The α2-Macroglobulin of Human Plasma: I. ISOLATION AND COMPOSITION

Abstract

Abstract The α2-macroglobulin of human plasma was isolated from Cohn Fraction III-0 by ultracentrifugation at a density of 1.065, followed by ammonium sulfate fractionation between 34 and 50% saturation and gel filtration on Sephadex G-200. Precipitation with zinc ions was an additional step for some preparations. The average yield of the purified α2-macroglobulin was 39% of that present in Fraction III-0. The purity of the final preparation was shown to be approximately 98% on the basis of immunochemical determination. The only impurity detected was the γ1a-immunoglobulin, which represented 1 to 3% of the total protein. On paper electrophoresis only a single component was observed. Starch gel electrophoresis revealed a major band corresponding to the S α2 of Smithies, and, in addition, a faint, slower band was observed. In the ultracentrifuge 94% of the protein sedimented with an s020,w of 20.6, and the remaining 6% of the protein was present in a peak with an s020,w of 30.8. The presence in these preparations of a component sedimenting more rapidly in the ultracentrifuge and migrating more slowly on starch gel electrophoresis suggests the occurrence of an additional physical form of the α2-macroglobulin, which may be an aggregate. The carbohydrate composition of the purified protein, in percentage of the dry weight of the molecule, was galactose, 1.36; mannose, 2.07; N-acetylglucosamine, 3.94; N-acetylneuraminic acid, 1.80; and fucose, 0.26. From acetyl analyses on the native and sialic acid-free protein, it was found that all of the hexosamine occurred in the N-acetyl form and that there was approximately one acetyl group for each sialic acid residue. Amino acid and amide analyses indicated that all of the amino acids common to globular proteins were present in the α2-macroglobulin. The combined monosaccharide and amino acid residue weights accounted for 96.6% of the weight of the protein. In 1 molecule of the α2-macroglobulin (mol wt 820,000), there are approximately 363 monosaccharide residues and 6,463 amino acid residues.