Mechanism and kinetics of merocyanine 540 binding to phospholipid membranes

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Abstract
The physicochemical mechanism for merocyanine 540 (M540) binding to unilamellar phosphatidylcholine (PC) vesicles was examined by steady-state and dynamic fluorescence and fluorescence stopped-flow methods. At 530-nm excitation, aqueous M540 has an emission peak at 565 nm, which red shifts to 580 nm with formation of membrane-bound monomers (M); bound dimers (D) are nonfluorescent. Equilibrium fluorescence titrations show that 50% of total M540 partitions into the membrane to form D at [M540]/[PC] (Rm/p) .apprx. 0.6. M and D concentrations are equal at Rm/p .apprx. 0.05. For Rm/p < 0.1, M540 has a single fluorescence lifetime (.tau.), which decreases with Rm/p [.tau.-1 (ns-1) = 0.48 + 3.3 Rm/p], indicating a rapid collisional rate between M to form D. Dynamic depolarization studies show that hindered rotation of M (r .infin. = 0.13 at Rm/p = 0.006) becomes more rapid (rotational rate 0.2-1.9 ns-1) with increasing Rm/p (0.006-0.075). The efficiences of energy transfer between n-(9-anthroyloxy) fatty acid probes (n = 2, 6,9, 12, 16) and bound M540 suggest that M is oriented parallel to the phospholipids near the membrane surface; studies of efficiences of n-AF quenching by D are consistent with an orientation of D perpendicular to the phospholipids. In stopped-flow fluorescence measurements in which M540 is mixed with PC vesicles, there is a rapid (1 ms) followed by a slower (10-50 ms) concentration-dependent fluorescence increase. Taken together with previous temperature-jump results [Verkman, A.S., and Frosch, M. P. (1985) Biochemistry 24, 7117-7122], these data suggest that solution M540 binds to the PC vesicle membrane in < 1 ms to a position parallel to the phospholipids where it undergoes 90.degree. reorientation in .apprx. 1 ms and dimerization in < 10 ns to lie perpendicular to the phospholipids deep in the membrane. M540 then undergoes a 10-ms translocation to a site at the opposite membrane surface.
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