Characterization of cholesterol-binding globulin by modified zone electrophoresis and O-(diethylaminoethyl) cellulose chromatography.

Normal human serum has been shown' to exhibit cholesterol-binding properties upon immunoelectrophoresis on agar gel. The evidence clearly supports the concept of a specific type of binding of cholesterol by alpha-beta globulin. Accordingly, the name "transcholesterin" was proposed for the reactive component. In order to evaluate the mechanism of in vitro interaction of cholesterol with transcholesterin, purified components are desirable. Sober and co-workers2 introduced the use of O-(diethylaminoethyl) (DEAE)-cellulose chromatography for the fractionation and identification of serum proteins. However, DEAEcellulose chromatography of serum proteins failed to resolve them into components homogeneous as judged by the criteria of paper electrophoresis.3 Therefore, it has been necessary to combine electrophoretic separation with column chromatography in the fractionation of serum proteins. The present communication describes the initial findings in an attempt to isolate and characterize transcholesterin by the combination of zone electrophoresis and the modified chromatographic procedure and also by chemical analysis and immunodiffusion studies. Materials and Methods.-Human sera: Pooled and individual fresh sera were obtained from healthy volunteers. The details of the procedure have been described elsewhere' for the following items: (a) agar-gel immunoelectrophoresis, (b) cholesterol solution in Teepol "L" (an alkyl sulfate detergent), and (c) transcholesterin assay involving immunoelectrophoresis. Lipoprotein and lipoprotein-free serum (LFF) preparations: Lipoprotein fractions at densities 1.063, 1.10, and 1.21 were obtained in the Spinco preparative ultracentrifuge according to the method of Havel et al.4 When total lipoproteins and LFF were desired, the preparative ultracentrifugation was carried out according to the procedure of Lewis et al.' at solvent density 1.21. All the fractions were adjusted to original serum volume with isotonic saline. Tests such as paper electrophoresis6 and starch-gel electrophoresis' 8 were performed to determine the purity of the lipoproteins and LFF samples. Further characterizations of these samples were by the Ouchterlony double-diffusion technique.9 Antisera against human serum lipoproteins: Antisera against human alpha-1, alpha-2, and beta lipoproteins produced separately in rabbits were obtained from Behringwerke, A. G., Marburg-Lahn, West Germany. Total cholesterol determination: A modified Liebermann-Burchard procedures using the Bausch and Lomb spectronic 505 spectrophotometer to read the density of the color at wavelength 640 mys was adopted for cholesterol quantitation. The reproducibility of the analytical procedure was good with a standard deviation of +5%. Vertical zone electrophoresis: Buffer preparation was described elsewhere (Fig. 5),1 except that the pH was adjusted to 8.6 instead of 8.1. Potato starch powder (Baker analyzed reagent) suspended in the buffer was slurried into a glass column (2.5 cm in diameter X 60 cm in length) stoppered at the bottom with a rubber stopper and center-