Separation of ricin A- and B-chains after dithiothreitol reduction.
After complete cleavage of ricin interchain disulfide bridge by 0.05 M dithiothreitol in nondenaturing conditions at 37 degrees C during 1 h 30 min, A- and B-chains were separated on a lactosaminyl-aminoethyl Biogel P-150 column at 4 degrees C, in the presence of 0.01 M dithiothreitol and 0.5 M MgCl2. A- and B-chains have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunology. Their specific activities have been tested by protein synthesis inhibition in a cell-free assay (rabbit reticulocyte lysate) and on whole cells (Zajdela hepatoma cells) and by hemagglutination. From these tests, the apparent cross contamination of the chains was about 0.1%.
Emmanuel
F
F
CNRS ER 64, UER Biomédicale des Saints-Pères, Paris, France.
Turpin
E
E
Alfsen
A
A
Frénoy
J P
JP
D000818:Animals; D002846:Chromatography, Affinity; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D006384:Hemagglutination / Q000187:drug effects; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D008970:Molecular Weight; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D012276:Ricin / Q000302:isolation & purification* / Q000494:pharmacology
D012276:Ricin; D004229:Dithiothreitol