A MODIFIED SPECTROSCOPIC METHOD FOR THE DETERMINATION OF THE TRANSBILAYER DISTRIBUTION OF PHOSPHATIDYLETHANOLAMINE IN SOYBEAN ASOLECTIN SMALL UNILAMELLAR VESICLES

A spectroscopic kinetic approach for determining the relative concentr ations of phosphatidylethanolamine (PE) exposed on the external and in ternal layers of small unilamellar vesicles (SUVs) used as a model sys tem and prepared by sonication of purified soya-bean asolectin is prop osed, based on the use of 2,4,6-trinitrobenzenesulphonic acid (TNBS) a nd N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The known reac tions between PE and TNBS and/or SDPD were used, separately or in comb ination, to derivatize PE in preformed vesicles. We have observed that mixing SUVs with excess TNBS results in a biphasic time course. Kinet ic analysis of the data supports the conclusion that external PE is ra pidly derivatized (fast phase) with a half-time of 2 min, In the next (slow) phase (half-time 70 min), TNBS permeates the vesicle membrane a nd also reacts with PE molecules facing the internal liposomal compart ment. Under the experimental conditions chosen, SPDP reacted with only the external PE molecules. The reaction of SUVs first derivatized wit h SPDP and then with TNBS further demonstrates that the two phases, ob served with TNBS, are due to modification of external and internal PE. Approx. 30% of PE was found to be facing the external bulk phase, thu s confirming the asymmetric distribution of the molecules in SUVs. The maximum number of thiol arms covalently linked by means of SPDP modif ication of PE on the surface of a single liposome was estimated at abo ut 10(2).