Mechanism of exchange of cytochrome b5 between phosphatidylcholine vesicles

The intervesicle exchange of cytochrome b5 has been studied by fluorescence quenching. The binding of cytochrome b5 to 1,2-bis(9.10-dibromostearoyl)-sn-glycerol-3-phosphorylcholine vesicles results in a quenching of cytochrome b5 fluorescence whereas the fluorescence is enhanced upon binding to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine vesicles. This difference in cytochrome b5 fluorescence upon binding was used to study the kinetics of cytochrome b5 intervesicle exchange between the "quenching" and "enhancing" vesicles. Separation of the two cytochrome b5-vesicle complexes by density gradient centrifugation provided direct evidence for cytochrome b5 intervesicle exchange. Both the fluorescence assay and the density gradient assay yield the same value for the extent of cytochrome b5 exchange, obtained after equilibration, between the two types of vesicles. Both experiments also indicate that cytochrome b5 binds in a reversible fashion and has an equal affinity for the two types of vesicles. The kinetics of the exchange process are consistent with a mechanism involving the transfer of cytochrome b5 through the aqueous phase and rule out a mechanism involving vesicle collision.