Tetralinoleoyl cardiolipin

Dimyristoylphosphatidylcholine (PC), tetramyristoyl cardiolipin (CL) and tetralinoleoyl cardiolipin were purchased from Avanti Polar Lipids. Unless otherwise indicated, studies reported herein employed tetramyristoyl CL. Five mg aliquots were dissolved in 200 μL CHCl3:CH3OH (3:1 v/v), and dried under a stream of N₂ gas, creating a thin film on the walls of the vessel. Samples were lyophilized overnight to remove residual solvent. To formulate ND, 750 μL 20 mM HEPES buffer, pH 7.2, was added to a 5 mg aliquot of dried phospholipid. The sample was vortexed to disperse the phospholipid which ultimately appeared as an opaque suspension. Subsequently, 2 mg recombinant human apoA-I [22 (link)] in 500 μL HEPES buffer was added to the lipid suspension. The final volume of the mixture was 1.25 ml. PC ND were formulated by bath sonication of the PC/apoA-I mixture at 25 °C until the solution cleared (< 10 min). Tetramyristoyl CL ND (hereafter referred to as CL ND) were formulated in a similar manner, with the exception that bath sonication was performed at 48 °C. Tetralinoleoyl CL ND were formulated by bath sonication at 25 °C under an N₂ atmosphere.

See also the Key Resource Table below. Common chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO). Horse heart cytochrome c (without isotopic labeling) was purchased from Sigma-Aldrich (catalog number C7752). Dioleoyl phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC; C18:1), 1-Palmitoyl(D31)-2-oleoyl-sn-glycero-3-phosphocholine, (16:0D31-18:1 PC), tetramyristoyl-cardiolipin (1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt), TMCL), monounsaturated tetraoleoyl cardiolipin (1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt), TOCL; C18:1), and bovine heart cardiolipin (BHCL; natural mix with mostly C18:2) were obtained from Avanti Polar Lipids (Alabaster, Alabama). Tetralinoleoyl-cardiolipin (1',3'-bis[1,2-dilinoleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt) TLCL (C18:2) was obtained as a custom synthesis from Avanti Polar Lipids. Control samples of oxygenated TLCLs (containing 1-4 oxygen) were biosynthesized from TLCL in the reaction catalyzed by soybean lipoxidase (LOX, Sigma-Aldrich) in 50 mM HEPES buffer containing 100 μM DTPA (for transition metals chelation) and saturated with oxygen (before addition of LOX) at pH 7.4. Oxygenated TLCL molecular species were purified by preparative reverse phase HPLC.

1,2-Dioleoyl-sn-glycero-3-phosphocholine (PC) and tetralinoleoylcardiolipin (CL) were obtained from Avanti Polar Lipids. Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) was obtained from InvivoGen. Liposomes were prepared as previously described (4 (link)) and added at 300 μM to lysates unless otherwise stated. Unless specifically stated, liposomes used were at a 50:50 mol% PC:CL ratio.