Purification and characterization of A[beta] and probing the interactions between myricetin and A[beta] using various spectroscopic methods
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Worldwide, millions of people suffer from symptoms of memory loss and confusion associated with Alzheimer's disease (AD). Mounting evidence suggests a close relationship between AD and aggregation of a naturally occurring protein fragment, the amyloid [beta]-peptide (A[beta]). Soluble oligomers of A[beta] are now considered to be the primary neurotoxic agent. Naturally occurring flavonoid compounds have been reported to inhibit/reverse A[beta] aggregation and reduce the A[beta]-induced neurotoxicity both in vitro and in vivo. Previous spectroscopic studies suggest multiple forms of interaction between A[beta] and myricetin, a flavonoid compound. In this study, two A[beta] fragments, A[beta](1-16) and A[beta](25-40), together with A[beta](1-42) were investigated. HPLC purification was conducted on crude A[beta](1-16) and A[beta](25-40) using a Beckman System Gold HPLC system, a Vydac C18 column and a combination of acetonitrile and water as mobile phase. The purified A[beta](1-16) and A[beta](25-40) has a purity of 91% and 90%, respectively. CD, UVRR spectroscopy and DLS have been employed to study the different aggregation states of A[beta](1-42) obtained by using 10 kDa and 30 kDa molecular weight cut-off filters or unfiltered (referred to LMW, IMW and HMW A[beta](1-42)). The studies have shown that LMW A[beta](1-42) is fully unstructured, containing oligomers up to tetramers; IMW A[beta](1-42) is likely a combination of disordered and some [beta]-sheet structure, containing large aggregates ranging from 30 kDa to 60 kDa; whereas HMW has high content of large aggregates. The solution conformation of A[beta] and A[beta] fragments were characterized using CD and UVRR spectroscopic methods. Both A[beta](1-42) and A[beta](25-40) are fully unordered, whereas A[beta](25-40) is likely has higher content of [beta]-strand structure. A[beta](1-16) is predominantly PPII structure. Various spectroscopic methods were employed to investigate the binding interactions between disordered A[beta] and myricetin, one of the flavonoid compounds. A[beta]-myricetin complex formation was visualized by myricetin's intrinsic fluorescence. ThT fluorescence assay and CD study showed that myricetin can inhibit A[beta](25-40) and A[beta](1-42) from forming [beta]-sheet structured fibril. CD and UVRR studies further evidenced conformational changes from both A[beta](25-40) and A[beta](1-42) induced by myricetin. In parallel, UV resonance Raman spectra indicate that the environment around one or more of the aromatic residues is altered when myricetin is present. These findings suggest that both hydrophobic interaction and hydrophilic interaction play a role in the binding of myricetin with A[beta].
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