FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDY OF MIXTURES OF PALMITIC ACID WITH DIPALMITOYLPHOSPHATIDYLCHOLINE USING ISOTOPIC-SUBSTITUTION

Fourier transform infrared spectroscopy has been used to study [1-C-13]palmitic acid and L-alpha-[dipalmitoyl-D312]phosphatidylcholine mixtures. The specific isotope labelling of these molecules has made possible the individualized study of the most characteristic bands originating from both lipids in the mixture. It has been possible, thanks to the labelling of palmitic acid, to directly determine its apparent pK in dDPPC, which was found to be approximately 8.7 at 25-degrees-C by measuring the intensities of the unionized and ionized carboxylic bands of [1-C-13]palmitic acid in the infrared spectra. The phase transitions of both L-alpha-[dipalmitoyl-D31(2)]phosphatidylcholine and [1-C-13]palmitic acid in aqueous mixtures at different molar ratios were observed to occur at very similar temperatures at bulk pH 7.4, i.e., when the fatty acid was protonated by more than 95%, providing evidence of a nearly ideal mixing behavior. However, they occurred at well differentiated temperatures at bulk pH 11.0, i.e., when the fatty acid was ionized by more than 99%, showing that a phase separation between the two types of molecules occurred. Hence, the protonation state of the fatty acid affects its miscibility with the phospholipid. On the other hand, the fatty acid did not appear to establish hydrogen bonds with the phospholipid as monitored through the absorption bands due to the ester carbonyl and phosphate groups of the phospholipid. A significant effect that was noticed on the phospholipid was the increase in the proportion of the ester carbonyl groups in the dehydrated state as a consequence of the presence of palmitic acid. The dehydration effect was more considerable at neutral than at alkaline pH.