Probing the effects of oxidative stress on biomimetic membrane interactions

This thesis studies the effects of oxidative stress on biomimetic membrane systems including their interaction with proteins relevant to oxidative stress. Two oxidation methods were used, in situ oxidation initiated using Fe(II) and ascorbate, and the incorporation of a synthetic “oxidised” phospholipid, PoxnoPC, into biomimetic membranes. The biomimetic systems employed included multi-bilayer stacks, tethered bilayers and phospholipid monolayers studied using a combination of reflectometry, attenuated total reflection infrared spectroscopy, electrochemical impedance spectroscopy, neutron diffraction and surface pressure-molecular area isotherms. Oxidation with Fe(II) and ascorbate, had a mild effect on membrane structure causing an increase in the order of the membrane, attributed to cross-linking of the phospholipids, but no significant impact on the thickness or completeness of the membrane. Incorporation of PoxnoPC, on the other hand, had a larger impact on the structure of the membrane. Inversion of the aldehyde-terminated truncated sn-2 chain of PoxnoPC into the head group region was observed, along with a slight decrease in the thickness and order of the membrane. Phospholipase A2 maintained activity on all oxidised membranes but did not show a lag phase for oxidised systems. There was, however, a decrease in the rate and extent of activity on oxidised membranes, with a larger decrease observed with the PoxnoPC incorporated membranes. Moreover, PLA2 was shown not to preferentially hydrolyse oxidised phospholipids. Plant annexin Gh1 was shown to bind strongly to membranes in a calcium dependent manner, contrary to previous reports, including penetration of the hydrophobic region. It was, however, found to bind with less affinity to oxidised membranes, with no binding to membranes containing 30 mol% PoxnoPC. There was also no evidence of protective crystalline domains. α-Synuclein also bound strongly to the head groups of the unoxidised membranes as well as penetrating into the hydrophobic region. Reduced binding was observed with the Fe(II) and ascorbate oxidised membranes as well as the 5 mol% PoxnoPC containing membranes, however, no binding was observed for the 30 mol% PoxnoPC containing membrane. ©, The Author, 2014