USE OF OCTADECYLRHODAMINE FLUORESCENCE DEQUENCHING TO STUDY VESICULAR STOMATITIS VIRUS FUSION WITH HUMAN AGED RED BLOOD CELLS*

Human erythrocytes were separated into five fractions representing different age groups. In each group phospholipid inside-outside translocation was determined by quantitation of the amino phospholipids phosphatidylserine and phosphatidylethanolamine and their lyso-derivatives by thin layer chromatography. To assess the role of transbilayer phospholipid distribution in the recognition and fusion of vesicular stomatitis virus (VSV) and human aged erythrocytes, we monitored the fusion kinetics using the octadecylrhodamine dequenching assay. Fusion of VSV with each single group of red blood cells (RBC) was not detectable with the youngest cells (Fl group) but increased with RBC aging (F2-F5 groups). The same increase in fusion was observed with microvesicles generated from RBC in which aging was mimicked by incubating the cells with Ca²⁺ in the presence of the Ca²⁺ ionophore A23187. Conversion of the aminophospholipids to the trinitrophenyl derivative by reaction with trinitrobenzensulfonate completely inhibits fusion on ghosts in which aging was artificially induced by translocation of aminophospholipids in the outer leaflet (symmetric ghosts). These results indicate that RBC become susceptible to VSV fusion during aging and in all pathology related to the aging process.     

Atmospheric pressure photoionization mass spectrometry of oligodeoxyribonucleotides

Small oligonucleotides (di- and trimers) were investigated by atmospheric pressure photoionization (APPI) with focus on the fragmentation mechanisms. The fragmentation patterns of these biomolecular ions have been monitored under dopant-assisted photoionization (DA-APPI) conditions. Our results reveal new aspects of the gas- phase chemistry of ions formed from such biomolecules. They illustrate that the reaction between low-energy electrons released from photoionization processes and di- and trinucleotides lies in dissociative electron attachment processes leading to phosphodiester bond cleavages and to the formation of numerous fragments in the ion source. The conditions of DA-APPI, which involve protic solvents and atmospheric pressure conditions, seem to be relevant for the study of radiation damages to biological molecules.     

Online coupling of high-resolution chromatography with extreme UV photon activation tandem mass spectrometry: Application to the structural investigation of complex glycans by dissociative photoionization

The activation of ions by extreme-energy photons (XUV) produced by a synchrotron radiation beamline is a powerful method for characterizing complex glycans using tandem mass spectrometry (MS). As previously described, this activation method leads to rich fragmentation spectra with many structurally valuable cross-ring cleavages while maintaining labile modifications on the glycan structures. However, until now, the tandem MS event was too long to be compatible with liquid chromatography elution times. In this work, the duty cycle of the activation and detection of fragments was shortened, and the background signal on the spectra was drastically reduced. Both improvements allowed, for the first time, the successful coupling of a UHPLC system to XUV-activated tandem MS. The approach was used to characterize a complex mixture of oligo-porphyrans, which are a class of highly sulfated oligosaccharides, in a fully automated way. Due to an enhanced dynamic range and an increased sensitivity, some hypothetical structures of low abundance have been unequivocally confirmed in this study and others have been revised. Some previously undescribed species of oligo-porphyrans that exhibit lateral branching have been fully resolved. This work contributes to the scarce knowledge of the structure of porphyrans in red algae and pushes the current capacities of XUV-activation tandem MS by demonstrating the possibility of a direct coupling with UHPLC. This study will considerably broaden the applicability and practicality of this method in many fields of analytical biology.     

Structural changes induced in proteins by therapeutic ultrasounds

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV–VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and α-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (α-helix or β-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.