HIGH-PRESSURE FREEZING CAUSES STRUCTURAL ALTERATIONS IN PHOSPHOLIPID MODEL MEMBRANES

The influence of high-pressure freezing (HPF) on the lipid arrangement in phospholipid model membranes has been investigated. Liposomes cons isting of pure dipalmitoylphosphatidylcholine (DPPC) and of DPPC mixed with a branched-chain phosphocholine ( 1,2 (4-dodecyl-palmitoyl)-sn-g lycero-3-phosphocholine) have been analysed by freeze-fracture electro n microscopy. The liposomes were frozen either by plunging into liquid propane or by HPE The characteristic macroripple-phase of the two-com ponent liposome system is drastically changed in its morphology when f rozen under high-pressure conditions. The influence of ethanol which a cts as pressure transfer medium was ruled out by control experiments. In contrast, no high-pressure alterations of the pure DPPC bilayer mem brane have been observed. We assume that the modification of the binar y system is due to a pressure-induced relaxation of a stressed and uns table lipid molecule packing configuration. HPF was performed with a n ewly designed sample holder for using sandwiched copper platelets with the high-pressure freezing machine Balzers HPMO10. The sandwich const ruction turned out to be superior to the original holder system with r egard to freeze-fracturing of fluid samples. By inserting a spacer bet ween the supports samples with a thickness of 20-100 mu m can be high- pressure frozen. The sandwich holder is provided with a thermocouple t o monitor cooling rates and allows exact sample temperature control. D espite a two-fold mass reduction compared to the original holder no HP F cooling rate improvement has been achieved (4000 degrees Cs-1). We c onclude that the cooling process in high-pressure freezing is determin ed mainly by cryogen velocity.