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 tra nslation assay. The presence of total yeast RNA reduced the covalent a ttachment of Procion blue MX-R to ricin A chain. Quantitatively modifi ed ricin A chain contained 2 mol Procion blue MX-R/mol 29-kDa subunit. Tryptic digestion and resolution of the peptides by reverse-phase hig h-performance liquid chromatography yields a blue peptide correspondin g to Gln5-Arg26 of ricin A chain. Thus, a Likely dye-binding site on r ecombinant ricin A was identified. This region is removed from the act ive-site deft of recombinant ricin A but may be involved in its substr ate binding.