Effective Toxins

Q: Why do your directions for SSP-SAP (Cat. #IT-11) state that it is to be used within hours after dissolution? To my knowledge, both proteins and peptides are stable in clean solution.

A: In fact, the two components of SSP-SAP (Stable Substance P and Saporin) are quite stable. However, we have found that many things happen in laboratories and some of them can impact stability. Probably the most severe is the loss of sterility. In that case, over time at room temperature or at 4°C, bacteria can grow on this rather excellent “medium.” This would cause inactivation. Because many laboratories, due to molecular biology work, have high levels of resident bacteria, we prefer to emphasize playing it safe.

Even if saporin is a stable protein, it is a protein and can suffer denaturation. This occurs more rapidly at room temperature than at 4°C, and hardly at all in the frozen state (really, it is stable for years when stored at -80°C). The maintenance of precise activity is of extreme importance to our customers who use these materials in vivo (their assays are very sensitive), and so we choose to advise the most conservative course.

Q: I understand that theoretically only one molecule of Saporin taken up by a cell is enough to induce cell death. I have been looking for literature on this topic but have not come across anything.

A: Definitely theoretical. The only article that we know of that states anything close to that is: Yamaizumi et al (1978) One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15(1): 245-250. As you can see, this article speaks to the enzymatic chain of diphtheria toxin, which has a slightly different mechanism of action for shutting down protein synthesis, but otherwise is similar to saporin. In fact, we test all sorts of toxins against cells in controlled conditions, and we have only one candidate that is in this range; all the rest are orders of magnitude away. It takes more than thousands per cell. Another question would be: how many actually get in?

In another FAQ, you addressed the question of one molecule of saporin killing a cell. Your response overlooked the data on ricin, abrin and modeccin (Eiklid, Olsnes and Pihl, Exp Cell Res, 126:321-326, 1980). In that paper, they showed that these RIP toxins applied to cells in culture produce all-or-none lethality. They used radioactive amino acid uptake and incorporation (as memory serves) and found only two types of cells, those with absolutely no uptake of label or those that were entirely normal – nothing in between. Also, if the data on ricin-induced apoptosis is correct (numerous authors), and I believe it is, then at low doses, the cells die from triggering apoptosis which seem possible with a single molecule of RIP free in the cytoplasm. To further complete your answer, someone (I haven’t found the article yet) showed that it took, on average, about 10,000 molecules of ricin/cell to kill cells in culture. This gives a hint at the efficiency of internalization and translocation in that cell type. I am not aware anyone else has looked at these issues with saporin conjugates.

A: Overlooking literature is actually a favorite sport of mine, but in this case I would respectfully point out conflicting information. There is a study of something that is quite between an all-or-none phenomenon: Barbieri et al. FEBS Lett, 2003 Mar 13;538(1-3):178-82. These authors document that ribosome-inactivating proteins have transforming activity on the classic FDA assay cell line: NIH3T3 cells. This would be a non-toxic activity that one presumes is due to internalization, and is somewhat on the none side of all or none, but hey, it’s an activity nonetheless. 

See: Targeted Toxins

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