Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

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Abstract
The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration and degree of hydration was studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44.degree. C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62.degree. C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least 2 less hydrated states, differing progressively by 1 H2O/phosphatidylserine, are observed at low pH with transition temperatures of 48 and 52.degree. C for dimyristoyl- and 65 and 68.5.degree. C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to .apprx. 15.degree. C for dimyristoyl- and 32.degree. C for dipalmitoylphosphatidylserine, and a pretransition is observed at .apprx. 6.degree. C (dimyristoylphosphatidylserine) and 21.5.degree. C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition its observed for the fully hydrated phosphate group down to pH .gtoreq. 0.5 but its affinity for water binding decreases steeply at pH .ltoreq. 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoylphosphatidylserine by .apprx. 8.degree. C at pH 7, by .apprx. 5.degree. C at pH 13 and by .apprx. 0.degree. C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of NaCl concentration to 5.5 M, the transition temperature increases by an additional 9.degree. C at pH 7, 13.degree. C at pH 13, .apprx. 7.degree. C in the fully hydrated state at pH 1 and .apprx. 4 and .apprx. 0.degree. C in the 2 less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of .apprx. 1 H2O/lipid; that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (.DELTA.pK2 = 2.85, .DELTA.pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant .epsilon. .simeq. 30, together with surface potentials of .psi. .simeq. -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by .apprx. 4.degree. C for each water/lipid molecule added, reaching a limiting value at a water content of .apprx. 9-10 H2O/lipid molecule.