Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids in model membranes.

The spectroscopic and ionization properties of various lipids labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group have been studied in model membranes using fluorescence, absorbance and electrophoretic mobility measurements. Electrophoretic measurements show that the NBD group is uncharged at neutral pH. However, at high pH, hydroxyl addition or deprotonation occurs with a pKa, depending upon conditions, of 11.5-11.8 for the NBD group of headgroup-labeled phosphatidylethanolamine (NBD-PE) and 11.1-11.5 for NBD labels placed at the end of one fatty acyl chain of a phosphatidylcholine (6-NBD-PC and 12-NBD-PC). This type of behavior is not observed in the case of a methylated NBD label placed in the flexible 'tail' of cholesterol (NBD-cholesterol). The similarity in pKa for NBD-PE and NBD-PCs suggests that in these cases the NBD group is at a similar depth in the membrane. This was examined further by comparison of the fluorescence emission maximum of the NBD group in model membranes with that in solvents of varying polarity. The apparent polarity experienced by NBD groups in model membranes indicates that for NBD-PE and 12-NBD-PC they are located at the polar region whereas the NBD group of NBD-cholesterol is deeply buried in a nonpolar region of the membrane. This conclusion is supported further by fluorescence quenching experiments measuring NBD exposure to the aqueous quencher Co2+. The results of this study confirm the tentative conclusions of our previous fluorescence quenching studies on the location of NBD groups in model membranes.