Radioiodinated, Photoactivatable Phosphatidylcholine and Phosphatidylserine: Transfer Properties and Differential Photoreactive Interaction with Human Erythrocyte Membrane Proteins

An isotopically labeled cross-linking reagent, succinimido 3-(3-[¹²⁵I]iodo-4-azidophenyl)propionate, has been synthesized and coupled to 1-acyl-2-(aminocaproyl)phosphatidylcholine according to previously described procedures [Schroit, A. J., & Madsen, J. (1983) Biochemistry 22, 3617–3623]. ¹²⁵I- and N₃-labeled phosphatidylserine (¹²⁵I-N₃-PS) was produced from the phosphatidylcholine (PC) analogue by phospholipase D catalyzed base exchange in the presence of L-serine. These phospholipid analogues are photoactivatable, are labeled with ¹²⁵I at high specific activity, completely incorporate into synthetic vesicles, and spontaneously transfer between membranes. When an excess of acceptor vesicles or red blood cells (RBC) was mixed with a population of donor vesicles containing the ¹²⁵I-N₃-phospholipids, approximately 40% of the analogues transferred to the acceptor population. After transfer in the dark to RBC, all of the ¹²⁵I-N₃-PC incorporated into the cells could be removed by washing with serum, whereas the ¹²⁵I-N₃-PS could not. After photolabeling of intact RBC, ~50% of the PC and 20% of the PS cross-linked to membrane proteins as determined by their insolubility in CHCl₃/MeOH. Analysis of probe distribution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ¹²⁵I-N₃-PS preferentially labeled a M_r 30000 peptide which contained ~30% of the protein-bound label.