| Ricin toxin is a plant toxin isolated from the seeds of Ricinus Communis. It is one of the most powerful plant toxins discovered. The protein toxin has a molecular weight of about 60000-65000, which consists of two chains linked by a disulfide bond. Ricin A chain (RTA) has enzymatical activity, whose molecular weight is about 31000. Ricin B chain is the binding subunit and has two specific binding sites, whose molecular weight is about 33000. The primary structure of ricin had been elucidated by Funatsu. RTA consists of 363 amimo acid residues, and has only two Lysine residues, which are very important to its activity. RTB has 259 amino acid residues, and can specifically recognize receptors on the cell membrane, which have a terminal galactose. RTB is responsible for the binding to the cell receptors, and the binding structure of RTB is related with Tyrosine and ?-NH2 of Lysine. The ricin enters into the cell through receptor-mediated endocytosis. RTA binds to protein L3 of ribosomal 60S subunit. It has specific N-glycosidase activity, and can catalytically cleave a specific adenine at the site of 4234 out of the 28S rRNA. This cleavage of adenine can lead to the disruption of ribosomal function, thereby, inhibits the protein synthesis, and then cause the death of cells. Ricin toxin has wide-range anti-tumor spectrum. For this reason, it attracts much interesting to many scientists. It can damage normal cells too.Because RTA is responsible for its enzymatical activity, if we attach RTA to specific monoclonal antibodies to synthesize conjugates, so called immunotoxins. Immunotoxins have been used for cancer-targeted therapy, and many of them now on clinical trials in USA.Our experiment is to isolate ricin toxin from seeds of Ricinus Communuis, and then to purify, identify and determine its bioactivity through the comparison of the different cytotoxicities to normal mice fibroblast cells (NIH 3T3) and colon cancer cells (colon205), We also use leukemia cells (K562) and large intestine cancer cells(SW480) to study its anti-cancer activity. I . The isolation and purification of Ricin toxinAccording to the method of Nicolson and Blaustein, Briefly, the seeds of Ricinus Communis was extracted with 0.01M, pH=7.2 phosphate -buffered saline (PBS). The solution was then centrifuged at 7000 rpm for 10 minutes. (NH4)zSO4 was added to the supernatant and reach the saturation of 40%. The solution was stored at 4癈 overnight, and then centrifuged at 7000 rpm for 10 minutes. The supernatant was added by (NH4)2SO4 to reach the saturation of 80%, and then centrifuged as the previous step. The pellet was dissolved in 0.01M, pH=7.2 PBS, and the solution was dialyzed against PBS. A Sepharose4B columnthis (2 X 25cm) was applied to separate the toxin. Firstly, the column was washed with 0.01M, pH=7.2 PBS to remove the unbound proteins. The bound proteins (ricinus agglutinin and ricin) were eluted with the gradient of lactose (0-0.5M). The proteins under the peak were collected, which contain ricin and ricinus agglutinin.7The molecular weight of ricinus agglutinin and ricin are 120000 and 60000-65000 respectively. We can further separate them by gel filtration by using the column of Sephadex-Gl00(2X 100 cm). II . The identification of Ricin toxinAccording to the literature, Ricin has a molecular weight of about 60000-65000, and has two subunits joinded by disulfide bridage. We use SDS-PAGE electrophoresis to estimate the molecular weight under the reduced condition. Two bands at the site around 31000 and 33000 were detected, under the non-reduced condition, only one band at the site around 65000 was visible, which indicates the molecular weight of the protein is about 65000. It consists of two different subunits. Ill. The activity of Ricin toxinSuccinate dehydrogenase is a very important enzyme in the citric acid cycle. It locates on the inner-membrane of mitochondria and is a sensitive indicator of the mitochondria function. Mitochondria provide energy for cells. The loss of its function m... |
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