Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether.
A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.
D000818:Animals; D001055:Apolipoproteins B / Q000097:blood*; D000440:Butanols; D004987:Ethers; D005260:Female; D006801:Humans; D006937:Hypercholesterolemia / Q000097:blood; D008055:Lipids / Q000097:blood; D008077:Lipoproteins, LDL / Q000097:blood; D008079:Lipoproteins, VLDL / Q000097:blood; D011817:Rabbits; D012997:Solvents; D013997:Time Factors
D001055:Apolipoproteins B; D000440:Butanols; D004987:Ethers; D008055:Lipids; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D012997:Solvents; C011779:diisopropyl ether