The application of NMR and MS methods for detection of adulteration of wine, fruit juices, and olive oil. A review.

This review covers two important techniques, high resolution nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), used to characterize food products and detect possible adulteration of wine, fruit juices, and olive oil, all important products of the Mediterranean Basin. Emphasis is placed on the complementary use of SNIF-NMR (site-specific natural isotopic fractionation nuclear magnetic resonance) and IRMS (isotope-ratio mass spectrometry) in association with chemometric methods for detecting the adulteration.

Determination of authenticity, regional origin, and vintage of Slovenian wines using a combination of IRMS and SNIF-NMR analyses.

The authenticity and geographical origin of wines produced in Slovenia were investigated by a combination of IRMS and SNIF-NMR methods. A total of 102 grape samples of selected wines were carefully collected in three different wine-growing regions of Slovenia in 1996, 1997, and 1998. The stable isotope data were evaluated using principal component analysis (PCA) and linear discriminant analysis (LDA). The isotopic ratios to discriminate between coastal and continental regions are the deuterium/hydrogen isotopic ratio of the methylene site in the ethanol molecule (D/H)(II) and delta(13)C values; including also delta(18)O values in the PCA and LDA made possible separation between the two continental regions Drava and Sava. It was found that delta(18)O values are modified by the meteorological events during grape ripening and harvest. The usefulness of isotopic parameters for detecting adulteration or watering and to assess the geographical origin of wines is improved only when they are used concurrently.

Identification of amino acids in wines by one- and two-dimensional nuclear magnetic resonance spectroscopy.

Amino acids are minor compounds in wines, but they have a profound influence on wine quality, and amino acids composition can be used to differentiate wines according to the vine variety, geographical origin, and year of production. The NMR signals of amino acids in NMR spectra are overlapped by the signals of other compounds present and especially by the signals of dominant compounds such as water, ethanol, and glycerol. In this work we used 1D (1)H and (13)C, 2D homonuclear COSY, TOCSY, and 2D heteronuclear HSQC and HMQC pulse sequences, also with an incorporated WET pulse sequence element that allows the simultaneous suppression of several frequencies. Complete (1)H and (13)C NMR assignments for 17 amino acids commonly present in wine and of gamma-aminobutyric acid at pH 3 have been achieved in wine sample of Sauvignon from the Coastal wine-growing region of Slovenia, vintage 1994.

Peptides neutralizing lipopolysaccharide - structure and function.

Lipopolysaccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research. In the last decade peptidic antagonists have become increasingly important in providing leads for treatment of LPS-mediated diseases. In this review an overview of the sources, functions and structures of antiseptic and antibacterial peptides that interact with LPS is presented.

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: an application to the production of a 15N-enriched fragment of lactoferrin.

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21-31 of human bactericidal protein lactoferrin.

Bacterial expression and refolding of different fragments of human CD14.

We have investigated bacterial expression of several fragments of CD14, a human cellular receptor for lipopolysaccharides (LPS). Despite binding of CD14 to the LPS, a vital constituent of bacterial outer membrane, we have succeeded in producing full length recombinant hCD14 in E. coli. High level of production of CD14 resulted in deposits of aggregated CD14 in bacteria in form of inclusion bodies, which made production of this protein possible. We have also produced N-terminal fragments consisting of 134 and 152 residues, which comprise N-terminal domain with 2 and 3 leucine rich repeats, respectively and a fragment that contains only leucine rich repeats. Production of the N-terminal domain consisting of 69 residues could not be detected, probably due to the degradation of the produced protein within the bacteria. Full length CD14 and a fragment of 152 residues from inclusion bodies were refolded, while the 134 residues fragment and the one with 10 leucine-rich repeats could not be refolded. Those results confirm that the minimal folding unit of CD14 must include N-terminal domain and at least 3 leucine rich repeats.

Solution structure of polymyxins B and E and effect of binding to lipopolysaccharide: an NMR and molecular modeling study.

The cyclic decapeptides polymyxin B (PmB) and E (PmE) (mo-K'TK'-cyclo-[K'K'XLK'K'T]; mo, methyl octanoate; K', diaminobutyric acid; X, D-Phe (PmB) or D-Leu (PmE)) display antimicrobial and lipopolysaccharide (LPS) antagonistic activities. We have investigated the conformational behavior of PmB and PmE in water solution, free and bound to LPS, by homonuclear NMR and molecular modeling methods. The free peptides exist in equilibria of fast exchanging conformations with local preferences for a distorted type II' beta-turn from residues 5-8, and/or a gamma-turn in residue 10. These two motifs are not present in the bound conformation of the peptides. The latter is amphiphilic separating the two hydrophobic residues in the cycle from the positively charged diaminobutyric acid side chains by an envelope-like fold of the cycle. The bound conformation is used for the derivation of a model of the PmB-lipid A complex based on electrostatic interactions and reduction of hydrophobic area. The proposed mode of binding breaks up the supramolecular structure of LPS connected with its toxicity. The model should contribute to the understanding of entropy-driven PmB-lipid A binding at the molecular level and assist the design of inhibitors of endotoxic activity.

Expression and refolding of functional fragments of the human lipopolysaccharide receptor CD14 in Escherichia coli and Pichia pastoris.

CD14 is a high-affinity cellular receptor specific for bacterial lipopolysaccharides (LPS), present in the bacterial cell wall. Binding of LPS to CD14 initiates the innate component of immune response and triggers a response that can lead to septic shock. In order to provide recombinant protein for the study of LPS-CD14 molecular interactions we have expressed human CD14 in Escherichia coli and Pichia pastoris. In bacteria, the protein was produced in high yield in the form of inclusion bodies. We have optimized the procedure for its refolding and generated correctly folded protein. A procedure to monitor the refolding efficiency by using conformation-specific anti-human CD14 monoclonal antibody has been established. A fragment of 152 amino acids of CD14 which retains the ability to bind LPS has been produced in a methylotrophic yeast, P. pastoris, expression system. The recombinant protein from yeast is glycosylated and secreted into the medium. The CD14 fragment was purified to homogeneity by immunoaffinity chromatography. Recombinant CD14 from both bacteria and yeast bind to LPS.

Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases.

Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C18 columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline. Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC50 in the range of 0.3-2.4 microM. IC50 values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 x 10(-7) M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase.

pH-induced conformational transitions of the propeptide of human cathepsin L. A role for a molten globule state in zymogen activation.

Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.

Influence of glycation on cis/trans isomerization and tautomerization in novel morphiceptin-related Amadori compounds.

The influence of N-glycation of the N-terminus on amide bond stereochemistry and tautomeric distribution has been explored via the synthesis and NMR analysis of novel N-(1-deoxy-D-fructos-1-yl) derivatives (Amadori compounds) of the exogenous, milk derived, opioid tetrapeptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH2). NMR analysis of the protected Amadori compounds revealed the presence of four configurational isomers in DMSO solution arising from cis/trans isomerization about Tyr1-Pro2 and Phe3-Pro4 peptide bonds. Comparison of the data obtained for protected Amadori compounds with those obtained for morphiceptin showed that equilibrium fraction of all-trans isomers in N-glycated peptide derivatives was smaller than in the parent peptide compound. Spectroscopic investigation of unprotected morphiceptin-related Amadori compound revealed the presence of multiple conformers in solution due to cis/trans isomerization of the peptide backbone and tautomerization of the sugar moiety. The equilibrium composition in DMSO is markedly shifted towards furanose forms, amounting to two-thirds of the mixture. The estimated equilibrium of the tautomeric forms in water solution revealed the beta-pyranose form as the major tautomer (66%).

Bioactive conformations of small peptides: a method for selection of candidates based on conformations of active and inactive analogues and its application to muramyl dipeptide.

A method has been developed that selects a subset of candidates for the bioactive conformation based on the comparison of conformation clusters of the active and inactive ligands. Those conformations of the active ligand that cannot be reached by inactive ligands in spatially presenting preselected "target" atoms (conformations "unique" to the active ligand) are extracted. The method is applied to muramyl dipeptide (MDP); the selection of candidates was performed using two of its diastereomers that have no immunostimulative properties. A third diastereomer and a rigidified analogue, both inactive, and the protein-bound conformation of a related peptidoglycan containing the MurNAc-L-Ala-D-Glu sequence are used as test cases; the latter was found to be most similar to one member of the set of unique conformations of MDP.

The biologically active conformation of ergot alkaloids.

Molecular mechanics and NMR studies of the D ring conformation of ergot alkaloids demonstrate that both D1 and D2 forms may exist in solution. The comparison of the geometric parameters defining the spatial relations between the aromatic moieties and the basic nitrogen of conformationally restricted dopamine analogs, and that of ergolene, shows the D1 conformation to be the bioactive one.