Binding characteristics and thermal behaviour of cytochrome-C oxidase, inserted into phospholipid-coated, magnetic nanoparticles.
A strategy for the immobilization of cytochrome-c oxidase, used as a representative membrane-bound enzyme, into so-called magnetoliposomes has been developed. The latter structures consist of a phospholipid bilayer which covers nanometer-sized Fe3O4 colloids. Incorporation of the enzyme into the phospholipid envelope is facilitated by a short sonication step. Upon adsorption, the reaction characteristics of the lipid-depleted enzyme are drastically changed. With double-layered phosphatidylcholine (PC) magnetoliposomes the activity increases by a factor of approximately 5. After a first magnetic fractionation step, approximately 67% of the activity remains with the magnetoliposome retentate. Subsequent magnetophoresis cycles show that the adsorbed enzyme is firmly fixed into the phospholipid coat. Upon immobilization, the thermal behavior is also profoundly affected. The heating inactivation curves show two sigmoidal transition zones. Irrespective of the PC type used, a first inflection point is located near 39 degrees C, whereas a second one, which is located at higher temperatures, clearly depends on the acyl chain length (56 degrees C with dimyristoyl-PC and 60 degrees C for dioleoyl-PC and Ovothin-200). An identical behavior is observed with classical proteoliposomes with an equal phospholipid composition. By contrast, monolayer-coated dimyristoyl-PC magnetic structures are inferior with respect to both their reactivation potency and their ability to strongly affix cytochrome-c oxidase and to improve the thermal stability of the enzyme.
de Cuyper
M
M
Interdisciplinary Research Center, Katholieke Universiteit Leuven, Kortrijk, Belgium.
Joniau
M
M
D003102:Colloids; D004134:Dimyristoylphosphatidylcholine; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004800:Enzymes, Immobilized; D052203:Ferrosoferric Oxide; D006358:Hot Temperature; D007501:Iron; D008051:Lipid Bilayers; D008081:Liposomes; D010087:Oxides; D010743:Phospholipids
D003102:Colloids; D004800:Enzymes, Immobilized; D008051:Lipid Bilayers; D008081:Liposomes; D010087:Oxides; D010743:Phospholipids; D007501:Iron; D003576:Electron Transport Complex IV; D004134:Dimyristoylphosphatidylcholine; D052203:Ferrosoferric Oxide