A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm.

A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gram-negative bacteria. It is based on the conversion of 3-oxo AHLs to their pentafluorobenzyloxime derivatives followed by gas chromatography-mass spectrometry (electron capture-negative ion). The method used [13C16]-N-3-oxo-dodecanoyl homoserine lactone ([13C16]-OdDHL) as the internal standard, and its validity was tested by spiking the supernatant and cell fractions with three levels of 3-oxo AHLs, i.e. 1, 10 and 100 ng per sample. These showed the method to be both sensitive (S/N ratio >10:1 for 1 ng) and accurate. The assay was applied to the biofilm and effluent of a green fluorescent protein (GFP)-expressing strain of Pseudomonas aeruginosa (6294) culture grown in flow cells. Biofilm volume was determined for three replicate flow cells by confocal scanning laser microscopy. OdDHL was detected in the biofilm at 632 +/- 381 microM and the effluent at 14 +/- 3 nM. The biofilm concentration is the highest level so far reported for an AHL in a wild-type bacterial system. The next most abundant 3-oxo AHL in the biofilm and effluent was N-3-oxo-tetradecanoyl homoserine lactone (OtDHL) at 40 +/- 15 microM and 1.5 +/- 0.7 nM respectively. OtDHL is unreported for P. aeruginosa and has an activity equivalent to OdDHL in a lasR bioassay. Two other 3-oxo AHLs were detected at lower concentrations: N3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 microM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).

A covalent thymine-tyrosine adduct involved in DNA-protein crosslinks: synthesis, characterization, and quantification.

A thymine-tyrosine adduct, (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine), was synthesized using a simple, single-step condensation between 5-(hydroxymethyl)uracil and L-tyrosine. This approach provides access to useful quantities (mg-g) of analytically pure reference material, and with minor modification, to stable isotope-labeled analogues (isotopomers). With reference material and a suitable internal standard available, isotope-dilution liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS/MS) was used to assay the adduct in a model system purged of oxygen, i.e., a gamma-irradiated N2O-saturated aqueous solution of thymine and tyrosine. The convenient synthetic route to standards and the method for quantification reported here will prove useful in assessing the significance of the adduct in biological systems. These studies also highlight the potential for artefactual adduct formation if the appropriate substrates are present under acidic conditions.

Chlorination of tyrosyl residues in peptides by myeloperoxidase and human neutrophils.

Hypochlorous acid is the major strong oxidant generated by human neutrophils, and it has the potential to cause much of the tissue damage that these inflammatory cells promote. It is produced from hydrogen peroxide and chloride by the heme enzyme myeloperoxidase. To unequivocally establish that hypochlorous acid contributes to inflammation, a stable and unique marker for its reaction with biomolecules needs to be identified. In this investigation we have found that reagent hypochlorous acid reacts with tyrosyl residues in small peptides and converts them to chlorotyrosine. Purified myeloperoxidase in combination with hydrogen peroxide and chloride, as well as stimulated human neutrophils, chlorinated tyrosine in the peptide Gly-Gly-Tyr-Arg. Rather than reacting directly with the aromatic ring of tyrosine, hypochlorous acid initially reacted with an amine group of the peptide to form a chloramine. The chloramine then underwent an intramolecular reaction with the tyrosyl residue to convert it to chlorotyrosine. This indicates that tyrosyl residues in proteins that are close to amine groups will be susceptible to chlorination. Peroxidases are the only enzymes capable of chlorinating an aromatic ring. Furthermore, myeloperoxidase is the only human enzyme that produces hypochlorous acid under physiological conditions. Therefore, chlorotyrosine will be a specific marker for the production of hypochlorous acid in vivo and for the involvement of myeloperoxidase in inflammatory tissue damage.

Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins.

Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.