STUDY OF TRANSVERSE DIFFUSION OF SPIN LABELED PHOSPHOLIPIDS IN BIOLOGICAL-MEMBRANES .1. HUMAN RED BLOOD-CELLS

Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the .beta. acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22.degree. C with sonicated spin-labeled phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the .beta. chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0.degree. C allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creating a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was stable at 22 and 37.degree. C for more than 4 h. The rate of outside-inside and inside-outside transition is apparently so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.