RESUMO
A method for the analytical isoelectric focusing of Nonidet-P40-delipidated apolipoprotein B of human plasma low-density lipoproteins has been developed. Isoelectric focusing was performed in the presence of the zwitterionic nondenaturing detergent Chaps, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, and the nonionic surfactant Nonidet-P40, polyoxyethyleneglycol p-t-octylphenol with a mean of 9.0 ethylene oxide units per molecule. Low-density lipoprotein (LDL) apolipoprotein B (apo-B) entered 3.75% polyacrylamide gels without precipitation at the sites of sample application, permitting apoprotein recoveries of greater than 90% in the migrating bands. LDL apo-B exhibited 10 distinguishable bands with apparent isoelectric points of 7.34 (band 1), 7.27 (band 2), 7.16 (band 3), 7.02 (band 4), 6.88 (band 5), 6.70 (band 6), 6.61 (band 7), 6.48 (band 8), 6.40 (band 9), and 6.24 (band 10), respectively. Bands 3 and 4, 6 and 7, as well as 8 and 9 could be identified as major double bands. When the focused apo-B was run in a second dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same relative molecular weight of B-100 was obtained for all focused bands. After electrotransfer to nitrocellulose paper, all bands reacted with polyclonal anti-human LDL antibody. Furthermore, the detergent-solubilized apo-B retained the immunological properties of native low-density lipoproteins when tested by double immunodiffusion against polyvalent anti-human LDL sera.
