Characterization of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresis/electrospray (ionspray) mass spectrometry and 252Cf plasma desorption mass spectrometry

A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass spectrometry and by 252Cf plasma desorption-time of flight mass spectrometry. The measured average molecular mass of the synthetic peptide was 4627.16 Da, which was only 0.08 Da higher than the calculated value (4627.08 Da). The plasma desorption mass spectrum of the synthetic peptide showed a protonated molecule at m/z 4624.1, which was 4 Da lower than the calculated one (4628.09 Da). The amino acid sequence of the peptide was confirmed in part by electrospray (ionspray) mass spectrometry using a high nozzle skimmer voltage difference. Five impurities were separated and identified by capillary electrophoresis/mass spectrometry and two of them also appeared in the plasma desorption mass spectrum.     

Chiral separation of alpha-amino acids by ligand-exchange capillary electrophoresis using N-(2-hydroxy-octyl)-L-4-hydroxyproline as a selector

The direct chiral resolution of underivatized alpha-amino acids by capillary zone electrophoresis (CZE) based on the principle of ligand exchange is described. An N-(2-hydroxyoctyl)-L-4-hydroxyproline/Cu(II) complex was used as a chiral selector. Besides amino acids containing aromatic residues, the basic amino acid histidine was resolved. Baseline separations were obtained for all amino acids investigated. The influence of selector concentration, electrolyte composition and pH on the resolution was investigated. It was found that there is a correlation between pI of the amino acids and the optimal pH.     

Comparison of the actions of glycine and related amino acids on isolated third order neurons from the tiger salamander retina

Whole cell voltage and current clamp recordings were obtained from third order neurons isolated from the salamander retina. Using cross desensitization, the structure-function relationship of short chain amino acids on the glycine receptor were examined. L-Serine, L-alanine, beta-alanine and taurine all cross desensitized with glycine, but did not show significant cross desensitization with GABA. This indicates that these amino acids act at the glycine receptor. The order of potency was glycine > beta-alanine > taurine > L-alanine > L-serine. TAG, a reputed selective taurine antagonist, was equally effective in blocking taurine and glycine currents. There is no evidence for distinct receptors for taurine. Amino acids with larger moieties at the alpha carbon, such as threonine and valine, produced inactive ligands. Placing a methyl group on the amine of glycine or esterification of the carboxyl group also greatly reduced activity. Based on these modifications of the glycine molecule, it appears that selectivity at the glycine receptor results in part from steric restrictions at all three sites in the glycine chain. The steric interference is most critical at the carboxyl and amino ends, and less limiting at the alpha carbon. Doses of L-serine that had only slight effects in voltage clamp experiments, nevertheless produced large effects in current clamp experiments. This indicates that several endogenous amino acids can have significant effects on membrane voltage, even when their shunting activity may be small. High concentrations of agonists produced desensitization in the voltage clamp records, but there was little evidence of desensitization in the current clamp experiments. These results indicate that several endogenous amino acids can activate the glycine receptor, but there is no evidence for a discrete receptor for taurine, beta-alanine, L-alanine or L-serine. Since all these endogenous amino acids have similar amino and acid terminals, reduction in potency results from steric interference around the alpha carbon. This graded potency may have functional significance in mediating inhibition.     

Production of anti-Ro/SS-A and anti-La/SS-B autoantibodies is closely coordinated in systemic lupus erythematosus and independent of anti-dsDNA production

Capillary zone electrophoresis (CZE) was utilized to identify a synaptobrevin-thioredoxin fusion protein (TSB-51). TSB-51 is a substrate for cleavage by botulinum toxin B at the Q(76)-F(77) site. TSB-51 was derivatized with a fluorophore, CBQCA [3-(4-carboxy-benzoyl)-2-quinoline-carboxaldehyde], for 4 h at room temperature. Optimal conditions for CZE separation of the TSB-51-CBQCA complex were determined: buffer (sodium borate), pH (9.0), applied voltage (25 kV), temperature (25 degrees C) and forward polarity. SDS-PAGE showed that TSB-51 had a molecular mass of approximately 19 kDa. The protein was transferred to PVDF membrane and sequenced by the Edman degradation method verifying the first twelve amino acids as SDKIIHLTDDSF. TSB-51 was also collected during CZE separation and subsequently sequenced yielding the first three amino acids as SDK. This CZE-LIF method coupled with the CBQCA derivatization, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilization of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botulinum toxin B on TSB-51.     

Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.     

Interaction of fluorescent probes with membranes of sarcoplasmic reticulum in AMP deamination

Gentamicin and ampicillin were dissolved in an L-amino acid solution especially prepared for newborn infants and infused intravenously over 24 h in 7 babies with serious neonatal surgical problems. Serum concentrations of the antibiotics were maintained rather constant and well above the minimal inhibitory concentration for most bacterial strains. One very sick newborn infant died with overwhelming Klebsiella pneumoniae septicemia. No signs of renal toxicity or ototoxicity were found. The serum amino acids remained within the normal range, except in 1 child with cytomegalovirus infection and liver insufficiency.     

Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (DeltaD13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and DeltaD13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys877 to Arg894.     

Free amino acids and peptides in the muscles of 1- and 2-day-old cadavers

High-voltage electrophoresis and the analytic procedure combination of high-voltage electrophoresis--paper chromatography were used for the analysis of protein-free extracts of muscle tissue in one-- to two--day old corpses. The protein-free extracts from both types of tissue specimen were found to contain also peptides in addition to free amino acids. The amino acids composition of isolated fractions is not mutually comparable. There were also differences as regards peptides. In the two--day corpse a new peptide appeared in the acid part and disappeared in the weak base part. The changes suggest that not only proteolysis but also peptidolysis are in progress on the second post-mortem day.     

Double coupling Edman chemistry for high-sensitivity automated protein sequencing

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10(-21) mol) limits of detection (3 sigma) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.     

Roles of amino acids 161 to 179 in the PSE-4 omega loop in substrate specificity and in resistance to ceftazidime

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Omega loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Omega loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.     

Development of a high-volume in situ mRNA hybridization assay for the quantification of gene expression utilizing scintillating microplates

The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family. In this study, we expressed the barley (Hordeum vulgare L.) GRP94 and the alpha isoform of human HSP90 (HSP90 alpha) in Escherichia coli and compared their dimer-forming abilities. Native polyacrylamide gel electrophoresis revealed that GRP94 (amino acids 69-809) and the full-length form of HSP90 alpha existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-terminal 200 amino acids of HSP90 alpha were sufficient for the dimerization. Limited proteolysis of the C-terminal half of GRP94 with thrombin revealed a 16-kDa fragment, which was derived from the C-terminus of GRP94 through the cleavage of either the Arg710-His711 or the Arg735-Leu736 bond. These cleavage sites were nearly, if not completely, equivalent to the proteolyzed region of HSP90 alpha. Their structural similarity prompted us to investigate, by use of a coexpression system, the possibility that the two proteins form a heterodimeric complex. A two-step affinity chromatography that specifically trapped only the complex revealed that the C-terminal 200 amino acids of HSP90 alpha and the C-terminal 326 amino acids of GRP94 associated with HSP90 alpha and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90 alpha. In conclusion, these results indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.     

Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit

alpha-Scorpion toxins and sea anemone toxins bind to a common extracellular site on the Na+ channel and inhibit fast inactivation. Basic amino acids of the toxins and domains I and IV of the Na+ channel alpha subunit have been previously implicated in toxin binding. To identify acidic residues required for toxin binding, extracellular acidic amino acids in domains I and IV of the type IIa Na+ channel alpha subunit were converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for 125I-alpha-scorpion toxin binding. Conversion of Glu1613 at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable alpha-scorpion toxin binding, but did not affect the level of expression or saxitoxin binding affinity. Conversion of individual residues in the IVS3-S4 extracellular loop to differently charged residues or to Ala identified seven additional residues whose mutation caused significant effects on binding of alpha-scorpion toxin or sea anemone toxin. Moreover, chimeric Na+ channels in which amino acid residues at the extracellular end of segment IVS3 of the alpha subunit of cardiac Na+ channels were substituted into the type IIa channel sequence had reduced affinity for alpha-scorpion toxin characteristic of cardiac Na+ channels. Electrophysiological analysis showed that E1613R has 62- and 82-fold lower affinities for alpha-scorpion and sea anemone toxins, respectively. Dissociation of alpha-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels. alpha-Scorpion toxin binding to wild type and E1613R had similar voltage dependence, which was slightly more positive and steeper than the voltage dependence of steady-state inactivation. These results indicate that nonidentical amino acids of the IVS3-S4 loop participate in alpha-scorpion toxin and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in alpha-scorpion toxin binding affinity between cardiac and neuronal Na+ channels. The results also support the hypothesis that this region of the Na+ channel is important for coupling channel activation to fast inactivation.     

Simple sheath flow reactor for post-column fluorescence derivatization in capillary electrophoresis

A system for post-column fluorescence derivatization in capillary electrophoresis is described. The post-column reactor uses a sheath flow detection cell where the reagents, o-phthaldialdehyde and beta-mercaptoethanol, are added to the sheath buffer and mix by diffusion with the analytes effusing from the separation capillary. Reaction progress is monitored and optimized by imaging a large portion of the sheath flow cuvette using an extended UV source and a CCD camera. Significantly, this design provides the ability to switch between the analysis of pre- and post-column derivatized amino acids and peptides easily and without sacrificing system performance. The lack of turbulent flow in this system minimizes post-separation band broadening. The limit of detection for glycine is 9.4 x 10(-8) M (110 amol) with a separation efficiency of 190,000 theoretical plates, without stacking. The performance of the system for a series of amino acids was evaluated using post-column and pre-capillary derivatization.     

Clinical significance of plasma chlorpromazine levels. I. Plasma levels of the drug, some of its metabolites and prolactin during acute treatment

An unidentified ninhydrin-positive substance found in uremic sera but not found in normal sera was isolated by gel-filtration through Sephadex G-75 followed by high voltage paper electrophoresis (pH 3.5), and identified as beta-aminoisobutyric acid using paper chromatography and automated amino acid analyzer. The quantitative determination of beta-aminoisobutyric acid in serum revealed that the level of beta-aminoisobutyric acid in uremic sera was much higher than that of normal sera. Gas chromatographic determination of the enantiomorphs of beta-aminoisobutyric acid showed that uremic sera contain R- and S-isomers of the amino acid, but with the R-isomer as the dominating form.     

The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded beta-sheet proteins by its sequence and properties

Omp21, a minor outer membrane protein of the soil bacterium Comamonas acidovorans, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene omp21 encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of beta structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic beta strands, which includes virulence proteins, such as the neisserial opacity proteins, Salmonella typhimurium Rck, and Yersinia enterocolitica Ail, as well as the major outer membrane proteins OmpA from Escherichia coli and OprF from Pseudomonas aeruginosa.     

Transfer RNA of a collagen producing tissue, chick-embryo calvaria

Transfer RNA was isolated from calvaria prepared from chick embryos incubated for 15-17 days. The chargeability of the unfractionated tRNA with ten amino acids tested was very similar to that of unfractionated tRNA from adult chicken liver when data were expressed on the basis of pmoles of amino acceptance per A260 unit of tRNA. However, the relative amount of tRNA in calvaria is only about one-fourth the amount in liver. Analysis of individual species of tRNA by two-dimensional electrophoresis on polyacrylamide gels shows that there are fewer isoaccepting species of tRNA in calvaria than in liver.     

The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic chitodextrinase

Chitin catabolism in Vibrio furnissii comprises several signal transducing systems and many proteins. Two of these enzymes are periplasmic and convert chitin oligosaccharides to GlcNAc and (GlcNAc)2. One of these unique enzymes, a chitodextrinase, designated EndoI, is described here. The protein, isolated from a recombinant Escherichia coli clone, exhibited (via SDS-polyacrylamide gel electrophoresis) two enzymatically active, close running bands ( approximately mass of 120 kDa) with identical N-terminal sequences. The chitodextrinase rapidly cleaved chitin oligosaccharides, (GlcNAc)4 to (GlcNAc)2, and (GlcNAc)5,6 to (GlcNAc)2 and (GlcNAc)3. EndoI was substrate inhibited in the millimolar range and was inactive with chitin, glucosamine oligosaccharides, glycoproteins, and glycopeptides containing (GlcNAc)2. The sequence of the cloned gene indicates that it encodes a 112,690-kDa protein (1046 amino acids). Both proteins lacked the predicted N-terminal 31 amino acids, corresponding to a consensus prokaryotic signal peptide. Thus, E. coli recognizes and processes this V. furnissii signal sequence. Although inactive with chitin, the predicted amino acid sequence of EndoI displayed similarities to many chitinases, with 8 amino acids completely conserved in 10 or more of the homologous proteins. There was, however, no "consensus" chitin-binding domain in EndoI.     

Physical properties and chemical compositions of cytoplasmic and mitochondrial malate dehydrogenase from Physarum polycephalum

The malate dehydrogenase isoenzymes from Physarum polycephalum have been purified to homogeneity as confirmed by gel filtration chromatography, polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. Certain physical and chemical parameters of the malate dehydrogenase isoenzymes reported here include sedimentation, molecular weight and subunit molecular weight. Most unique of the differences between the isoenzymes were the widely separate isoelectric points of 9.83 for mitochondrial malate dehydrogenase and 6.14 for the supernatant malate dehydrogenase. The amino acid analyses of each form were done revealing the isoenzymes were unquestionably unique proteins differing in the content of ten amino acids.     

Fluorescence detection of proteins and amino acids in capillary electrophoresis using a post-column sheath flow reactor

A simple laser-induced fluorescence detection method for proteins and amino acids in capillary electrophoresis is reported. A sheath flow cell is utilized as a post-column reactor for fluorescence derivatization of proteins and amino acids by addition of o-phthaldialdehyde-2-mercaptoethanol to the sheath fluid. With the use of a 50 microns I.D. capillary, the limits of detection for carbonic anhydrase are 0.73 nM or 1.8 amol which represents a five- and two-fold improvement, respectively, over the best results previously reported for post-column detection. In addition, separation efficiencies up to 8.07 x 10(5) are achieved and the detector response is linear over three-orders of magnitude. These results demonstrate that mixing is adequate and the reaction kinetics are rapid enough to provide sensitive detection with this approach. Also, because this post-column derivatization scheme requires no instrumental changes to a typical sheath flow cell detector, the system can be used for detection of pre-column labeled analytes and for native fluorescence detection.     

Separations of derivatized amino acid enantiomers by cyclodextrin-modified capillary electrophoresis: mechanistic and molecular modeling studies

The enantiomers of 5-dimethylamino-1-naphthalene sulfonyl (DNS)-derivatives of selected amino acids were successfully separated using capillary electrophoresis (CE) employing cyclodextrins (CD) as enantio-selective running buffer additives. A previously described model for retention and chiral recognition in CD-modified CE is shown to adapt well in this application. Resolution of the isomers is strongly influenced by the type and concentration of cyclodextrin employed, as predicted by the model. Although data indicates differences in the electrophoretic mobilities for some of the completely complexed enantiomer pairs, selectivity generally requires exploiting differences in the amino acid-CD complexation constants for enantiomer pairs. In this work, the D-enantiomers exhibit larger formation constants and are complexed to a greater degree (elute first) at moderate CD concentration. When mixtures of amino acids are analyzed, the effects of separation conditions on general elution behavior must be considered or separated enantiomer pairs will co-elute with other enantiomers. Preliminary results aimed at predicting the strength of DNS-amino acid enantiomer-CD interactions based on molecular modeling studies are presented. A statistical mechanical approach to treating computationally derived enantiomer-CD interaction energies is shown to provide reasonable correlation with separation performance.