Alzheimer's disease (AD) is the most common type of dementia characterized by intracellular appearance of neurofibrillary tangles, synaptic and neuronal loss; and extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques. The initial causes leading to AD are unknown, and the available treatments are only effective at slowing the degeneration process. The accumulation of arginine in the brain of Alzheimer patients indicates a possible disruption of enzymes responsible for its metabolism. One such enzyme is neuronal nitric oxide synthase (nNOS) and controlling its activity by interacting with nanoparticles may lead to a delay in the onset of the disease. Neuronal nitric oxide synthase was purified using DEAE-Sephacel ion exchange resulting in 10 % yield, 0.43 fold recovery and specific activity 0.09 U/mg. The enzyme was found to be a dimer with a molecular mass of 150 kDa. Characterisation of the nNOS showed an optimum temperature and pH of 50°C and 7.5 respectively, and it was relatively stable at the optimum conditions (t½ = 100 min). The purity was analysed by SDS-PAGE followed by Western blot. Purified nNOS was challenged with 3-7 nm silver and 4-15 nm gold nanoparticles of between synthesized chemical using AgNO3 and either sodium borohydride or sodium citrate. Results showed that gold nanoparticles are more effective at low concentration (5 μM) than silver nanoparticles due to their size difference. Incubation of different concentration of nanoparticles (5, 15, 25, 50 μM) with the purified nNOS showed an initial decrease of 5% in enzyme activity which over time was restored to 80%. This suggests that different nanoparticles are produced in different sizes and interaction over a given time may result in enzyme association–dissociation mechanism. Inhibition studies showed a strong binding of both nanoparticles with Ki values of 1.4 μM and 0.2 μM for silver and gold, respectively. Both nanoparticles inhibited the activity of nNOS extensively as they bound strongly to the inhibition site on the enzyme and were more in contact with fluorophores nanoparticles. This was confirmed by fluorimetry with binding constants of 0.0084 μM and 0.01092 μM for silver and gold, respectively. Results of this study suggest that silver and gold nanoparticles competitively inhibit nNOS.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:rhodes/vital:3875 |
| Date | January 2013 |
| Creators | Ngqwala, Nosiphiwe Patience |
| Publisher | Rhodes University, Faculty of Science, Biochemistry, Microbiology and Biotechnology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | 134 leaves, pdf |
| Rights | Ngqwala, Nosiphiwe Patience |
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