Affinity labeling of recombinant ricin A chain with Procion blue MX-R.

Recombinant ricin A chain was irreversibly modified by Procion blue MX-R, a dichlorotriazinyl analogue of Cibacron blue F3G-A, at pH 7.5 and 4 degrees C in 90 h with over 95% loss of activity in an in vitro translation assay. The presence of total yeast RNA reduced the covalent attachment of Procion blue MX-R to ricin A chain. Quantitatively modified ricin A chain contained 2 mol Procion blue MX-R/mol 29-kDa subunit. Tryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a blue peptide corresponding to Gln5-Arg26 of ricin A chain. Thus, a likely dye-binding site on recombinant ricin A was identified. This region is removed from the active-site cleft of recombinant ricin A but may be involved in its substrate binding.