Purification of ricin A1, A2, and B chains and characterization of their toxicity.

This paper describes a protocol for the preparation of highly purified A (A1 and A2) and B chains of the plant toxin, ricin, and biochemical and biological characterization of these proteins. Intact ricin was bound to acid-treated Sepharose 4B and was split on the column into A and B chains with 2-mercaptoethanol. The A chains were eluted with borate buffer containing 2-mercaptoethanol. A1 and A2 were then partially separated by cation exchange chromatography and the contaminating B chain was removed by affinity chromatography on Sepharose-asialofetuin and Sepharose-monoclonal anti-B chain. The B chain was eluted from the Sepharose 4B column by treatment with galactose and was further purified by cation and anion exchange chromatography; contaminating A chains were removed by affinity chromatography on Sepharose-monoclonal anti-A chain. The purified A and B chains were active as determined by their ability to inhibit protein synthesis in a cell-free assay and their binding to asialofetuin, respectively. Furthermore, by polyacrylamide gel electrophoresis, toxicity in mice, and toxicity on several different cell types, both A and B chains were shown to be minimally cross-contaminated. Finally, it was shown that ammonium chloride significantly enhanced the nonspecific toxicity of B chains for cells in vitro. In contrast, ammonium chloride did not enhance either the nonspecific toxicity of A chains in vitro or the specific toxicity of A chain-containing immunotoxins prepared with the highly purified A1, A2 chains.