摘要
植物白头翁（ａｍｅｎｏｎｅ）茎的抽提液经ＣＭ－ＳＦＦ柱和ＳｅｐｈａｃｒｙｌＳ－２００柱分离纯化，得到一种毒蛋白，用高效凝胶蛋白柱和反相高效液相色谱法结合光电二极管阵列检测器确认分离峰的纯度，在高效凝胶蛋白柱上制备了少量毒蛋白纯样，测定了蛋白分子量和氨基酸组成。
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℃. 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 1mol/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 conneted Protein 125 columns with mobile phase of 0 2mol/L phosphate buffer (pH 6 5), the eluate was monitored at 280nm on photodiode array detector. The protein presents typical the absorption spectrum of protein in ultraviolet region with the strong absorption at 280nm and the weak absorption at 260nm. 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 000D. The composition of amino acids was determined with OPA post column derivatization/fluorescence detection.
出处
《色谱》
CAS
CSCD
北大核心
1998年第5期414-416,共3页
Chinese Journal of Chromatography
关键词
白头翁
凝胶柱
毒蛋白
分析
核糖体失活蛋白
amenone, gel column, high performance protein column, amino acid
