α₁-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of α₁-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14–0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36–0.72 pH unit. After treatment with 0.025m-H₂SO₄ at 80°C for 1h, which removed all the sialic acid, the increase was 0.40–0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.