Abstract

Fetuin was isolated by alcohol-metal ion fractionation. Although the homogeneity of the preparation has been demonstrated by many conventional techniques, microheterogeneity was clearly demonstrated by resolution into eight components on starch gel electrophoresis at pH 4.2. The microheterogeneity did not result from the method of preparation because normal fetal calf serum and fetuin prepared by ammonium sulfate fractionation had the same electrophoretic pattern. Furthermore, the identical bands, characteristic of the purified fetuin, were present in sera collected from five fetal calves. Treatment with neuraminidase reduced the number of bands from eight to three, indicating that sialic acid is primarily responsible for the microheterogeneity observed. Fetuin which was denatured in 8 m urea or reduced with 2-mercaptoethanol in 8 m urea failed to show definite electrophoretic bands; the importance of tertiary structure as a contributory factor to the microheterogeneity is suggested. Purified fetuin was resolved into several fractions by chromatography on DEAE-Sephadex; each fraction contained 2–3 of the bands found on electrophoresis and reveals that the fetuin variants arise from discrete structural differences. The fractions showed similar immunochemical properties, sedimentation constants, and absorption coefficients at 278 mμ. The sialic acid, hexose and hexosamine content increased significantly according to the order of elution. The increase in carbohydrate was accompanied by a small decrease in peptide content. Amino acid analysis revealed virtually no relative difference between the fractions; minor differences were demonstrated by peptide mapping. It was concluded that the microheterogeneity results primarily from differences in the number and arrangement of the sialic acid residues at the surface of the molecule. Conformational variations of the polypeptide portion of the fetuin molecule may also play a contributing role.

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