Purification and chemical characterization of papain-solubilized histocompatibility-2 antigens from mouse liver.
A large scale purification of histocompatibility-2 (H-2) antigens from mouse liver is described. The antigens were solubilized by a limited papain digestion of a crude preparation of liver membranes (strain A/J) and purified by ion exchange chromatography, gel filtration, affinity chromatography, and isoelectric focusing. The overall degree of purification of H-2Kk was 1,300-fold and that of H-2Dd was 1,500-fold; approximately 8 mg of purified H-2a antigens were obtained from 1 kg of liver. The purification was followed by a sensitive radioimmunoassay in which H-2a-containing fractions were used to inhibit the binding of 125I-labeled H-2a to appropriate antisera. H-2Dd and H-2Kk co-purified through all the steps but the concentration of H-2Kk was 2- to 3-fold higher than that of H-2Dd in the liver homogenate as well as in the purified H-2 preparation. beta 2-microglobulin was initially present in a 3- to 10-fold excess over H-2 in the liver homogenate, but the purified H-2 preparation contained approximately 2 mol of alloantigenic heavy chain/mol of beta 2-microglobulin. Isoelectric focusing and disc-gel electrophoresis showed a charge heterogeneity of H-2, with a mean isoelectric point of pH 4.9. Electrophoresis on sodium dodecyl sulfate gels showed one band. Denaturing conditions were required to remove beta 2-microglobulin and small amounts of impurities from H-2. The amino acid sequence of the first 27 residues of the isolated heavy chains was determined.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000276:immunology; D005260:Female; D006649:Histocompatibility Antigens / Q000302:isolation & purification; D008099:Liver / Q000276:immunology*; D008297:Male; D051379:Mice; D010206:Papain; D011233:Precipitin Tests; D011863:Radioimmunoassay; D013154:Spleen / Q000276:immunology
D000596:Amino Acids; D006649:Histocompatibility Antigens; D010206:Papain