Spin-label studies on the aqueous regions of phospholipid multilayers

Abstract

H2O-soluble spin labels were used to study dimyristoyllecithin (DML) phospholipid multilayers. Previous studies reported a bound water region associated with dimyristoyllecithin containing about 10 molecules of H2O per phospholipid, a trapped H2O region located between the lamellae containing approximately 11 molecules per phospholipid and a free H2O region external to the lamellae. This investigation showed that certain H2O-soluble spin-label molecules have their motional properties differentially modified by these 3 H2O environments. The labels also reveal the onset of lipid-phase transitions even though they have high H2O solubility. A phosphate-containing spin label demonstrated strong anisotropic motion in the lipid-H2O system above the phase transition but not below. The addition of cholesterol to the DML-H2O system removed the anisotropic motion of 2,2,6,6-tetramethyl-4-phosphopiperidine-N-oxyl (Tempophosphate) and obscured the detection of bound, trapped and free H2O. In addition to the charge-charge interactions between Tempophosphate and DML, 2 other spin labels were used in the charged and uncharged states. 2,2,6,6-Tetramethyl-4-aminopiperidine-N-oxyl (Tempamine) in the charged state showed extremely strong anisotropic motion, presumably due to the interaction between the charged amine and the phosphate group of DML. When only partially charged, Tempamine showed much less anisotropic motion. PCA was analyzed at pH values where the carboxyl group was protonated and unprotonated. The resulting interaction was different at the 2 pH values. These H2O-soluble spin labels mimic ionic or nonionic solutes. Upon freezing, the spin labels are expelled from the ice regions into remaining aqueous regions. The usefulness of this approach in studying solute behavior when freezing occurs and potential studies involving aqueous regions of cytoplasm are considered.