A novel SPR based method for measuring diffusion coefficients: From small molecules to supramolecular aggregates

Investigating peptides and proteins conformations in vitro is of paramount importance in biochemistry and cell biology, as the understanding of many physiological pathways and pathological processes, which underscore the onset and progression of “conformational diseases”, is closely dependent on the actual possibility of monitoring the conformation, oligomerization and the specific properties, such as metal-binding features, that proteins adopt in complex systems.In this work, we report a newly designed Surface Plasmon Resonance (SPR) based dispersion analysis, hereby named D-SPR, which allows to measure the diffusion coefficients of molecules with unprecedented easiness and precision. Several small molecules have been tested with this new approach, and the diffusion coefficients obtained are in accordance with the values reported in the literature. A theoretical background is given and the newly designed method has been also applied to carnosine, a dipeptide which has recently attracted great attention by virtues of its anti-oxidative, anti-aggregating and enzyme activating properties. Results clearly show the high performance of the new method, which is based on the newly unveiled SPR capability to measure diffusion coefficients, to give information on carnosine metal-induced oligomerization, expanding the potentiality of commonly used SPR instruments well over the canonic investigation of biomolecular interactions

IDE Degrades Nociceptin/Orphanin FQ through an Insulin Regulated Mechanism

Insulin-degrading enzyme (IDE) was applied to catalyze hydrolysis of Nociceptin/Orphanin 1-16 (OFQ/N) to show the involvement of the enzyme in degradation of neuropeptides engaged in pain transmission. Moreover, IDE degradative action towards insulin (Ins) was inhibited by the OFQ/N fragments, suggesting a possible regulatory mechanism in the central nervous system. It has been found that OFQ/N and Ins affect each other degradation by IDE, although in a different manner. Indeed, while the digestion of OFQ/N is significantly affected by the presence of Ins, the kinetic profile of the Ins hydrolysis is not affected by the presence of OFQ/N. However, the main hydrolytic fragments of OFQ/N produced by IDE exert inhibitory activity towards the IDE-mediated Ins degradation. Here, we present the results indicating that, besides Ins, IDE cleaves neuropeptides and their released fragments act as inhibitors of IDE activity toward Ins. Having in mind that IDE is present in the brain, which also contains Ins receptors, it cannot be excluded that this enzyme indirectly participates in neural communication of pain signals and that neuropeptides involved in pain transmission may contribute to the regulation of IDE activity. Finally, preliminary results on the metabolism of OFQ/N, carried out in the rat spinal cord homogenate in the presence of various inhibitors specific for different classes of proteases, show that OFQ/N proteolysis in rat spinal cord could be due, besides IDE, also to a cysteine protease not yet identified