Biochemistry of the amatoxins: preparation and characterization of a stably iodinated alpha-amanitin.

Iodination of alpha-amanitin at the 7-position in the 6-hydroxy-2-sulfoxytryptophan moiety is effected with 1 equiv of iodine monochloride in methanol. The isolated product shows a lambdamax in methanol at 301 nm, compared with 305 nm for the parent alpha-amanitin; in methanolic 0.01 M NaOH the lambdamax are 330 and 332 nm for the product and parent, respectively. Spectrophotometric titration of the phenolic hydroxyl shows a decrease in pKa from 9.72 (alpha-amanitin) to 7.94 (7 iodo-alpha-amanitin). Appropriate spectrophotometric examination therefore distinguishes between parent and product. Proton magnetic resonance shows two aromatic protons (v4H = 7.57; V5H = 6.90 ppm; j4,5 = 9) in the 7-iodo-alpha-amanitin and three aromatic protons (v4H = 7.64; V5H = 6.78; V7H = 6.94 ppm; j4,5 = 9; J5,7 = 2) in alpha amanitin thus establishing the extent and position of iodine substitution. The 7-iodo-alpha-amanitin effectively inhibits RNA polymerase activity with half-maximal inhibition at 2 X 10(-9) M and 10(-4) M for the sea urchin RNA polymerases II and III, respectively. Addition of [125I]-7-iodo-alpha-amanitin (200 Ci/mmol) to crude extracts from sea urchin blastula, MOPC 315 plasmacytoma, and adult Oregon R Drosophila melanogaster followed by resolution on DEAE-Sephadex demonstrates that the radioactive ligand binds stably and specifically with RNA polymerase II in each of these extracts.