5-IODONAPHTHYL-1-AZIDE LABELING OF PLASMA-MEMBRANE PROTEINS ADJACENT TO SPECIFIC SITES VIA ENERGY-TRANSFER

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.