Structural and activity characterization of irradiated antimicrobial peptide (WLBU2) for use in blood processing and biomedical applications Public Deposited

http://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/bn999870q

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  • Sepsis is a life-threatening blood infection which leads to an uncontrolled systemic inflammatory state. In North America alone, sepsis annually afflicts approximately 750,000 individuals and kills 38-50% of these (more than HIV/AIDS, breast cancer, and colon cancer combined). Current treatment involves administration of IV fluids, antibiotics and hemoperfusion, requiring prolonged ICU/hospital stays. High-throughput microfluidic devices have been proposed to remove the circulating endotoxin and pathogens from blood. A coating based on polyethylene oxide (PEO)-based triblock copolymers will prevent protein adsorption and cell adhesion. Engineered cationic amphiphilic peptides (WLBU2) tethered on the PEO chains are expected to bind circulating endotoxin and inactivate Gram-positive and Gram-negative bacterial cells. These PEO-triblock tethers will be most economically immobilized on device surfaces using γ-irradiation, and immobilization of pre-formed WLBU2-triblock constructs would greatly simplify the coating process. However, the effect of irradiation on the structure and antimicrobial activity of WLBU2 was previously unknown. In this work, we used proton nuclear magnetic resonance (¹H-NMR), ultraviolet (UV) spectroscopy, fluorescence spectroscopy, and circular dichroism (CD), and mass spectrometry to characterize chemical and structural changes caused by γ-irradiation. The bioactivity of γ-irradiated WLBU2 was also tested against Escherichia coli and Pediococcus pentosaceus in a radial diffusion bacterial inhibition assay. Irradiation of WLBU2 appears to cause oxidative ring-opening of the tryptophan side-chains and a decrease in α-helicity. However, the irradiated peptide was no less effective at inhibiting the growth of E. coli and P. pentosaceous than native WLBU2. These results suggest that using γ-irradiation to immobilize pre-formed WLBU2-triblock constructs will not destroy the antimicrobial activity of the complex and thus offers an economically viable and highly efficient means to impart biocompatibility and bioactivity in microfluidic devices.
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