Hi Mansour,

Yes, AmpC β-lactamase is an important target for developing novel effective antibacterial therapies. This enzyme, however, is among those many biocatalytic molecules, which form large colloid-like aggregates. So, it might follow recently discovered mechanism explaining the effects of many promiscuous inhibitors: some organic molecules form large colloid-like aggregates that sequester and thereby inhibit enzymes (Nature Chemical Biology 1, 146–8, 2005). To this end, numerous promising inhibitors of AmpC β-lactamase all are added in the presence of 0.01% (or less) freshly prepared Triton X-100 detergent. Nonionic Triton-X100 detergent is added in order to suppress the formation of aggregates in solution and exclude aggregation-based promiscuous inhibitors (J. Med. Chem. 45, 1712−22, 2002). This aggregate-suppressing constitutes Triton-X100’  stabilizing effect you asked about. In general, colloidal aggregation of organic molecules is the dominant mechanism for artifactual inhibition of proteins, and controls against it are widely deployed. Active molecules that act via aggregation continue to be found in early discovery campaigns, and a precedent-based approach is developed to match known aggregators with new molecules that may be expected to aggregate and lead to artifacts (J. Med. Chem.,  58, 7076–87, 2015).

Best wishes,

Ilya

Mansour:

Not my area of expertise, but here's what is available in RG just in case you missed these:

https://www.researchgate.net/publicliterature.PublicLiterature.search.html?type=keyword&search-keyword=Triton+X-100+stabilize+an+enzyme&search-abstract=&search=Search

Best regards,

Debra

That is an extremely low concentration. I typically use 0.005-0.01% Triton X-100 in enzyme assays in microplates. The purpose is mainly to prevent the protein from sticking to the plastic surface of the plates.

TritonX-100 is a non ionic detergent and it can only increase the flexibility of protein structure and enhance the activty of some enzymes. but is is not similar in all cases.

Hi Mansour,

Yes, AmpC β-lactamase is an important target for developing novel effective antibacterial therapies. This enzyme, however, is among those many biocatalytic molecules, which form large colloid-like aggregates. So, it might follow recently discovered mechanism explaining the effects of many promiscuous inhibitors: some organic molecules form large colloid-like aggregates that sequester and thereby inhibit enzymes (Nature Chemical Biology 1, 146–8, 2005). To this end, numerous promising inhibitors of AmpC β-lactamase all are added in the presence of 0.01% (or less) freshly prepared Triton X-100 detergent. Nonionic Triton-X100 detergent is added in order to suppress the formation of aggregates in solution and exclude aggregation-based promiscuous inhibitors (J. Med. Chem. 45, 1712−22, 2002). This aggregate-suppressing constitutes Triton-X100’  stabilizing effect you asked about. In general, colloidal aggregation of organic molecules is the dominant mechanism for artifactual inhibition of proteins, and controls against it are widely deployed. Active molecules that act via aggregation continue to be found in early discovery campaigns, and a precedent-based approach is developed to match known aggregators with new molecules that may be expected to aggregate and lead to artifacts (J. Med. Chem.,  58, 7076–87, 2015).

Best wishes,

Ilya

Thank you all for your informative answers. 

Dear Mansour

Your question is regarding the mechanism by which, Triton X-100 stabilizes the enzyme beta-lactamase in vitro.

Triton X-100 is a non ionic detergent, composed of polyethylene glycol moiety (hydrophilic head) and octylphenyl moiety (hydrophobic tail). At a concentration equal to its CMC (around 0.015%) or less (0.005% - 0.015%, Shapiro comment) the detergent does not form stable micelles but rather fixes at protein hydrophobic sites and inhibits hydrophobic surface-protein adhesion in addition to protein-protein hydrophobic interaction; thust  the protein remains active. At a concentration > CMC, the detergent forms stable micelles, and in the present of aggregating protein (e.g. beta lactamase) the micelles integrate  the protein  to form colloidal detergent-protein suspension (see the comment by Ilya Tsyrlov). Triton X-100 at a concentration ≤ 0.005 (your question; 0.00006% is negligible amount) fixes at hydrophobic domains of protein (e.g. your example, AmpC beta lactamase) with its hydrophobic tail which results in minimization of enzyme-enzyme hydrophobic interaction and maintaining of a more stable active conformation of the protein, as the protein-fixed hydrophilic detergent head forms hydrogen bonds with water and other chemicals in the enzyme environment.

I hope this comment can be useful

which paper？ Cloud you share it？

The concentration you were referring  to is an extremely low concentration, with this very low concentration, Trition X-100 is having no effect on the protein composition and nature.  The purpose is majorly to prevent the protein from sticking to the surface of the vessels used for the experiment.

By antagonising hydrophobic interactions, this detergent can either oppose protein-protein aggregation or equally oppose protein interaction with other hydrophobic surfaces. It sounds as if your effect is likely to be the first one, but, as Adam and John have stated, there can be important effects in preventing adhesion to the plastic of microtitre plates, spectrophotometer cuvettes, plastic test-tubes etc. This can become very noticeable for some proteins at low concentrations and means, of course, that you get false results. For us, the initial warning comes when you notice your enzyme rates are not strictly proportional to how much enzyme you put in! In these situations a little bit of Triton can be very beneficial.

 Hello Xinkuan,

Regarding you request about the paper, I found a similar one that they've used 0.00002% Triton X-100 for the same reason in the link below.

Best

http://www.nature.com/nchembio/journal/v1/n3/full/nchembio718.html

Dear Mansour

Thank you for your article. I will read it carefully.

Best