The stability of vesicular pH gradients (between the inner and outer aqueous solutions) depends on processes that can allow protons to permeate across phospholipid barriers. Phospholipid bilayers are thought not to be permeable to charged species (per the pH partition hypothesis). However, some studies suggest H+/OH~ permeability to be surprisingly high, as high as 10~4 cm/s, greatly exceeding that of about 10~12 cm/s for Na+ [409-419]. Biegel and Gould [409] rapidly changed the pH (acid pulse measurements) of a suspension of small unilamellar vesicles (SUVs; soybean PC) from the equilibrated pH 8.2 to the external pH 6.7, and monitored the rate of influx of H+ into the vesicles. (The pH inside of vesicles can be measured by fluorescent probes [409,419].) It took several minutes for the internal pH to drop from pH 8.2 to 7.4. This time was long because charge transfer led to buildup of a potential difference across the membrane (Donnan potential), which was slow to dissipate. The time was dropped to about 300 ms in the presence of a K+ ionophore, valinomycin, an antiporter type of effect. The proton ionophore, bis(hexafluoroacetonyl)acetone, dropped the reequilibration time down to <1 ms.

Discussions of the possible mechanisms of H+ transport ensued. It was pointed out that the solubility of water in n-alkanes was high enough to suggest the participation of membrane-dissolved water in the transport mechanism. Biegel and Gould [409] predicted that the SUVs used in their study could have 30-40 H2O molecules dissolved in the bilayer hydrocarbon (HC) core. Meier et al. [411] measured the concentration of water in the HC interior of bilayes to be about 100 mM. Two short reviews discussed proton conductance: Nagle [412] defended the position that''water wires'' inside the HC core can explain H+ conductance; Gutknecht [413] questioned that view, proposing that fatty acid impurities can also explain the phenomenon, in a flip-flop movement of the neutralized weak acid. Proton carriers such as CO2 or H2CO3 could also be involved [415]. The last word has not been said on this topic.

Using liposomes made from phospholipids as models of membrane barriers, Chakrabarti and Deamer [417] characterized the permeabilities of several amino acids and simple ions. Phosphate, sodium and potassium ions displayed effective permeabilities 0.1-1.0 x 10"12 cm/s. Hydrophilic amino acids permeated membranes with coefficients 5.1-5.7 x 10"12 cm/s. More lipophilic amino acids indicated values of 250-410 x 10"12 cm/s. The investigators proposed that the extremely low permeability rates observed for the polar molecules must be controlled by bilayer fluctuations and transient defects, rather than normal partitioning behavior and Born energy barriers. More recently, similar magnitude values of permeabilities were measured for a series of enkephalin peptides [418].


Working with liposomes requires considerable care, compared to octanol. Handling of liposomes is ideally done under an inert atmosphere at reduced temperatures. Prepared suspensions ought to be stored frozen when not used. Air oxidation of cis double bonds is facile; hydrolysis of esters to form free fatty acids (FFAs) is usually a concern. The best commercial sources of phospholipids have <0.1% FFA. Procedurally, a dry chloroform solution of a phospholipid is placed in a round-bottomed glass flask. Argon is allowed to blow off the chloroform while the flask is vortexed; a thin multilamellar layer forms on the glass surface. After evacuation of the residual chloroform, a buffer is added to the flask, and the lipid is allowed to hydrate under vortexing agitation, with argon gas protecting the lipid from air oxidation. A suspension of multilamellar vesicles (MLVs; diameter >1000 nm) forms in this way. [162] Small unilamellar vesicles (SUV, 50 nm diameters) can be made by vigorous sonication of MLVs. [385,386] Hope and coworkers developed procedures for preparing large unilamellar vesicles (LUV, 100-200 nm diameter) by an extrusion technique (ET), starting from the MLV suspension [389-391]. Freeze-and-thaw (FAT) steps are needed to distribute buffer salts uniformly between the exterior aqueous solution and the aqueous solution trapped inside vesicles [390]. Methods for determining volumes of liquid trapped inside the vesicles have been discussed [392]. When liposome surfaces are modified by covalent attachment of polyethylene glycol (PEG) polymer, the so-called stealth liposomes can evade the body's immune system, and stay in circulation for a long time, acting like a Trojan horse bearing drugs [393]. Such systems have been used in drug delivery [391,393]. Ordinary liposomes carrying drugs are quickly dismembered by the immune system.

For partition studies, only SUV [385,386] or LUV [149] should be used; MLVs have many layers of trapped solution, which usually cause hysteresis effects [162].

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