Analytical Methods for the Study of Opioid Peptides

Abstract

Dynorphin A 1-17 [Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln] is an endogenous opioid peptide. It is widely distributed in blood and CNS tissue and exhibits a high affinity to the kappa (κ) opioid receptors and binds with less affinity to both delta (δ) and mu (µ) opioid receptors. This peptide has been found to show both antinociceptive and analgesic effects within the central nervous system. Dyn A is also involved in the body’s immune response, in addition to control of heart rate, blood pressure, body temperature and feeding behavior. However, upregulation of Dyn A due to disease states has been shown to cause nonopioid activity such as hyperalgesia, allodynia, and excitotoxicity. In addition, altered levels of the neuropeptide have been linked to neurological disorders, including Alzheimer’s and Parkinson’s disease. Capillary electrophoresis is a powerful technique that can achieve high-efficiency separations of charged analytes. However, CE has limited use for the analysis of basic proteins and peptides, due to their adsorption onto the inner surface of the fused silica at pHs below their pI. This adsorption can lead to a loss of efficiency, irreproducibility of migration times, and peak tailing. This thesis describes the separation of dynorphin A 1-17 and its key metabolites using fused silica capillaries coated with positively charged polydiallyldimethylammonium chloride (pDDA) and pDDA stabilized gold nanoparticles modified capillaries. The coating material minimized unwanted adsorption of the positively charged peptides onto the surface of the fused-silica capillary. The studies with capillary electrophoresis were performed using UV detection at 214 nm. However, the detection limits are too high to determine endogenous concentrations of these bioactive peptides in brain microdialysis samples. To improve the detection limits, while still maintaining small sample volume requirements, the development of glass and glass/polydimethylsiloxane microchip electrophoresis (ME) system with fluorescence detection is described. Several fluorescence tags were evaluated including the fluorescent probe, fluorescein isothiocyanate, and the fluorogenic probe, naphthalene-2,3-dicarboxaldehyde with sodium cyanide, which produces a fluorescent 1-cyanobenz[f]isoindole (CBI) derivative. The ME-LIF system was shown to separate CBI-peptides of derivatized fragments of dynorphin A.