The process of biological membrane fusion can be analysed by topological methods. Mathematical analysis of the fusion process of vesicles indicated two significant facts: the formation of an inner, transient structure (hexagonal phase - HII) and a translocation of some lipids within the membrane. This shift had a vector character and only occurred from the outer to the inner layer. Model membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) was studied. 31P- and 1H-NMR methods were used to describe the process of fusion. 31P-NMR spectra of multilamellar vesicles (MLV) were taken at various temperatures and concentrations of Ca2+ ions (natural fusiogenic agent). A 31P-NMR spectrum with the characteristic shape of the HII phase was obtained for the molar Ca2+/PS ratio of 2.0. During the study, 1H-NMR and 31P-NMR spectra for small unilamellar vesicle (SUV), which were dependent on time (concentration of Pr3+ ions was constant), were also recorded. The presence of the paramagnetic Pr3+ ions permits observation of separate signals from the hydrophilic part of the inner and outer lipid bilayers. The obtained results suggest that in the process of fusion translocation of phospholipid molecules takes place from the outer to the inner layer of the vesicle and size of the vesicles increase. The NMR study has showed that the intermediate state of the fusion process caused by Ca2+ ions is the HII phase. The experimental results obtained are in agreement with the topological model as well.
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