| Abstract
| - We establish high-sensitivity isothermal titration calorimetry (ITC) as a fast, reliable, and versatiletool for assessing membrane translocation of charged compounds. A combination of ITC uptake and releasetitrations can discriminate between the two extreme cases of half-sided binding and complete transbilayerequilibration on the experimental time scale. To this end, we derive a general fit function for both assaysthat allows for incorporation of different membrane partitioning models. Electrostatic effects are taken intoaccount with the aid of Gouy−Chapman theory, thus rendering uptake and release experiments amenableto the investigation of charged solutes. This is exemplified for the flip−flop of the anionic detergent sodiumdodecyl sulfate (SDS) across the membranes of 100-nm-diameter unilamellar vesicles composed of1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in aqueous solution (10 mM phosphate buffer,154 mM NaCl, pH 7.4). If repulsive electrostatic forces are accounted for adequately, SDS binding to POPCmembranes can be evaluated on the basis of ideal mixing in all phases. At 25 °C, the intrinsic partitioncoefficient between the interfacial aqueous phase and the membrane amounts to 3.5 × 106; however,detergent flip−flop is negligibly slow under these conditions. Raising the temperature to 65 °C lowers theintrinsic partition coefficient to 1.4 × 106 but enables rapid transbilayer distribution of the detergent and,therefore, binding to or desorption from both membrane leaflets. Thus, combining a surface partitionequilibrium with simple electrostatic theory appears highly useful in monitoring transmembrane movementof ionic compounds by ITC, thereby eliminating the need for specific reporter groups.
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