Abstract
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.
Highlights
Vitro and in vivo cytotoxicity of both chains
The toxin most widely used is ricin which is a disulfidebonded heterodimeric glycoprotein consisting of a B chain and an A chain [7,8]
Yields o f A and B Chain-Table I summarizes the isolation scheme described under “Materials and Methods,” the yields obtained at each step, and the concentrations required for 50%inhibition of protein synthesis (Em) of the material on Daudi cells in vitro
Summary
Yields o f A and B Chain-Table I summarizes the isolation scheme described under “Materials and Methods,” the yields obtained at each step, and the concentrations required for 50%inhibition of protein synthesis (Em) of the material on Daudi cells in vitro. It should be noted that for the A chain, the largest variation in yield among experiments occurred during and following elution from the Sepharose 4B column. During this step andoccasionally during the elution from the Portions of this paper (including “Materials and Methods” and Footnote 4) are presentedinminiprint at the end of this paper. Miniprint is easiIy read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Full size photocopies are dso included in themicrofilm edition of the Journal thataviasilable from Waverly Press. The abbreviationsused inthe Miniprint are: HEPES, 442hydroxyethy1)-1-piperazineethanesulfonic acid; SPDP, succinimidyl 3-(2-pyridyldithio)propionate
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
