Fluorescence quenching of cytochrome b5 in vesicles with an asymmetric transbilayer distribution of brominated phosphatidylcholine.
Several fluorescence techniques have been used to estimate the depth, in the membrane, of the endogenous tryptophans of membrane-bound proteins. We reported recently the use of phosphatidylcholines specifically brominated at different positions of the sn-2 acyl chain for this purpose (Markello, T., Zlotnick, A., Everett, J., Tennyson, J., and Holloway, P. W. (1985) Biochemistry 24, 2895-2901). The membranes made from these brominated lipids will have the brominated lipid in both monolayers, and so the estimated depth of the fluorophore will be relative to either the inner or outer surface of the membrane, but will not distinguish between these two extremes. To differentiate between these two models vesicles have now been made with an asymmetric distribution of brominated lipid, by use of phosphatidylcholine exchange protein. The asymmetric vesicles were isolated by virtue of their density, and their asymmetry was established by addition of an amphipathic fluorescent carbazole compound. With these vesicles it was shown that the tryptophan in the membrane-binding domain of cytochrome b5 which is quenched by bromolipid is located 0.7 nm below the outer surface of the membrane vesicles, rather than 0.7 nm from the inner surface.
Everett
J
J
Zlotnick
A
A
Tennyson
J
J
Holloway
P W
PW
D000818:Animals; D001966:Bromine; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D007700:Kinetics; D008051:Lipid Bilayers; D008099:Liver / Q000378:metabolism; D010713:Phosphatidylcholines; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D015786:Cytochromes b5; D001966:Bromine