Purification and activity study of the A- and B-chains of cinnamomin, a type II ribosome-inactivating protein.

The strong hydrophobic interaction between the A- and B-chains of cinnamomin, a type II ribosome-inactivating protein, makes it difficult to separate A- and B-chains after the disulfide bond is broken. We failed to separate the A-chain from B-chain of cinnamomin using methods under usual conditions. A convenient method for purification of the A- and B-chains of cinnamomin on a large scale has been developed. We chose urea to weaken the non-covalent interaction between the A- and B-chains. In the presence of 4M urea, the A- and B-chains of the reduced cinnamomin are separated effectively by DEAE-cellulose chromatography. The purified A-chain still displays the RNA N-glycosidase activity and the B-chain loses the lectin activity. After refolding in vitro in the presence of lactose, the B-chain is renatured and the active B-chain with lectin activity can be further purified by Sepharose 4B affinity chromatography. From 80 mg of cinnamomin, 10 mg of A-chain (25%) and 38 mg of the B-chain (95%) were obtained. In addition, the intrinsic fluorescence spectra of the A- and B-chains were employed to study the structural changes in the active and the non-active forms of cinnamomin A- and B-chains.