[Isolation, purification and determination of the constituents of a toxic protein from plant amenone].
A toxic protein constituent named AME from the stems of plant amenone, have been isolated and purified. Through CM-SFF column chromatography and gel filtration on Sephacryl S-200 column with phosphate saline buffer as mobile phase. All of the operations were performed at 4 degrees C. The pulverized plant amenone material was soaked in phosphate saline buffer, homogenized, left standing overnight and then squeezed through coarse cloth by wringing. The supernatant was applied on the S-SFF column. Then, the column was eluted with the phosphate buffer containing 1 mol/L NaCl. The eluate was collected and dialyzed against water and phosphate buffer. The chromatography of the crude toxin dialyzed was carried out on the CM-SFF column with gradient elution of phosphate buffer containing NaCl. The fourth peak was collected and then applied on a gel filtration Sephacryl S-200 column using neutral phosphate buffer as mobile phase. The protein was further separated on two connected Protein 125 columns with mobile phase of 0.2 mol/L phosphate buffer (pH 6.5), the eluate was monitored at 280 nm on photodiode array detector. The protein presents typical the absorption spectrum of protein in ultraviolet region with the strong absorption at 280 nm and the weak absorption at 260 nm. The purity of peak of the protein was judged from the spectrum. The molecular weight of AME measured by two connected protein columns was approximately 35,000 D. The composition of amino acids was determined with OPA post-column derivatization/fluorescence detection.