MURINE HISTOCOMPATIBILITY-2 (H-2) ALLOANTIGENS . PURIFICATION AND SOME CHEMICAL PROPERTIES OF SOLUBLE PRODUCTS FROM H-2B AND H-2D GENOTYPES RELEASED BY PAPAIN DIGESTION OF MEMBRANE FRACTIONS

Membrane-located murine H-2 alloantigens were solubilized by limited papain digestion of crude membrane fractions of mouse spleen cells of H-2b and H-2d genotypes. The major active fragments of the papain digest were purified by the following procedures: ammonium sulfate fractional precipitation, column chromatography on Sephadex G-150, CM-Sephadex, and DEAE-Sephadex, and polyacrylamide disc gel electrophoresis. The disc gel eluates were essentially homogeneous when reelectrophoresed on polyacrylamide at pH 4.3 or in sodium dodecyl sulfate. The final products from each mouse genotype were 300 to 700 times more active than the starting material in the quantitative inhibition assay. Chemical analyses showed that the active alloantigen fragments were glycoproteins containing about 90% protein. The electrophoretically pure H-2b material contained ca. 5% neutral carbohydrate, 4.5% glucosamine, and 1.2% sialic acid. The identical fractions of H-2d genotype contained ca. 4% neutral carbohydrate, 3% glucosamine, and 1% sialic acid. There was no phosphate nor detectable lipid. Amino acid analyses of these same fractions showed almost identical patterns except for a higher arginine and glutamic acid content for the H-2d alloantigen. An estimate of the molecular weight of these major alloantigen-bearing fragments was of the order of 65,000-75,000 as judged by Sephadex column chromatography and by sucrose gradient centrifugation. © 1969, American Chemical Society. All rights reserved.