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Isolation of Immune Globulins from Milk and Colostrum with Rivanol¹

Minnesota Agricultural Experiment Station, St. Paul

ABSTRACT

Rivanol (2-ethoxy-6,9-diamino-acridine lactate) has been shown to precipitate quantitatively from blood serum the albumin and most of the - and ß-globulins (1, 2, 4). When applied to plasma, rivanol precipitates fibrinogen as well as albumin and - and ß-globulins. The -globulins remain in solution (1, 2, 4). A simple technique is thus afforded for isolation and analysis of the -globulins. It seemed worth while to determine whether rivanol can be employed in a similar fashion to fractionate the proteins of milk.

The technique described for blood serum or plasma was applied to separated colostrum, to acid whey from skimmilk, and to a crude lacto-globulin that had been isolated from acid whey by precipitation with 43% saturated ammonium sulfate. Specifically, the method consisted of adding 3.5 volumes of 0.4% aqueous rivanol² solution to one volume of solution to be fractionated, adjusting the pH to 8.5, centrifuging or filtering off the precipitated protein, and removing rivanol from the supernatant by adsorption on activated charcoal (2). The protein in the supernatant was analyzed electrophoretically in the Klett apparatus, after suitable concentration by pervaporation and dialysis against veronal buffer of pH 8.6, ionic strength = 0.1.

¹ Paper No. 4071, Scientific Journal Series, Minnesota Agricultural Experiment Station. These studies were made possible in part by a grant from the Animal Disease and Parasite Research Division, Agricultural Research Service, USDA.

³ Department of Agricultural Biochemistry.

⁴ College of Veterinary Medicine.