Purification of Sepharose-Unbinding Ricin from Castor Beans (Ricinus communis) by Hydroxyapatite Chromatography

Sepharose-unbinding ricin was one-step separated and purified from a crude extract of castor beans (Ricinus communis) by affinity chromatography on hydroxyapatite. This purification method does not require the time-consuming and complicated steps, such as gel filtration and ion-exchange chromatography, that have been essential in the separation of Sepharose-unbinding toxins. Using this method, approximately 180 mg of ricin was obtained from 100 g of castor beans using a bed volume of 80 ml on a hydroxyapatite column. Weak affinity of the ricin on Sepharose was confirmed and compared with Sepharose-binding ricin (ricin D), using radioiodinated ricins. The molecular mass of the ricin was estimated to be 62 kDa by 10% SDS–PAGE under nonreducing conditions. Under reducing conditions, the purified ricin appeared to be two subunits, corresponding to the molecular masses of 30 and 32 kDa. The pIvalue was determined to be approximately 8.9 for the ricin. Uptake of the ricin by HeLa cells was measured as almost half of ricin D uptake. Similar results were observed on CEM cells as well.In vitrocytotoxicity of ricins on different cell lines was measured by the MTT method. When compared with ricin D, the purified ricin showed approximately 10-fold less cytotoxicity to HUT78 or K562 cells and 30-fold less toxicity to CEM cells. This lower cytotoxicity of the ricin may be due to its lower cell-binding properties as evidenced by its low affinity for the cell surfaces. From these results, the purified ricin was considered to be ricin E.