DPH acts as a probe for orientation of vesicles

be masked by any protein signal in an experiment, the other peaks are well clear of the protein region, and should allow interpretation of vesicle orientation.

200 250 300 350 400 450 500 -0 .1 0 -0 .0 8 -0 .0 6 -0 .0 4 -0 .0 2 0.00 LD spectrum, DPH on PE film Wavelength / nm LD

The LD spectra of vesicles containing DPH were measured at various concentrations in order to demonstrate the e↵ectiveness of DPH as a probe forLD. The orientation method was Couette flow. Representative spectra are shown in Figures 6.2 and 6.3.

200 250 300 350 400 450 500 -0 .0 10 -0 .0 05 0.000 0.005 0.010

LD spectra, DPH incorporated into lipid

Wavelength / nm LD BTLE POPC/POPS/chol SoyPC DMPC DMPC/POPS/chol

Figure 6.2: LDspectrum of DPH in lipid vesicles at approximately 50 mg/mL in aqueous bu↵er.

20 -0 .0 15 -0 .0 10 -0 .0 05 0.000 0.005 0.010

LD spectra, DPH incorporated into lipid

LD

BTLE POPC/POPS/chol SoyPC DMPC/POPS/chol

It was apparent from Figures 6.2 and 6.3 that DPH did not orient to the same extent in all cases, and that clear trends, or reproducibility, have not been achieved. However, the bands associated with DPH from Figure 6.1 are present to some extent in all case, and in some cases are extremely strong. Spectra gathered with BTLE and POPC/POPS/cholesterol, for example, are dominated by the DPH bands. In other cases, soybean PC for example, the bands are visible but not strong, and could be overwhelmed by introductions of other compounds. The bands for DPH have negative sign, indicating that the DPH orients as if it were another lipid in the membrane, i.e. it is parallel to the normal to the lipid surface.

The data shown in Figures 6.2 and 6.3 posed a problem, as it was necessary to determine standardised, optimised, methods for utilising DPH as an LD probe, and results from di↵erent lipid systems contradicted each other. The optimal bands forLD probes would be present and clear, but minimal, so as not to be confused with other analytes. It was, therefore, essential to reduce the strength of DPH LD bands, and also to reduce the amount of lipid required for the sample, so as to be comparable with the experiments presented in Chapters 4 and 5: i.e. 1 mM, or around that order of magnitude.

DPH was incorporated into revised vesicle systems, and theLD measured. These samples contained lipid at around 5 mg/mL. Figures 6.4, 6.5, 6.6 and 6.7 showLD spectra gathered for the lipid systems with DPH present. Figure 6.4 includes a spectrum without DPH to verify that DPH does not influence the position of the lipid band observed.

200 250 300 350 400 450 500 -0 .0 02 -0 .0 01 0.000 0.001 0.002 0.003

LD spectra, lipids incorporating DPH

Wavelength / nm

LD

POPC/POPS/chol/DPH POPC/POPS/chol

Figure 6.4: LD spectra of vesicles comprising POPC, POPS, cholesterol. Solid lines represent data gathered for vesicles also containing DPH, dotted line for vesicles without. Lipids at approximately 5 mg/mL. Path length of capillary was 500µm.

200 250 300 350 400 450 500 0.0000 0.0005 0.0010 0.0015 0.0020

LD spectra, lipids incorporating DPH

LD

200 250 300 350 400 450 500

0.000

0.002

0.004

0.006

LD spectra, lipids incorporating DPH

Wavelength / nm

LD

Soy PC/DPH

Figure 6.6: LD spectra of vesicles comprising soybean PC and DPH. Lipids at approximately 5 mg/mL. Path length of capillary was 500µm.

200 250 300 350 400 450 500 -0 .0 08 -0 .0 04 0.000 0.004

LD spectra, lipids incorporating DPH

Wavelength / nm

LD

BTLE/DPH

Figure 6.7: LD spectra of vesicles comprising brain total lipid extract and DPH. Lipids at approximately 5 mg/mL. Path length of capillary was 500µm.

Some of the spectra presented in Figures 6.4, 6.5, 6.6 and 6.7 show that DPH gives still gives discernible bands under these revised conditions. The proportion of DPH used was optimised such that minimal bands would be observed in the POPC/POPS/cholesterol sample, thereby reducing any potential impact on a signal from protein. However, it is clear that the soybean PC vesicles do not display DPH LD signal at this concentration. DPH may not be able to incorporate into the soybean lipids, or may have itsLDsignal quenched by other vesicle components. The work presented in this thesis does not answer why this is the case. Extensive work may be required to determine why soybean PC prevents DPH giving strongLD. Unpublished data from the Rodger laboratory does appear to show that if the concentration of DPH is very high (around 10 % w/w), or if the lipid concentration is very high (over 50 mg/mL) it will generate anLD signal, however, this was not compatible with the experimental aims in this case.

Another interesting result from this set of data is the strikingly di↵erent shape of the spectrum for BTLE (Figure 6.7). While the other three systems have strongly positive signals around 200 nm, BTLE has a very sharp negative band here. This is not easily explained by usual factors such as scattering of light by vesicles, however there is no evidence of an absorption band at this wavelength for BTLE. Some combination of factors, including some scattering, may be responsible for this LD band. It is believed that the BTLE is such a complex extract the some components of the lipid mixture are able to give very strong negative LD signal when oriented in Couette flow, with their head groups oriented perpendicular to the direction of the flow. There must be some other chromophore, as yes unidentified.

The orientation parameterSwas calculated for the DPH bands observed in these lipid/DPH

LD spectra. The spectra were first corrected for sloping between 500 and 400 nm, so that the region around the DPH bands was as uniform as possible. Table 6.1 shows the values for S calculated here. An example of the method used for this calculation is given in Appendix C.

Table 6.1: Values forS calculated from DPHLDsignal in lipid/DPHLD spectra (1 significant figure). An implementation of the method used for this calculation is given in Appendix C.

Lipid components S

POPC/POPS/cholesterol 0.01 DMPC/POPS/cholesterol 0.001 Soybean PC 0.0003 BTLE 0.01

It may be noted that these values are lower than those reported in Chapter 3. This may indicate that the model should be reformulated, or that more data is needed before calculatingS.