Bi. Meiklejohn et al., 5-IODONAPHTHYL-1-AZIDE LABELING OF PLASMA-MEMBRANE PROTEINS ADJACENT TO SPECIFIC SITES VIA ENERGY-TRANSFER, Biochimica et biophysica acta. Biomembranes, 1324(2), 1997, pp. 320-332
We have examined conditions optimal for 5-iodonaphthyl-1-azide (INA(+)
) labeling of membrane proteins proximal to known membrane sites. Memb
rane-bound INA can be indirectly activated by energy transfer from vis
ible chromophores. We demonstrate that the efficiency of this sensitiz
ed activation is enhanced by use of triplet-forming chromophores such
as eosin and by deoxygenation. Variation of sensitized activation effi
ciency with INA concentration indicates that the critical distance for
eosin-INA energy transfer in solution is 8-14 Angstrom. We suggest th
at photosensitization occurs through tripler exchange and present an i
mproved labeling protocol based on these findings. This protocol was u
sed to examine whether different accessory proteins are associated wit
h isolated and crosslinked Type I Fc(epsilon) receptors on 2H3 rat bas
ophilic leukemia cells. 2H3 cells were incubated with eosin-conjugated
IgE and irradiated at 514 nm yielding [I-125]INA derivatized peptides
at 53, 38, 34, and 29 kDa. Crosslinking IgE with mouse anti-rat IgE p
rior to irradiation labeled three additional proteins at 60, 54, and 4
3 kDa. These results demonstrate the utility of sensitized INA labelin
g in characterizing protein-protein interactions in membranes of intac
t cells and indicate the importance of considering photophysical facto
rs when selecting sensitizers and reaction conditions. We discuss esti
mation of the size of the membrane region surrounding a sensitizing ch
romophore within which INA labeling of membrane proteins occurs.