorter, since the pore size of these media is much smaller than the average distance between the beads in a packed bed. Accordingly, these separation media (which can be regarded as homogeneous continuous beds [monoliths]) should theoretically give very high resolution, which has been verified experimentally: Frontal analysis of a neutral marker, acetone, showed that electroendosmosis in a homogeneous gel displaced the boundary without any distortion except that caused by diffusion (the marker was selected not to interact with the gel). All other common disturbing phenomena in chromatography (for instance, Eddy diffusion and nonspecific adsorption) were, accordingly, negligible, indicating that electrochromatography in homogeneous gels may be the ideal chromatographic method. However, to fully utilize these desirable chromatographic properties of homogeneous continuous beds, one has to choose analyte/bed interactions with sufficiently high association-dissociation rate constants (i.e., the residence time of the analytes in the stationary phase must be kept very short), which will be the subject of forthcoming studies. The electrophoretic counterpart of capillary electrochromatography (CEC), electrophoresis of noncharged analytes in a solution of charged polymers, seems to give a somewhat larger zone broadening, probably due to their high viscosity and conductivity, with attendant longer analysis (= diffusional) times compared to gels. Since the mobile phase is propelled through the gels by electroendosmosis, the theoretical and experimental requirements for high electroendosmotic flow in gels, i.e., short analysis times, are discussed. The electroendosmotic velocity v(x) can be estimated by the simple equation v(x) = Vmax (1-e-kappa x) (1/kappa = the thickness of the double layer; Vmax = the plug flow velocity) when the distance from the channel wall, x, << the radius R of the channel (pore). For kappa R > or = 5, the equation obtains with good approximation for all R values. An initially straight zone in a gel pore should be heavily distorted by the electroendosmotic flow, according to this equation (see Figure 1). However, due to rapid diffusion and other leveling effects, the zone is transported as in perfect plug flow, as is shown experimentally. A plot of electroendosmotic mobility obtained by frontal analysis against 1/(1 + square root of mu) can be used to estimate roughly the pore size in a gel and permits quantitative examination of the current theory of electroendosmosis. It is not a trivial problem to synthesize ligand-containing gels with pores large enough to allow a high electroendosmotic flow. Therefore, we have described a universal method: a polymer containing phenylboronate and acrylic acid groups was synthesized and entrapped in a standard agarose gel (for automated runs, replaceable methoxylated agarose should be used). Both of these charged groups serve to generate the electroendosmotic flow required in electrochromatography. This gel was designed to have the dual property of separating compounds that contain vicinal OH groups in the cis-configuration (exemplified by ribonucleosides) by reaction with the boronate groups, and aromatic substances by virtue of the acrylic acid residues (and perhaps also the phenyl groups) in the polymer and the agarose chains. The latter interaction, the so-called aromatic adsorption, has the advantage that it does not require a time-consuming attachment of ligands. (ABSTRACT TRUNCATED)

© 1997 Federation of European Biochemical Societies.

Electrophoretic and chromatographic experiments performed under straightforward conditions do not always provide satisfactory resolution. An obvious approach, then, is to manipulate the magnitude of relevant separation parameters, such as charge (zeta potential), size, and hydrophobicity, all of which can be accomplished by complex formation. This alternative has been studied with both a neutral, an anionic, and a cationic derivative of beta-cyclodextrin in an attempt to increase the resolution of peptides and proteins in free-zone electrophoresis utilizing the capillary format. The investigation showed that charged beta-cyclodextrins are suitable for this purpose. As expected, the effect seems to be most significant for substances with a small net surface charge, i.e., low mobility. Consequently, it may be advantageous to choose a pH of the buffer that is not far from the isoelectric point of the solutes. It should be emphasized that changes in the electropherograms observed upon addition of any complexing agent to the buffer may involve improvement or worsening of the resolution. Only by experimentation can one determine whether complexation with cyclodextrins favors resolution, since our knowledge about the interactions taking place is limited. However, if a positively (negatively) charged beta-cyclodextrin decreases the resolution of an acidic (basic) protein, one can expect theoretically, a negatively (positively) charged beta-cyclodextrin to increase the resolution, as was verified experimentally. The difference in mobility between two peaks caused by the complexation with cyclodextrins need not be larger than 2-3% for satisfactory resolution because the peaks are sharp. We have introduced a new definition for the resolution of two very adjacent peaks--the most common and interesting case in real-world analyses--that does not require measurement of peak widths.

This paper gives a survey of recent methodological studies of capillary electrophoresis at the Department of Biochemistry, Uppsala University. Several methods for on-and off-tube concentration of solutes have been developed. These methods also permit desalting of the sample, which is necessary for the creation of narrow starting zones in zone electrophoresis and to reduce the risk of precipitation and narrowing of the separation window in isoelectric focusing (IEF). A unique method designed particularly for desalting of samples for IEF is also discussed. To obtain high resolution the adsorption of solutes to the capillary wall must be eliminated. A pH stable, hydrophilic polymer coating has, therefore, been developed. Although polymer solutions give lower resolution than do gels they are often used as molecular-sieving media because they are replaceable and thus permit repetitive automated analyses in the same capillary. We have shown that low-melting methoxylated agarose gels are also replaceable and give the same high resolution as do polyacrylamide and dextran gels. Following an electrophoresis the methoxylated agarose gels can be displaced in the stationary capillary past the detection window. This scanning technique has several advantages. A capillary with strong electroendosmosis permits, in principle, separation of both acidic and basic proteins in one run. However, depending on whether the capillary wall is negatively or positively charged either the basic or the acidic proteins most often migrate with strong tailing caused by the electrostatic interaction with the capillary wall. The distortion of the zones is much less if the electroendosmotic flow is replaced by a hydrodynamic flow and the electrophoresis is performed in a coated electroendosmosis-free capillary to suppress adsorption of both basic and acidic proteins. By a unique technique we can prepare chromatographic beds with diameters as small as 5-25μm. These beds, which are more homogeneous than conventional beds built up of preformed beads, can be used with advantage for electrochromatography and for different modes of capillary chromatography of fractions obtained from micropreparative HPCE experiments. There is a general trend and desire to decrease the analysis time without sacrificing resolution. Therefore, we have developed low-conductivity buffers with satisfactory buffering capacity which permit field strengths as high as 2, 000V/cm with attendant very short run times. Many enantioselective agents gave strong UV-absorption. Therefore, the experimental conditions must be chosen so that they do not pass the detection window. Otherwise, the noisy background is very disturbing. When a zone migrates from a straightinto a curved section of a capillary the molecules at the outer lane will lag behind those at the inner lane. The attendant loss in resolution, which all commercial apparatus exhibits, can be decreased considerably by coiling the capillary into the figure-of-eight or a serpentine path. A straight capillary is, however, preferable. Methods for the determination of pH and electrical conductivity in small volumes are discussed. A technique for micropreparative HPCE and HPLC is presented. An HPCE apparatus designed according to new principles is described (straight capillary; one electrode vessel is closed to avoid hydrodynamic flow in the capillary; electroendosmotic pump for washing of the capillary; thermostated slit for the UV beam to obtain a straight base line; only a small part of the capillary is not cooled actively; thermal application of the sample; the sample rests as a small droplet on a carousel; the droplet is covered by a cap which can be lifted by a magnet; effective insulation for field strengths up to at least 2, 000V/cm).

The polymer beds described are synthesized in aqueous solution directly in the column or batchwise in the form of large clusters of small particles. The conventional, expensive step involving prepreparation of beads in an organic solvent is thus omitted. Beds were synthesized from piperazine diacrylamide, methacrylamide and allyl glycidyl ether. The epoxy-activated beds thus obtained were used for covalent attachment of either nonpolar ligands (e.g. octadecanol) or polar OH-rich substances (e.g. dextran). The non-polar beds were used for reversed-phase chromatography, as were polar ones following coupling with 1,2-epoxyoctadecane. Coating with OH-rich substances serves two purposes: (I) the matrix becomes hydrophilic, decreasing nonspecific interactions (modifiers can be excluded) and hence increases resolution and (II) many—OH groups are available (e.g. for coupling to epoxides), a prerequisite for high ligand density. Resolution of proteins was high even at high flow rates. Depending on the method used for the synthesis of the bed, resolution of proteins either increased with an increase in flow rate or decreased slinghtly. Choice of the correct temperature was very important for high resolution of CNRr-digested peptides.

The three‐dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 Å resolution to a crystallographic R‐value of 16.5% with good stereo‐chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2s) but several parts of the molecule are distinctly different. The most significant differences from PLA2s from bovine pancreas and rattlesnake occur in the stretches 56–80 and 85–89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The Cα positions for residues 86–88 differ by about 6 Å from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site.

An optimization procedure for the separation of 24 PTH-amino acids by high-performance liquid chromatography on an inexpensive Merck Superspher Si 60 RP-8, (4.0 x 250 mm) column with PTH-Nle as an internal standard is described. The effects of pH, ionic strength, temperature and gradient were investigated. Using conventional HPLC equipment, the practical detection limit is about 5 pmol.

M. Jiang, J. Häggblad, E. Heilbronn, B. Rydqvist and D. Eaker. Some biochemical characteristics and cell membrane actions of a toxic phospholipase A2 isolated from the venom of the pit viper Agkistrodon halys (Pallas). Toxicon25, 785 – 792, 1987. — A toxic component (AgTx) from the venom of Agkistrodon halys (Pallas) was isolated using DEAE-cellulose DE11 and CM-Sephadex C50 column chromatography and finally purified to homogeneity by FPLC on a MonoQ column. The toxin is a neutral (pI 6.9) single chain polypeptide with a mol. wt of 14,100 and an amino acid composition (123 residues) roughly similar to that of notexin. AgTx was found to have phospholipase A2 activity which was dependent on calcium and stimulated by sodium deoxycholate. The toxin caused efflux of 2-deoxy-(1-3H)-glucose-6-phosphate (a cell membrane integrity probe) as well as of [3H]acetylcholine from rat brain synaptosomes. No cell membrane damage was induced by AgTx on cultured N1E 115 neuroblastoma cells and chick myotube cultures. The ld50 ws 150 μg/kg (i.p.) in mice. The main symptom observed was respiratory paralysis. The results obtained show that AgTx can be classified as a toxic phospholipase A2 with a presynaptic site of action.

An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor bound neurotoxin. Toxicon 21, 219-229, 1983--Free Naja naja siamensis neurotoxin was acetylated with non-radioactive and acetylcholine receptor-bound neurotoxin with radioactive acetic anhydride. The toxins from the two experiments were combined and the monoacetyl derivatives isolated by chromatography on Bio-Rex 70. The yields were determined by spectrophotometry and scintillation counting. To localize the acetyl group, a radioactive monoacetyl toxin was oxidized with performic acid, digested with trypsin and a peptide with the radioactive acetyl group was isolated by gel filtration on Sephadex G-25 and high voltage paper electrophoresis. Amino acid analysis indicated from which part of the molecule the peptide was derived. In free toxin, Ac-Lys 23 and 49 account for 56% and 12%, respectively, of the monoacetyl derivatives, and in bound toxin for only 25% and 8%. Lys 49 is as reactive as Ile 1 in free toxin and 50-150% more reactive than Lys 69, 35 and 12, but it has the lowest reactivity in bound toxin, being only about half as reactive as any of these three residues. The large decrease in reactivity of Lys 23 and 49 indicates that they interact with the receptor. The proximity of the receptor makes them less accessible to acetic anhydride. The reactivities are compared to that of Lys 12, which in free toxin has the least reactive amino group. The yield of Ac-Lys 23 relative to that of Ac-Lys 12 drops from 12.4 to 1.5, or by 88%, Lys 49, 2.6 and 0.5 (81%); Ac-Ile 1, 2.6 and 1.1 (58%); Ac-Lys 69, 1.9 and 0.9 (53%); Ac-Lys 35, 1.8 and 1.0 (44%). The drop in reactivity relative to that of Lys 12 indicates a real decrease, provided that Lys 12 does not become more reactive in bound toxin. This is unlikely, since sequence homology shows that Lys 12 corresponds to Lys 15 of the neurotoxin oxiana II of Naja naja oxiana, a residue known to interact with the receptor. Sequence homology also supports the conclusion that the drop in the reactivity of Ile 1 has the same cause. The receptor-binding region of the siamensis toxin is rather large, containing the residue Lys 23 and 49, Ile 1 and probably also Lys 69 and 35.

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.

The amino acid sequence of the alpha-subunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan has been determined. The very basic protein, by itself a moderately neurotoxic phospholipase, consists of a single polypeptide chain of 119 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease and trypsin. Chymotryptic peptides and cyanogen bromide fragments were used to align and complete the sequence, which was determined by automated Edman degradation. The taipoxin alpha-subunit is closely homologous to the other taipoxin subunits and to other elapid snake venom phospholipases A2.

The presynaptic neutrotoxin-phospholipase, Notechis II-5 from the venom of NotechisScutatus scutatus (Australian tiger snake) has been crystallized in a form suited for x-ray diffraction analysis. The crystals belong to the orthorhombic space group P21 21,21, with unit cell dimensions, a=146.1,b =43.5 and c =39.0 A. There are two molecules of Notechis II-5 in the asymmetric unit. The molecular weight is about 13,500. Notechis II-5 is highly homologous to Notexin, another presynaptic toxin from the venom of the Australian tiger snake, to bovine and porcine pancreatic phospholipases A and other venom phospholipases.

The complete amino acid sequence of E. schistosa VI:5b, a strongly myotoxic phospholipase A2 from the venom of the common sea snake, Enhydrina schistosa, has been determined. The sequence determination was done exclusively by automated Edman degradation on constituent peptides. The main fragmentation was done by cyanogen bromide cleavage yielding two fragments of approximately equal size. Peptides derived by cleavage with trypsin and Staphylococcus aureus V8 protease were used to complete and align the sequence. The results indicate that the myotoxin consists of a single chain of 119 amino acids cross-linked by 7 disulfide bridges and is closely homologous to other phospholipases A2 of elapid origin.

The complete amino acid sequence of Notechis II-1, a non-neurotoxic, non-enzymatic phospholipase A2 homolog from the venom of the Australian tiger snake Notechis s. scutatus has been determined. The protein consists of a single chain of 119 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined mainly by automated Edman degradation. Notechis II-1 contains all of the residues that appear to be invariant in elapid and pancreatic phospholipases A2 except at position 30 in the sesquence, where an otherwise invariant glycine is replaced by serine.