Continuous gradient temperature Raman spectroscopy from −100 to 40 °C yields new molecular models of arachidonic acid and 2-Arachidonoyl-1-stearoyl-sn-glycero-3-phosphocholine

Abstract

Despite its biochemical importance, a complete Raman analysis of arachidonic acid (AA, 20:4n-6) has never been reported. Gradient temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry (DSC) to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements that occur near and at phase transitions. Herein we utilize the GTRS technique for AA and 1-18:0, 2-20:4n-6 phosphatidyl choline (AAPC) from cryogenic to mammalian body temperatures. 20Mb three-dimensional data arrays with 0.2°C increments and first/second derivatives allowed complete assignment of solid, liquid and transition state vibrational modes. The AA DSC shows a large exothermic peak at −60°C indicating crystallization or a similar major structural change. No exothermic peak of this magnitude was observed in six other unsaturated lipids (DHA, n-3DPA, n-6DPA, LA, ALA, OA). Melting in AA occurs over a large range: (−60 to −35°C): very large frequency offsets and intensity changes correlate with premelting initiating circa −60°C, followed by melting (−37°C). Novel, unique 3D structures for both molecules reveal that AA is not symmetric as a free fatty acid, and it changes significantly when in the sn-2 phospholipid position. Further, different CH and CH2 sites are unequally elastic and nonequivalent.