Highly specific “sensing” of tryptophan by a luminescent europium(III) complex

The europium(III) complex 1-Cl3 (S,S-2,2′-(((1,10-phenanthroline-2,9-diyl)bis(methanylylidene))bis(azanylylidene))bis(3-methylbutanamide)europiumtrichloride) undergoes, only in the presence of the amino acid tryptophan, a change of emission at 615 nm. In the presence of few equivalents of tryptophan, emission of the europium complex is enhanced while it disappears upon addition of large amounts. This behavior can be assigned to displacement of the sensitizing phenanthroline ligand of 1-Cl2•Trp in the latter case.

Exploring the possibility of early cataract diagnostics based on tryptophan fluorescence

A novel route for early cataract diagnostics is investigated based on the excitation of tryptophan fluorescence (TF) at the red edge of its absorption band at 317 nm. This allows penetration through the cornea and aqueous humour to provide excitation of the ocular lens. The steepness of the red edge gives the potential of depth control of the lens excitation. Such wavelength selection targets the population of tryptophan residues, side chains of which are exposed to the polar aqueous environment. The TF emissions around 350 nm of a series of UV-irradiated as well as control lenses were observed. TF spectra of the UV cases were red-shifted and the intensity decreased with the radiation dose. In contrast, intensity of non-tryptophan emission with maximum at 435 nm exhibited an increase suggesting photochemical conversion of the tryptophan population to 435 nm emitting molecules. We demonstrate that the ratio of intensities at 435 nm to that around 350 nm can be used as a measure of early structural changes caused by UV irradiation in the lens by comparison with images from a conventional slit-lamp, which can only detect defects of optical wavelength size. Such diagnostics at a molecular level could aid research on cataract risk investigation and possible pharmacological research as well as assisting surgical lens replacement decisions.

Mode of Action of the Antimicrobial Peptide Aureocin A53 from Staphylococcus aureus

ABSTRACT We investigated the mode of action of aureocin A53 on living bacterial cells and model membranes. Aureocin A53 acted bactericidally against Staphylococcus simulans 22, with >90% of the cells killed within a few minutes. Cell death was followed by lysis, as indicated by a clearing of the cell suspension and Gram staining. Aureocin A53 rapidly dissipated the membrane potential and simultaneously stopped biosynthesis of DNA, polysaccharides, and protein. Aureocin A53 induced a rapid release of preaccumulated glutamate and Rb+. Experiments on model membranes demonstrated that aureocin A53 provoked significant leakage of carboxyfluorescein (CF) exclusively from acidic liposomes but only at relatively high concentrations (0.5 to 8 mol%). Thus, the bactericidal activity of aureocin A53 derives from membrane permeation via generalized membrane destruction rather than by formation of discrete pores within membranes. Tryptophan emission fluorescence spectroscopy demonstrated interaction of aureocin A53 with both acidic and neutral membranes, as indicated by similar blue shifts. Since there was no significant aureocin A53-induced CF leakage from neutral liposomes, its appears that the peptide does interact with neutral lipids without provoking membrane damage.

Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975

The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 °C, indicating that Asp-312 present in the ‘sucrose box’ was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a β-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius.