UBC Theses and Dissertations
Characterization of the very low density lipoproteins and apoproteins of egg yolk granules Kocal, James Thomas
The very low density lipoproteins (VLDL (MF)) from the granules of hen's egg yolk were isolated by a combination of preparative ultracentrifugation and agarose gel filtration. Ultracentrifugal, electrophoretic and chromatographic analyses were performed to characterize and assess the purity of the VLDL (MF) and apoVLDL. The Beckman Prep UV Scanner accessory to the ultracentrifuge was used in conjunction with an on-line data acquisition system for the determination of flotation/sedimentation coefficients. The software that was developed to permit rapid data analysis using this system is described. An agarose disc gel electrophoretic system was developed which, in combination with the use of sudan black B prestained lipoproteins, allowed for rapid electrophoretic analysis of large VLDL (MF) particles. Flotation velocity analysis of VLDL (MF) indicated a heterogeneous preparation with two major floating boundaries (Sf 75 and 207S). Alkylation of VLDL (MF) with iodoacetamide to prevent aggregation due to oxidation of sulfhydryl groups decreased the flotation rates of the macromolecules to Sf 41 and 108S. It is possible that the VLDL (MF) particles exist in a partially aggregated state in fresh egg yolk. Concanavalin A affinity chromatography of VLDL (MF) produced a retained and an unretained fraction. The retained fraction contained twice as much total carbohydrate as the unfractionated VLDL (MF), while the unretained VLDL (MF) contained half as much carbohydrate. All fractions were glycoproteins containing hexose, hexosamine and sialic acid sugar residues. VLDL (MF) was partially delipidated with n-heptane which preferentially extracted the neutral lipids and preserved the solubility of the phospholipid-protein residues. Two fractions were isolated by gel filtration and characterized by analytical ultracentrifugation and by their phospholipid/protein ratios. VLDL (MF) was delipidated using sodium deoxycholate (NaDOC) and ethanol-ether procedures. Removal of lipids by both methods produced aggregated apoprotein, but the NaDOC apoprotein remained soluble. That obtained from ethanol-ether was precipitated, but could be solubilized in sodium dodecylsulfate (SDS) or urea. Tetramethylurea (TMU) was used to delipidate VLDL (MF) and dissociate the apoprotein. Electrophoretic analysis indicated the presence of three apoprotein subunits, one of which was an aggregate of the other two. SDS electrophoresis of the proteins extracted from the TMU gels demonstrated that each band was heterogeneous, containing many polypeptides. ApoVLDL was dissociated into 22 polypeptides (MW ranging from 9,600-136,000 daltons) in the presence of urea, SDS and 2-mercapto-ethanol (BME). Fourteen of these bands were glycoprotein in nature, staining positively with Schiff reagent. In the absence of BME, only 18 bands were resolved, most of which contained higher molecular weight aggregates not present in the reduced samples. The apoVLDL was fractionated on 6% agarose columns containing 8M urea or 2mM SDS. Two apoprotein fractions were eluted from the SDS column, and three from the-urea column. The protein from the void volume of the urea column was an aggregate of the other two proteins. A correlation was found between the fractions eluted from the SDS column and the proteins extracted from the TMU gels. The molecular weights of the two apoproteins from the SDS column were analyzed by sedimentation equilibrium techniques. As plots of 1n c vs. r² were nonlinear, weight average molecular weights were calculated for each measured radial distance from the meniscus to the bottom of the cell. The high molecular weight component ranged from 33,000 to 166,000 daltons and the low molecular weight component ranged from 6,000 to 28,000. These values corresponded well with the molecular weight ranges of the polypeptides characterized by SDS gel electrophoresis.
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