Ricin removal

Ricin, an extremely toxic molecule isolated from the castor bean, inactivates eukaryotic 28S tibosomal RNA by providing the N-glycolytic cleavage or removal of a single adenine. 

Remove excess crosslinker from the activated ricin by gel filtration using a desalting resin. 

The solution is filtered and the ricin is washed on the filter with sodium sulfate solution to remove additional non-toxic nitrogen. 

The ricin and abnn A chains used to block high affinity binding sites on the column should be alkylated to remove the free sulfhydryl group that could interfere with conjugate purification... 

The principal use of the plant is lor the oil contained in the seeds, This oil is pressed out without healing the seeds. The particular properties make this oil valuable for specialized uses, such as low temperature lubrication. It is an important constituent of hydraulic brake fluid and other fluids where the degree of compressibility is important- Castor oil also finds medical uses, as an ingredient of special soaps, and in the preparation of some lexiile dyes. Ricin, an alkaloid present in castor oil. also has been used in insecticides. Prior lo the preparation of refined castor oil for medical purposes, ricin must be removed. 

Dissolve deglycosylated ricin A chain (dgA) in 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.5, at a concentration of 10 mg/ml. The buffer should be degassed under vacuum and nitrogen bubbled through it to remove oxygen. Prepare half the amount of A-chain solution as the amount of antibody prepared... 

Chain A loses its toxicity when the B chain is removed. In ricin immunotoxins, the MAb replaces the B chain function and takes the responsibility for both the target cell specificity and cell entry of the A chain Ricin immunotoxins using the anti-CD5 antibody (a marker on T cells and some B cells) have been investigated in T and B cell lymphomas ricin immunotoxins using the anti-CD 19 antibody (a B lymphocyte marker) have been investigated in non-Hodgkin s lymphoma. 

The toxic ricin is a small protein molecule consisting of two parts, chains A and B. The B chain is similar to proteins called lectins which recognize and bind to the membranes surrounding the cells in our bodies. The B chain attaches the ricin to the cell membrane which then folds inwards so that the ricin molecule is taken inside the cell inside a bag called a vacuole. There is only one bond between the A and B chains and this now breaks. The B chain then makes a hole in the vacuole through which the A chain passes into the cell. Here it heads straight for structures called ribosomes, where proteins, many of which are vital for the functioning of our bodies, are made. The A chain then selectively removes a specific molecule (the base adenine) from the RNA in the ribosomes. RNA contains the information required to make proteins, and removal of part of the information blocks the synthesis of proteins. The cell therefore dies. One molecule of ricin may be sufficient to kill one cell. This makes it the most potent toxin known. 

Abrin exerts its toxic action in the same way as ricin. The abrin B-chain avidly binds to a variety of cell types, in particular reticuloendothehal cells which bear the appropriate mannose receptors. The abrin B-chain also binds to the galactosyl-terminated receptors on the cell membrane to allow the entry of the abrin A-chain. Once internalized, the abrin A-chain is transported from the cell membrane to the ribosomes, where it catalytically inactivates the 60S ribosomal subunit by removing adenine from positions 4 and 324 of 28S rRNA, thereby inhibiting protein synthesis and causing cell death (Stripe and Barbieri, 1986). 

FIGURE 25.3. The effects of ricin on hepatic nonsulfhydryl content. Concentration of nonsulfhydryl content in the liver was compared between control (untreated) mice and mice treated with 25 pg ricin/kg. Protein was removed by precipitation with 2% TCA final concentration. Values are the averages of 3-7 animals SD. p < 0.05 with respect to corresponding control group. 

Ricin A-chain (Inland Labs) at 3 mg/mL in PBS was reduced for 30 min with 30 mM DTT at 30°C. Excess DTT was removed by Sephadex G-25 gel filtration in 5 mM sodium acetate buffer, pH 4.7, containing 50 mM NaCl and 0.5 mM EDTA. The thiol content was assessed by Ellman s assay (Ellman, 1959). MIANS (Molecular Probes) was added at a ratio of 0.9 mole MIANS 1.0 mole thiol. The reaction mixture was incubated at ambient temperature for 15 min after the pH was raised to 7.0 using 1 M Tris.HCl buffer, pH 7.4. The MIANS was quenched by addition of 1 mole equivalent of freshly prepared cysteine.HCl/mole MIANS and incubation for an additional 15 min. Any remaining thiol groups were alkylated by adding a 5-fold molar excess of iodoaceteimide over total thiol and after an additional 30 min, the protein (MIANS-ricin A-chain) was dialyzed against 0.1 M Tris.HCl buffer, pH 8.5. 

Hazard Contains ricin, which must be removed before internal use. 

Shiga toxin produced by Shigella dysenteriae has similar structural features. The toxin binds to a glycolipid (Gb3), undergoes endocytosis, and the enzymatie Ai fragment, which is a specific N-glycosidase, removes adenine from one particular adenosine residue in the 28S RNA of the 60S ribosomal subunit. Removal of the adenine inactivates the 60S ribosome, blocking protein synthesis. Ricin, abrin, and a number of related plant proteins inhibit eukaryotic protein synthesis in a similar manner (Chapter 25). 

Many clinically important antibiotics function by inhibiting protein synthesis. All steps of protein synthesis are susceptible to inhibition by one antibiotic or another. Diphtheria toxin inhibits protein synthesis by covalently modifying an elongation factor, thereby preventing elongation. Ricin, a toxin from castor beans, inhibits elongation by removing a crucial adenine from rRNA. 

Castor oil seeds contain a toxic glycoprotein called Ricin which remains in the pressed seed cake but which can be removed with water. 

The extract is then chromatographed on an acid-treated Sepharose 4B (2 wk at 37°C with 1 M propionic acid) column equilibrated with 50 mM borate buffer with 50 mM NaCl (borate-saline), pH 8.0. This binds to both RT and ricin agglutinin (RCA1) and removes all other seed proteins. 

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