Studies on the Apoproteins of the Major Lipoprotein of the Yolk of Hen's Eggs. IV. Aggregation in Urea of Proteins of Intermediate and High Molecular Weight and the Isolation of Four Electrophoretically Distinct Proteins

Abstract

The gel-filtration chromatography of the total apoprotein mixture from hen''s egg yolk lipoprotein in acidic 6 M was re-examined using the gel-filtration materials Sephadex and cross-linked Sepharose. Electrophoresis was used in attempts at separating the constituents of intermediate and high MW. On Sephadex G100 at high protein concentrations (> 0.8% w/v) there was apparent separation of proteins in the intermediate MW range (about 60,000), but this was a chromatographic artefact. On Sepharose CL-6B columns 4 fractions were resolved, including a fraction containing proteins that emerged later than expected from their MW, i.e., a retarded fraction. From these fractions 4 electrophoretically distinct proteins referred to as apovitellenins III-VI were isolated by further chromatography. The same proteins were isolated by electrophoresis in detergent. These proteins account for most intermediate and high MW apoproteins. Their amino acid compositions are similar but with significant differences, and the electrophoretic patterns of their cyanogen bromide digests are different. The composition of the retarded fraction depended on the conditions under which the lipoprotein was isolated and on the apoprotein concentration during chromatography. Rapid isolation of lipoprotein and/or apoprotein concentrations favored a large retarded fraction rich in the low-MW protein, apovitellenin I, suggesting the possibility that the retarded fraction contained aggregates related to those originally in the yolk.