Lipid Peroxides Promote Large Rafts:

Effects of Excitation of Probes in Fluorescence

Microscopy and Electrochemical Reactions
during Vesicle Formation
A. Ayuyan and F. Cohen. Biophys. J. 91: 2172-2183 (2006).
Short summary

Problem: for some lipid compositions, spectroscopy reports that only

small, nanometer-scale rafts should be present, whereas in fact large rafts
are observed by microscopy.

Hypothesis: excitation of fluorescent lipid probes leads to the generation

of lipid peroxides that break down into products which dramatically alter
membrane properties (increase membrane permeability and fluidity, alter
cholesterol organization and distribution within bilayer membranes).
 System: DOPC/SM/Chol, NBD-DPPE (lo) and Rho-DOPE (ld) as probes.
Methods: electroswelling (ITO-glasses or titanium plates as electrodes);
fluorescent microscopy.

In the presence of photoprotector (NPG) rafts

do not appear at 250C (A) or 100C (B), but
some rafts do appear at 10C (C). GUVs were
formed from a 40:20:40%
DOPC/eSM/cholesterol mixture. NBD-DPPE
was used as probe for each of these panels.

In the absence of antioxidants, large

rafts formed within 15 s after
illumination (250C). GUVs were
formed from a 40:20:40%
DOPC/eSM/Chol mixture. Rho-DOPE
was used as probe. The times after
starting illumination are shown (in
units of seconds) in the upper left
corners.

A. Ayuyan and F. Cohen. Biophys. J. 91: 2172-2183 (2006).

Photoexcitation of a fluorescent probe results in a cascade of reactions. These
reactions occur without any destruction of the excited fluorescent lipid probe that
decays back to its ground state, from which it can again be photoexcited.

A. Ayuyan and F. Cohen. Biophys. J. 91: 2172-2183 (2006).

Forming GUVs with ITO-glass electrodes, but not with titanium
electrodes, generates peroxides.

Bar graph: ITO-glass and titanium

electrodes were used to prepare
GUVs composed of 60:20:20%
DOPC/eSM/cholesterol. The use
of ITO-glass led to peroxides, but
titanium electrodes did not
generate peroxides.

Images: when ITO-glass

electrodes were used to prepare
GUVs composed of 40:20:40%
DOPC/eSM/cholesterol, some
large rafts formed at 250C (A);
more formed at 40C (B).
Preparing GUVs under precisely
the same conditions, but using
titanium electrodes, did not lead
to rafts, shown here at 100C (C).
NBD-DPPE was used as probe for
all images.
A. Ayuyan and F. Cohen. Biophys. J. 91: 2172-2183 (2006).

Electrochemical production of peroxides during electroswelling
with ITO-glass as electrodes.

Photochemical production of peroxides under photoexcitation of
fluorescent probes.

USE ANTIOXIDANTS!

• NPG (N-propyl gallate)

• BHT (butylated hydroxyl toluene)
• DTT (dithiothreitol)

A. Ayuyan and F. Cohen. Biophys. J. 91: 2172-2183 (2006).