Acid dissociation constants of diphytanylglycerolphosphorylglycerol-methylphosphate, and diphytanylglycerolphosphorylglycerophosphate and its deoxy analog

Abstract Acid dissociation constants of 2,3-diphytanyl- sn -glycero-1-phosphoryl- sn -3′-glycero-1′-methylphosphate (PGP-Me), the major phospholipid in extreme halophiles (Halobacteriaceae) , and of the demethylated 2,3-diphytanyl- sn -glycero-1-phosphoryl- sn -3′-glycero-1′-phosphate (PGP) and its deoxy analog 2,3-diphytanyl- sn -glycero-1-phosphoryl-1′-(1′,3′-propanediol-3′-phosphate) (dPGP), were calculated by an original mathematical procedure from potentiometric titration data in methanol/water (1:1, v/v) and found to be as follows: for PGP-Me (and presumably PGP), p K 1 =3.00 and p K 2 =3.61; for PGP, p K 3 =11.12; and for dPGP, p K 1 =2.72, p K 2 =4.09, and p K 3 =8.43. High-resolution 31 P NMR spectra of intact PGP-Me in benzene/methanol or in aqueous dispersion showed two resonances corresponding to the two P-OH groups, each of which exhibited a chemical shift change in the pH range 2.0–4.5, corresponding to acid dissociation constants p K 1 =p K 2 =3.2; no further ionization (p K 3 ) was detected at higher pH values in the range 5–12. The present results show that PGP-Me titrates as a dibasic acid in the pH range 2–8, but above pH 8, it titrates as a tribasic acid, presumably PGP, formed by hydrolysis of the methyl group during the titration with KOH. Calculation of the concentrations of the ionic molecular species of PGP-Me, PGP and dPGP as a function of pH showed that the dianionic species predominate in the pH range 5–9, covering the optimal pH for growth of Halobacteriaceae. The results are consistent with the concept that the hydroxyl group of the central glycerol moiety in PGP-Me and PGP is involved in the formation of an intramolecular hydrogen-bonded cyclic structure of the polar headgroup, which imparts greater stability to the dianionic form of PGP-Me and PGP in the pH range 5–9 and facilitates lateral proton conduction by a process of diffusion along the membrane surface of halobacterial cells.