Measurement of conformational changes in the structure of transglutaminase on binding calcium ions using optical evanescent dual polarisation interferometry.

The conformational changes occurring when the protein transglutaminase binds calcium ions have been studied using the optical evanescent technique of dual polarization interferometry (DPI) implemented via a dual slab waveguide structure. Immobilized transglutaminase layers of 4-5 nm in thickness were obtained, which when challenged with calcium ions underwent a contraction of approximately 0.5 nm (depending on the concentration of calcium) and an increase in refractive index of approximately 1 x 10-2. The affinity constant for the calcium binding was found to be in the range of 0.95 +/- 0.2 mM. The results reported are in good agreement with those found in the literature obtained by other techniques. It has also been shown that the structural changes occurring during the binding event are considerably larger than the mass changes that take place; thus, DPI offers a potentially valuable method to study real-time structural changes occurring to proteins when they bind metal ions.

Quantifying the effects of melittin on liposomes.

Melittin, the soluble peptide of bee venom, has been demonstrated to induce lysis of phospholipid liposomes. We have investigated the dependence of the lytic activity of melittin on lipid composition. The lysis of liposomes, measured by following their mass and dimensions when immobilised on a solid substrate, was close to zero when the negatively charged lipids phosphatidyl glycerol or phosphatidyl serine were used as the phospholipid component of the liposome. Whilst there was significant binding of melittin to the liposomes, there was little net change in their diameter with melittin binding reversed upon salt injection. For the zwitterionic phosphatidyl choline the lytic ability of melittin is dependent on the degree of acyl chain unsaturation, with melittin able to induce lysis of liposomes in the liquid crystalline state, whilst those in the gel state showed strong resistance to lysis. By directly measuring the dimensions and mass changes of liposomes on exposure to melittin using Dual Polarisation Interferometry, rather than following the florescence of entrapped dyes we attained further information about the initial stages of melittin binding to liposomes.

Temporal and spatial profiling of the modification of an electroactive polymeric interface using neutron reflectivity.

Electropolymerized films of the functionalized pyrrole, pentafluorophenyl-3-(pyrrol-1-yl)propionate (PFP), were reacted with a solution-phase nucleophile, ferrocene ethylamine. This reaction was chosen as a model representative of a postdeposition modification of the polymer membrane's properties. For the first time, a nondestructive method for direct chemical analysis of the reaction profile within the electrodeposited polymer membrane after nucleophilic substitution is presented. This was achieved through the application of in situ neutron reflectivity with supplementary analytical information concerning the film's chemical composition obtained from XPS, FT-IR, and electrochemical measurements. The results presented illustrate how, for a partially reacted film resulting from a short reaction time, the extent of reaction with ferrocene ethylamine is not homogeneous throughout the thickness of the film, but occurs predominantly at the polymer/solution interface. We show that the progress of the reaction within the polymer film is limited by the transport of reacting species in the dense regions of the membrane that are furthest from the solution interface. The data do not fit an alternative model in which there is spatially homogeneous progression of the reaction front throughout the bulk of the thin film polymer. Guided by the neutron reflectivity measurements, suitable modifications were made to the electrodeposition method to prepare films whose architecture resulted in faster rates of reaction.

Refractometric discrimination of void-space filling and swelling during vapour sorption in polymer films.

Thin polymeric films have been deposited as upper cladding layers on a new integrated optical interferometer fabricated from layers of silicon oxynitride on a silicon wafer. The evanescent field of the probing waveguide mode transduces refractive index changes in the polymer layer into the measured phase changes in the device. Real-time measurement of index change and its sign is obtained. Upon exposure to humid air, we record water sorption by films of poly(vinyl pyrrolidone) by a rapid positive index change for void-space filling followed by a slow negative index change for swelling. Sorption of water vapor into a thin film of the viscous liquid polymer polyethylenimine shows only swelling mode behaviour and a simple constitutive model can be applied to give the fractional water occupied volume.