Solubilization of low-density lipoprotein with sodium deoxycholate and recombination of apoprotein B with dimyristoylphosphatidylcholine.
Apoprotein B (apoB) of human plasma low-density lipoprotein (LDL) (d 1.025-1.050 g/mL) has been solubilized with solid sodium deoxycholate (NaDC) above its critical micellar concentration. ApoB is isolated by gel-filtration chromatography as a mixed micellar complex of protein and detergent in high yield in a lipid-free form. A soluble apoB-dimyristoylphosphatidylcholine (DMPC) complex has been prepared by incubation of aqueous solutions of apoB-NaDC and DMPC-NaDC (2/1 w/w) at room temperature with detergent removal by extensive dialysis. A combination of gel chromatographic and density gradient fractionation of DMPC-apoB incubation mixtures demonstrates that a reasonably well-defined complex of DMPC and apoB is formed with a 4:1 w/w lipid:protein ratio. Negative-stain electron microscopy shows these particles to be single-bilayer phospholipid vesicles with a diameter of 210 +/- 20 A into which the apoB is incorporated. Circular dichroic spectra of NaDC-solubilized apoB show apoB to have similar conformation to that seen in the native LDL particle. However, apoB that has been complexed with DMPC exhibits more alpha-helix. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band (apparent Mr 366000) for apoB after solubilization, purification, and interaction with phospholipid. The behavior of apoB during its reassociation with phospholipid and the structural features of the DMPC-apoB particle are similar to those observed in the interaction of solubilized membrane proteins with lipid rather than that of other apo-lipoproteins.
D001053:Apolipoproteins; D001055:Apolipoproteins B; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D003840:Deoxycholic Acid; D004134:Dimyristoylphosphatidylcholine; D008077:Lipoproteins, LDL / Q000032:analysis; D008854:Microscopy, Electron; D008970:Molecular Weight; D010713:Phosphatidylcholines; D011485:Protein Binding; D012995:Solubility
D001053:Apolipoproteins; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D010713:Phosphatidylcholines; D003840:Deoxycholic Acid; D004134:Dimyristoylphosphatidylcholine