Hydrophobic Photolabeling with ¹²⁵I-TID of Red Blood Cell Membranes

Abstract

Reagents such as 3-trifluoromethyl, 1–3(m-iodophenyldiazirine) (TID) have been introduced with the aim of identifying integral protein in a membrane and to study the hydrophobic sector (Bayley 1983). To reach this goal the reagents have been made:

• 1 Hydrophobic, in order to make them readily partition in the hydrophobic domain of the membrane; this property will determine their localization (however, by the same property they will also be able to occupy hydrophobic pockets present on the hydrophilic domain of an integral protein);

• 2 Photoactivatable, in such a way that they can be activated in a very short time only when all the biological manipulations are ended. This most important property of photolabels allows one to start the labeling reaction only when needed; moreover in this way intermediates can be generated with such a reactivity that they will be able to form covalent derivatives even with the aliphatic side chains frequently found in the lipid-exposed surface of integral proteins;

• 3 Radioactive, in order to be able to trace the labeling at the level of the protein or the subunit (mainly by SDS-gel electrophoresis as in the present experiment) and even further at the level of the modified residues (by sequencing). In this sense ¹²⁵I is one of the best isotopes to be used because of its high specific radioactivity, which lowers the amount of probe needed (and parallely the perturbation of the system), and because of its easier detectability in autoradiography.