Identification of a biological excimer involving protein-protein interactions: A case study of the α-synuclein aggregation.

Excimer formation based on pyrene derivatives stacking has been used to probe conformational changes associated with a variety of protein interactions. Herein, in search for the nature of the protein interactions involved in amyloid proteins aggregation we studied the spectroscopic features of the Nα-acetyl-l-tyrosinamide (NAYA) parent compound and of a well-known aggregate amyloid protein, the α-synuclein (Syn). The aggregation of this amyloid disordered protein has been implicated in the development of Parkinson's disease, which is an increasingly prevalent and currently incurable neurodegenerative disorder. Also, Syn aggregation has been widely investigated but, information concerning the conformational alterations in the diverse protein aggregated species at the molecular level, is still scarce. Three different molecular configurations of the NAYA parent compound were at least found to exist in its solutions containing 1,4-dioxane. Two of these NAYA molecular configurations were found to produce a more efficient excimer fluorescence. For Syn solutions containing 1,4-dioxane, one molecular configuration involving the intermolecular interaction between the protein tyrosyl group and the protein peptide bond was found to exhibit excimer fluorescence. This study is the first one reporting the formation of a biological excimer exhibiting fluorescence. Although very weak, this can be used as a signature of protein-protein interactions and, ultimately, enabling to access the complex interactions network existing in the amyloid aggregated species.

Mass spectrometric studies of the reaction of a blocked arginine with diketonic α-dicarbonyls.

The modification of arginine residues by diketonic α-dicarbonyls, in structural proteins and enzymes studies, is a process known for decades. The chemistry of these reaction processes is, however, not fully understood. Moreover, modification of arginine residues by reaction with α-dicarbonyls in glycation has also not been completely elucidated. Aspects related to the early stages of the condensation of arginine with one dicarbonyl molecule, leading to the formation of dihydroxyimidazolidines and their dehydrated forms, in particular, are here approached in more detail. Taking into consideration the usually rapid kinetics involved in the formation of the early reaction product species, we decided to use fast, sensitive and selective analytical techniques, such as electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS(n)) to monitor the reactions of a blocked arginine (acetyl-arginine) with several selected diketonic α-dicarbonyls, to identify and characterize the mentioned transient species and to probe the reaction mechanism involved. Compounds grouped into two different classes according to their structural similarity were identified, namely acetyl-dihydroxyimidazolidines and acetyl-bis(dihydroxyimidazolidines), together with their dehydrated species. The former compounds are known to exist in solution. The reactivity of acetyl-bis(dihydroxyimidazolidines) seems to be different from that of acetyl-dihydroxyimidazolidines. To note that dehydration appears to be reinforced in acetyl-bis(dihydroxyimidazolidines) chemistry with respect to acetyl-dihydroxyimidazolidine chemistry, while both structurally related compounds involve mostly dihemiaminals reactivity. Two different ion structures are proposed for single dehydrated acetyl-bis(dihydroxyimidazolidines), concerning the two more symmetrical and two more asymmetrical dicarbonyls reacted. In acetyl-bis(dihydroxyimidazolidines) formation, we concluded that the importance of single dehydration relies on the rapid minimization of sterics and energetics of the reaction moieties formed. These reactions occur also in a selective way, regarding the two compound structures proposed for single dehydrated acetyl-bis(dihydroxyimidazolidines). Further considerations are also established for the formation of single dehydrated acetyl-bis(dihydroxyimidazolidines). An explanation for the reversible nature of the reaction of arginine with diketonic dicarbonyls is also provided. This study reinforces the potential of the fast, sensitive and selective electrospray ionization mass spectrometry techniques for the investigation of transient species and their mechanistics, that might otherwise not be feasible by means of the most commonly used spectroscopic techniques.

Plant extracts with anti-inflammatory properties--a new approach for characterization of their bioactive compounds and establishment of structure-antioxidant activity relationships.

Geranium robertianum L. (Geraniacea) and Uncaria tomentosa (Willd.) DC. (Rubiaceae) plant extracts, frequently used in traditional medicine for treatment of inflammatory and cancer diseases, were studied to identify potential bioactive compounds that may justify their therapeutic use and their underlying mechanisms of action. Since some of the pharmacological properties of these plant extracts may be linked to their antioxidant potential, the antioxidant activity, in relation to free radical scavenging, was measured by the ABTS/HRP and DPPH() assays, presenting U. tomentosa the higher activity. The antioxidant activity was also evaluated by scavenging of HOCl, the major strong oxidant produced by neutrophils and a potent pro-inflammatory agent. U. tomentosa was found to be a better protector against HOCl, which may justify its effectiveness against inflammatory diseases. SPE/LC-DAD was used for separation/purification purposes and ESI-MS/MS for identification/characterization of the major non-volatile components, mainly flavonoids and phenolic acids. The ESI-MS/MS methodology proposed can be used as a model procedure for identification/characterization of unknowns without the prerequisite for standard compounds analysis. The ESI-MS/MS data obtained were consistent with the antioxidant activity results and structure-activity relationships for the compounds identified were discussed.