Protein concentrations:Can it drastically change its effect?

[face]

I'm doing some experiments where im testing the toxic effects of a certain chemical on some tissue. my results however just seem very illogical but im sure they are accurate.

basically ive been doing toxic assays using a protein at a range of concentrations. in each assay i have a control which contains no protein and should reflect the normal state of the cells.

I'll use the free radical assay as an example. the smaller concentrations of the protein added to the cells seem to lower the generation of these superoxides below the control value. this suggests that its actually benefitial to the cells by lowering the toxic supreoxides, but the higher concentrations greatly increase the levels way above the control, and hence the toxicity.

i just cant figure out why different concentrations of the same protein can give two completely different outcomes. is there some kind of protein theory that im missing? as far as im aware this doesnt make sense. i would really appreciate any insight

thanks

[Cat]

Without knowing what your protein is there is no way of forming a theory. However, if your protein happens to be, let’s say, a transcription factor, then your results are highly probable. Also, your scenario reminds me of vitamin toxicity – beneficial in low doses but harmful in high.

[face]

Hi Cat thanks for replying

why do you think my results are highly probable if it is a TF? there was one theory that seems plausable, in that the protein activates cell repair mechanisms that counteract the effects. at low concentrations this is would be very noticable and would appear to increase the cells health, but high concentrations may signficantly overcome this.

i like that theory, and i may leave it at that. but i also have results, that show the opposite. on a test measuring the cell cycle activity ( S phase) higher concentrations seem to sigficantly increase activity but low concentrations inhibit it the activity - well below the control. It's these particular results that have me the most stomped. any ideas/theories?

[MrMistery]

did you do some determinations at various intervals of time after treatment? Here is an idea: inoculate your protein on your culture, then after 6 hours take the flasks out of the incubator and freeze the cells. After 12 hours take the other flasks out and freeze them. Same after 24 and 48. Then unfreeze the cells and do MTT. You might discover that the superoxid levels rise at first and then decrease, which would make sense, as sometimes the cell will synthesize a large amount of scavenger enzymes that will make the superoxide level even lower than normal. If that is indeed the case, you can check for the level of oxidative stress combating enzymes like superoxid dismutase, catalase etc using a spectophotometer.

[Cat]

Well let me research this particular protein and I’ll get back to you on that.

As far as what I said about TF activity (hypothetically):

If you introduce a little extra TF that causes activation if genes encoding for products responsible for removal of ROSs - it would be beneficial.

If you introduce too much, however, it would be very likely to trigger cellular response to negate its own activity that is also very likely to result in overcompensation and suppression of target genes, thus, leading to increase of ROSs and cell damage.

[face]

thanks for the input guys.

[Cat]

I provided an answer for your question on my web. Please, visit:

http://science-ed.fullsubject.com/1-on- ... t18.htm#45

[MichaelXY]

Um, I had a comment, but on second thought, never mind.