Analysis of membranes photolabeled with lipid analogues. Reaction of phospholipids containing a disulfide group and a nitrene or carbene precursor with lipids and with gramicidin A.

Analogues of fatty acids have been synthesized which contain an S-S bridge in the aliphatic chain and at the omega-carbon the photoactivatable p-(3-trifluoromethyldiazirino)phenyl or p-azidophenyl group as a carbene and nitrene precursor, respectively. These acids were used to acylate 1-palmitoyl phosphatidylcholine by the procedure described by Gupta et al. (Gupta, C.M., Radhakrishnan, R., and Khorana, H.G. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4315-4319). Multilamellar liposomes prepared from various radioactively labeled lipids and 2 to 10 mol% of one of the photosensitive phospholipid analogues were photolyzed and the extent of cross-linking was determined. The carbene-generating probe labeled dipalmitoyl phosphatidylcholine in yields of 15 to 20%, while labeling with the nitrene probe was about 10 times less efficient. In contrast these probes were found to react with the unsaturated fatty acid of 1-palmitoyl-2-oleoyl phosphatidylcholine with almost equal efficiency. The nitrene was 2 to 3 times more efficient than the carbene in cross-linking to gramicidin A. Amino acid analysis and Edman degradation of the labeled peptide indicated that both probes predominantly attacked tryptophan residues. No geometrical correlation was found between the expected penetration of the reactive group of the probe within the bilayer and the labeled sites of the peptide. This result is discussed in terms of chemical selectivities of the photoactivated intermediates. After photocross-linking, the disulfide bridge can be cleaved to generate a free sulfhydryl group which subsequently can be utilized for preparative or analytical purposes. Photolabeled lipids and peptides can be separated easily from the bulk of the unlabeled material. These phospholipid analogues are substrates for the phospholipid exchange protein isolated from calf liver. Transfer from sonicated dispersions of the analogues into an acceptor membrane was demonstrated. Individual monolayers can thus be labeled separately with these reagents.