Gertsch Group

Institute of Biochemistry and Molecular Medicine, University of Bern, Switzerland

On concentration

On concentration
Jurg Gertsch - Sat Jan 24, 2015 @ 01:47PM
Comments: 5

The basic biological phenomena rely on molecular interactions which are mostly non-covalent. When it comes to providing evidence that two molecules interact with each other, say e.g. two proteins that might communicate via binding to each other or a ligand and a receptor, an inhibitor and its target, etc. then we usually measure a dissociation constant. a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex  falls apart into its component molecules, or when a salt splits up into its component ions. 

The dissociation constant is commonly used to describe the affinity between a ligand (such as a drug or substrate) and a protein i.e. how tightly a ligand binds to a particular protein. Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic and hydrophobic interactions and Van der Walls forces. 

Reading manuscripts and published papers, there are cases where binding is used as an argument or proof, but often completely neglecting the underlying dissociation constant. E.g., when it comes to protein-protein interactions proteins are over-expressed and then co-immunoprecipitated, often without any control over the copy numbers of these proteins. Since binding is a function of concentration, anything will bind to anything when the concentration is high enough. The other case is that apparently specific inhibitors are used at such high concentrations (exceeding the dissociation constant originally measured) to prove that a phenomenon is mediated via a particular pathway or receptor. It is surprising how little concentration is taken into account! If you want to see what you want to see it does not seem to be a crime to rise the inhibitor concentration such that you finally see it. In order words, you have 50% chance that your inhibitor blocks your pathway and by rising its concentration, the chance will increase. To my opinion, many small organic molecules will inhibit a protein when its concentration is high enough. That is exactly why we need nanomolar inhibitors and also apply them at nanomolar conentrations.

Comments: 5

Comments

1. Liu M   |   Sat Oct 24, 2015 @ 12:04PM

Concentration is indeed important and we all struggle with papers claiming apparently specific interactions, or making conclusions thereof, which are artefacts. We would need a global standard, especially for biological work. Do you know of any such initiatives? Best, Mei

2. Tim   |   Fri Apr 01, 2016 @ 09:37PM

I see this over and over again: paper on some natural product that displays interesting inhibition of something, like NF k B for example, and then looking at the details, we see the activity is in the dozens of uM. In other words, too weak to be really useful, not something that can be dosed. With compounds active in the tens of uM, often correlates to dosing with grams of product. Single point of action can be lost with high dosages.

That being said, there are some non-toxic natural products in the borderline region of low single digit uM, that can be dosed, corresponding to a gram or less dosages. So there is a bit of a trade off between activity and toxicity, that gives a gray area in the low uM region.

Generally, nM activity is required for sensible dosing, to allow for losses of compound in vivo, serum protein binding, first and second pass metabolism, uneven distribution in organs/tissues and other such ADME issues.

Unfortunately, a global standard seems to be not really possible because of the wide ranging, variable ADMEs and variable off target issues.

3. Philippe   |   Mon Sep 12, 2016 @ 05:04PM

Hi, I do not really fully agree with your comment. I think that higher concentrations used in research can still uncover relevant targets and sometimes you just need to increase your concentration to make things visible. Also, the in vitro situation is artificial too and you cannot simulate the real physiological situation in any case.

4. Kumar   |   Thu Jun 01, 2017 @ 04:09PM

Do not agree with Philippe - you see things in vitro that are non-physiological because you increase your concentration. The misuse of doses and concentrations are a big problem in biological research.

5. joseph   |   Tue Sep 19, 2017 @ 07:01AM

Content composition is very fascinating work who are in this work are truly appreciate the work. You ought to have huge learning about all the common things and subjects. The https://usawriters.org/ discloses to us the estimation of this work for the future in light of the fact that numerous authors are getting a charge out of good life.

Post a Comment




powered by Doodlekit™ Free Website Maker