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.
Pu
Z
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Laboratory for Ribosome Research, Shanghai Institute of Biochemistry, Academia Sinica, China.
Xie
L
L
Wang
E
E
Liu
W Y
WY
D020418:Algal Proteins; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects; D006386:Hemagglutination Tests; D008099:Liver / Q000187:drug effects; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D054790:Ribosome Inactivating Proteins, Type 2; D012270:Ribosomes / Q000187:drug effects*; D013050:Spectrometry, Fluorescence
D020418:Algal Proteins; D010940:Plant Proteins; D011506:Proteins; D054790:Ribosome Inactivating Proteins, Type 2; C061930:cinnamomin, Phytophthora cinnamomi