Synthesis of Cardiolipin Derivatives with Protection of the Free Hydroxyl: Its Application to the Study of Cardiolipin Stimulation of Cytochrome c Oxidase

Cardiolipin derivatives retaining the free hydroxyl on the polar head group were synthesized. With the use of a tetrahydropyranyl ether to protect this hydroxyl, fatty acyl substitutions were made at both of the 2-positions of cardiolipin (CL). The disubstituted derivatives were obtained in high yields. The stimulation of delipidated cytochrome c oxidase activity shows a hyperbolic dependence on the concentration of these CL derivatives. Both activation parameters, the apparent dissociation constant (&K_d,app) and the maximum change in molecular activity (ΔAct_max), depend on the chain length of the tails, with less dependence on the degree of saturation. Natural CL (92% C_18;2, 8% C_18:1) and CL disubstituted with oleic acid (47% C_18:2, 52% C_18;1) were equally effective at stimulating cytochrome c oxidase activity, with an apparent dissociation constant of approximately 1 μM when incubated in 0.3% Triton X-100 and assayed in lauryl maltoside. CL disubstituted with hexanoic acid, however, is a poorer activator, with an apparent dissociation constant of 6.8 μM and a ΔAct_maxthat is 50% of that achieved with natural CL. Dilysocardiolipin, with complete removal of two of the fatty acid tails, shows negligible stimulation of cytochrome c oxidase activity.