Capillary zone electrophoresis separation of tryptophan and its metabolites, including quinolinic acid.

Capillary zone electrophoresis with UV absorbance detection was used to separate tryptophan and ten of its metabolites. Run buffers of pH 4.0-10.0 were evaluated for their effect on resolution; a pH 9.6 buffer was found to give optimum separation of all components. Ethylenediaminetetraacetic acid (EDTA), which prevents complexation of some analytes with polyvalent cations, was included in the run buffer to insure good peak shape and reproducible mobilities. The resulting method was used to detect the presence of quinolinic acid in a urine sample.

Capillary electrophoresis with pulsed amperometric detection of carbohydrates and glycopeptides.

The utility of capillary electrophoresis with pulsed amperometric detection (CE-PAD) for the analysis of carbohydrate-containing samples from a variety of biological sources is described. CE-PAD was used to separate a mixture of oligosaccharides obtained from bovine fetuin and to monitor the desialylation process used in the characterization of the oligosaccharides. Additionally, the high resolving power of the system was demonstrated using a series of glycopeptides obtained from recombinant coagulation factor VIIa, which possess the same decapeptide core but differ in the extent of sialylation. Deglycosylation of these glycopeptides for characterization purposes resulted in a mixture of carbohydrates and peptides. Unlike CE with UV detection, this system gave good responses for all analytes, demonstrating the unique ability of PAD to respond to the electrochemical features of diverse classes of biomolecules such as carbohydrates and peptides. Finally, CE-PAD was applied to the analysis of a tryptic digest of recombinant tissue plasminogen activator. The use of different detection potentials in sequential runs on a sample gave structural information about the peptides, such as glycosylation. A brief review of prior applications of CE-PAD to the analysis of standard mixtures of simple saccharides is also presented.

Computer-assisted assignment of peptides with non-standard amino acids.

A comprehensive peptide assignment program and its application to a cyclic peptide, cyclosporin A, are presented in this paper. A group of graph theoretical algorithms using fuzzy logic are discussed with the aid of examples from cyclosporin A. The algorithms deal with heavily overlapped peaks, recover disjointed and distorted spin coupling networks, and include strategies for sequence-specific assignment. A procedure to extend the Protein Knowledge Base for automatically assigning non-standard amino acid residues is also presented. The program is capable of completely automated assignment for small peptides (approximately 20 residues). For such molecules, it is insensitive to whether the peptide chain is cyclic or acyclic, and to whether amide protons are present or absent. For larger peptides/proteins, more user interaction is required and the sequence-specific assignment step usually must proceed through fragments smaller than the full length to avoid problems due to occurrence of a combinatorial explosion. The program can be applied as a rigorous tool to check manual assignments. The fuzzy graph theoretical concepts built in the program are illustrated with 2D proton spectra of a peptide, but may be extended to higher-dimensional spectra, other biopolymers, natural products and other organic structures.

Characterization of glycopeptides from recombinant coagulation factor VIIa by high-performance liquid chromatography and capillary zone electrophoresis using ultraviolet and pulsed electrochemical detection.

Four glycopeptide (GP) fractions containing glycosylated Asn 322 were isolated from a tryptic digest of recombinant coagulation factor VII by reversed-phase-HPLC (RP-HPLC). Characterization of the GPs by enzymatic desialylation and RP-HPLC as well as by enzymatic deglycosylation, RP-HPLC, and high-pH anion-exchange chromatography indicated that the four GPs consisted of the same decapeptide but with 0, 1, 2, or 4 residues of sialic acid. In comparison to HPLC, capillary zone electrophoresis (CZE) using uv and pulsed electrochemical detection (PED) afforded improved separation of GPs from each other and from contaminants. CZE-uv and CZE-PED of the desialylated GPs and deglycosylated GPs corroborated the results obtained with the chromatographic methods.

Determination of the number and distribution of oligosaccharide linkage positions in O-linked glycoproteins by capillary electrophoresis.

O-Linked oligosaccharide chains are found in a wide variety of glycoproteins. Determination of the number of O-linked serine and threonine residues is an important step in glycoprotein characterization. Alkaline release of the oligosaccharide chains from the peptide chain followed by bisulfite addition to the unsaturated residues generates sulfonates. Hydrolysis of treated samples followed by derivatization with naphthalene 2,3-dicarboxyaldehyde and cyanide results in analytes which are separated and quantitated by capillary electrophoresis with UV detection. The utility of the method was demonstrated using bovine submaxillary mucin yielding results that agreed with previously published values.

Separation and quantitation of the amino acid neurotransmitters in rat brain by capillary electrophoresis.

Capillary electrophoresis (CE) has been used to separate the CBI derivatives of the neurotransmitters gamma-aminobutyric acid, glycine, glutamate, aspartate, norepinephrine and dopamine from 18 other amino acids present in the rat brain. The procedure, which requires an injection volume of < 10 nl, gave detection limits of 24-40 fmol using UV detection at 420 nm. Efficiencies for the derivatized amino acids varied from 344,000 to 444,000 theoretical plates. The method was applied successfully to the quantitation of the amino acid neurotransmitters and several other amino acids in a rat brain homogenate.

Monitoring excitatory amino acid release in vivo by microdialysis with capillary electrophoresis-electrochemistry.

Capillary electrophoresis (CE) with electrochemical detection (ED) was used to determine extracellular levels of aspartate, glutamate and alanine in samples from the frontoparietal cortex of the rat which were obtained by microdialysis. The method was used to monitor the effect on the overflow of the excitatory amino acids aspartate and glutamate of an influx of high concentrations of potassium ion. Samples were derivatized with naphthalenedialdehyde-cyanide prior to analysis. Detection limits for aspartate and glutamate were 80 and 100 nM, respectively. CE-ED is extremely useful for the analysis of microdialysis samples because of the very small sample volumes required by this analytical technique. The use of ED provides the requisite sensitivity and allows verification of peak purity by voltammetry.

Ubiquitin function studied by disulfide engineering.

Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active. The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin. The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis.

Determining stereo-specific 1H nuclear magnetic resonance assignments from distance geometry calculations.

Stereo-specific 1H nuclear magnetic resonance assignments can be obtained following distance geometry structure calculations. The key to this method is to allow stereo-related atoms or methyls to float between pro-R and pro-S configurations, the final configuration being determined by the experimental constraints. Resonances from stereo-related pairs are given initial random assignments (either pro-R or pro-S) for identifying nuclear Overhauser effects (NOEs). A list of distance constraints using these assignments is compiled and a series of structures calculated where the chirality of non-C alpha chiral centers is not constrained; no pseudoatom corrections are required. Calculated structures are both locally and globally well-determined since the assignments rely upon the structure determination rather than the structure quality relying upon stereo-specific assignments. The method represents a global approach to determining stereo-specific assignments versus previously reported methods where only intraresidue NOEs and J-coupling information are used.

Sequential 1H NMR assignments and secondary structure identification of human ubiquitin.

1H NMR assignments of human ubiquitin (76 amino acids, Mr 8565) have been made by a combination of DQF-COSY, DQF-RELAY, NOESY, DQ, and isotropic mixing experiments. Complete NH, C alpha H, and C beta H assignments were obtained; resonances not yet assigned are the side-chain amides of Q-40, Q-41, Q-49, N-60, and Q-62 and the peripheral protons (C gamma H and outward) of M-1 and K-27. A total of 558 out of 579 (96%) potentially observable protons were assigned. Particular attention was directed toward obtaining complete assignments of the aliphatic residues (seven Ile, nine Leu, four Val) since these residues form an extensive hydrophobic core and NOEs from these residues are invaluable for structure calculations. The secondary structure elements were also identified from the sequential NOE data and differ slightly in description from the published 2.8 A resolution crystal structure [Vijay-Kumar, S., Bugg, C. E., Wilkinson, K. D., & Cook, W. J. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3582-3585]; the NMR data suggest that residues 48-50 form a short fifth strand in the beta-sheet and that residues 56-61 form a helical turn. The sequential assignment results presented here are in agreement with the main chain directed assignments presented in the preceding paper [Di Stephano, D., & Wand, A. J. (1987) Biochemistry (preceding paper in this issue)].

Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin.

To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.

1H NMR studies of lambda cro repressor. 1. Selective optimization of two-dimensional relayed coherence transfer spectroscopy.

Two-dimensional relayed coherence transfer NMR spectroscopy (RELAY) has been used to corroborate side chain spin system identities in crowded regions of the 1H NMR spectrum of the lambda cro repressor protein. The mixing time in the RELAY experiments was optimized for specific preselected spin systems by using recently developed methods [Bax, A., & Drobny, G. (1985) J. Magn. Reson, 61, 306-320], which utilize the transverse relaxation time (T2) of the molecule and relevant J couplings for the defined spin system. We demonstrate that a mixing time of 26 ms gives rise to strong C alpha H-C gamma H3 RELAY cross peaks for all valine, threonine, and isoleucine residues, while RELAY cross peaks for other spin systems are weak or are not observed. This allows for rapid and unambiguous identification of the side chain resonances for valine, isoleucine, threonine, and alanine (by elimination). The use of optimized RELAY for analyzing and identifying spin systems in complex spectra is discussed.

1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum.

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.

Sensorimotor therapy: its effect on electroencephalograms of acute comatose patients.

This study investigated the effect of sensorimotor therapy on cortical activity of acute comatose patients. Frequency changes in delta, theta, and alpha bandwidths taken before and after sensorimotor therapy of three comatose patients were quantified by Compressed Spectral Array and compared to similarly measured changes before and after nontreatment sessions. The two therapy sessions per day consisted of 30 minutes of selected sensory stimulation with requested or elicited motor responses. Two nontreatment sessions daily consisted of 30 minutes of various types of stimulation or rest. Each patient served as an independent control. Results of statistical analyses indicated a differential response to treatment by cerebral hemisphere and by bandwidth in all three subjects, with significant change seen in the more damaged hemisphere and in frequency coinciding with a more alert neurologic state. Results support the hypothesis that sensorimotor therapy has a significantly greater effect on cortical activity of a comatose patient than does passive stimulation.

Structural and immunochemical characterization of the acidic arabinomannan of Mycobacterium smegmatis.

A serologically active, acidic arabinomannan has been isolated from Mycobacterium smegmatis. The polysaccharide contains approximately 56 arabinosyl and 11 mannosyl residues, and 2 phosphate, 6 monoesterified succinate, and 4 ether-linked lactate groups. After saponification to remove succinyl groups, the polysaccharide can be separated into phosphorylated (55%) and nonphosphorylated (45%) forms, the former containing a little more arabinose and a little less mannose than the latter. The structures of these polysaccharides were investigated by 1H- and 13C-n.m.r. spectroscopy and methylation analysis, before and after selective cleavage of furanosyl linkages. The phosphorylated and nonphosphorylated forms of the polysaccharide were found to have similar, if not identical, structures. The main structural feature of the polysaccharides is the presence of chains of contiguous arabinofuranosyl residues linked alpha-(1 leads to 5). These chains are attached at 0-4 of arabinopyranosyl residues that are present in a core region of the polysaccharide that also contains mannopyranosyl residues. Immunochemical studies demonstrated that the polysaccharide is an effective, precipitating antigen with antisera from rabbits immunized with cell walls or heat-killed cells of M. smegmatis. The polysaccharide is, however, more effective as a precipitating antigen after removal of the succinate groups, and completely ineffective after removal of arabinofuranosyl residues. The polysaccharide therefore contains an important antigen in common with the arabinogalactan lipopolysaccharide of the cell wall of the bacterium, i.e., chains of contiguous alpha-(1 leads to 5)-linked arabinofuranosyl residues.

Amino acid requirements of Aeromonas.

Four of the 12 cultures of Aeromonas hydrophila, 5 of the 10 A. shigelloides, and 9 of the 10 A. salmonicida that were studied required arginine and lysine, among other amino acids, for their growth.