I want to show the effect of some detergent on enzymatic activity in immobilized form and free form. The data shows that I get different results. Tritton has bad effect on immobilized form(as increasing concentration of triton0.1%-0.5% decreases activity ) although enzymatic activity increase in free form of enzyme at same concentration. I did this test for SDS and I get a good result from it. Can I say SDS denatures my enzyme as it's concentration increases but it's denaturation effect on immobilized form is in further concentration!How can I vindicate triton results?
Can you explain how the activity is measured for immobilized and free enzyme?
One reason why Triton may increase the activity of free enzyme is by preventing surface adsorption of the enzyme. This is often done when measuring enzyme activity in polystyrene plates. This would not be an issue with immobilized enzyme, of course. Detergent could also prevent protein aggregation, which is not a problem with immobilized enzyme. At a sufficiently high concentration of the detergent, however, it might become inhibitory by binding up the substrate and/or protein in micelles. Also, Triton can accumulate peroxides which can damage proteins.
Unlike the non-ionic detergent Triton, which does not tend to denature proteins, SDS is an ionic detergent and does denature proteins. It would be expected to inhibit both free and immobilized enzyme.
Hi Parastou, just to follow up on Adam's answer, if your TX-100 is yellow then it has oxidation products in it.
Thanks Mr.Adam and Mr.Steingrimur.
No,triton that i have used is viscose and colorless liquid,aother question,how can i say protein become aggregate?!I produce enzyme with sonication and enzyme band on SDS gel is very sharp!but activity it is not very high!can I say some enzyme molecules get aggregate?!
One way to tell if your protein is aggregated is to perform gel filtration chromatography and compare the elution volume to standard proteins. Dynamic light scattering is another way, if you have the instrument available. A third way is analytical ultracentrifugation, which also requires specialized equipment.
I agree with Adam and I can say that Triton shows a bad effect on immobilized lipase because of the mass transfer limitation. While on a presence of free enzymet this detergent can form micelles and promote more contact surface between substratos and biocatalyst, in an immobilized enzyme, the detergent can deposit on support surface.
Hi Parastou, based on all the comments above, I think you really need to tell us what enzyme activity you are looking at.
Your TX-100 might not have oxidation products, OK.
But detergents generally do not affect enzymes that react with substrates that are soluble in liquid phase. Detergents really affect enzymes that react with substrates that are only soluble in lipid phase.
Thanks Mr.Stefansson and Mr.Adam B Shapiro for your guidance. can you give me a reference about SDS and triton effect on protein activity? thanks alot.
Hi Parastou, comparing activities of a soluble enzyme to an immobilized enzyme is going to be difficult.
Again, it depends on what enzyme you are measuring.
Hi Mr. Adam B Shapiro , do you know the concentration limits of Triton X-100 for enzyme analysis in general before it becomes inhibitory?
Dear Parastou,
Hi, you need to consider four independent points.
1) The effect of each detergent on enzymatic activity is different.
2) The effect of detergent on free and immobilized enzymes is different.
3) The effect of detergent varies with different concentrations.
4) The effect of a detergent with a constant concentration on different enzymes is different.
Therefore, for each enzyme, the type of detergents and the concentration of detergents in the free or stabilized state must be optimized.
Best
Mehdi
In our experiments, Triton X 100 has an inhibitory effect for some hydrolases and oxidoreductases in the non- immobilised system.
Yes, dear Mahmoud, but it depends on Triton X100 concentration, at low concentration, it has no inhibitory effect on hydrolases in the free enzyme system.
Yes, it is depending on the type of the enzyme even at low concentration.
Concerning Triton X-100 and other detergents containing polyoxyethylene, care should be taken to make sure the detergent quality has not deteriorated during long-term storage in air, which results in the formation of destructive peroxides. I always use Triton X-100 that comes as a 10% solution in ampules under inert gas. After opening the ampule, I store the unused portion at -80oC.
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