@Rajeev, IC50 does depend on enzyme concentration. IC50 is an operational parameter defined as the concentration of inhibitor (ligand) required for achieving 50% inhibition (binding saturation) of the enzyme (receptor). Therefore, independent of the binding affinity, there is a lower limit for IC50: 50% of enzyme (receptor). Let's say you have a very potent inhibitor (e.g. with an inhibition constant of 1 pM). If you perform an inhibition assay with 20 nM enzyme concentration, your IC50 will be 10 nM; but if you perform an inhibition assay with 200 nM enzyme concentration, your IC50 will be 100 nM. The reasoning is that, in order to inhibit 50% of the enzyme you must add, at least, the same amount (concentration) of inhibitor.

Why would you like to know the enzyme concentration? Comparing ic50 values from different experiments, could lead to false interpretation of the results. If you consider using ic50 of one substance from the article you should, for the consistency of the results, rather plan an experiment where you deternine their ic50 by your protocol, in your hands.

By the way, if the article is published in good (IF) scientific journal, their policy and good scientific writing practice tells that the experimental section should be written in the way that the experiment could be repeated by another research scientist. So all you need is an article, or supplemental info.

Hi Kamil Brozewicz, Thank you very much for your reply.

I have seen IC50 values in some research paper and incase if i would like to repeat it and would like to confirm that the IC50 mentioned is true what should i do? If IC-50 value changes with enzyme concentration whats the significance in finding IC-50. If i report IC-50 value of an inhibitor with very less concentration of enzyme I will get IC50 in nanomolar concentration similarly if i found IC-50 value for same inhibitor with high concentration of enzyme I should get a different IC-50 value. I am really confused with this IC-50 values. This problems happens while finding EC-50 values of anticancer compounds too. Many authors take 10 raise 6 cells in culture wells for MTT assay and some other take different cell number while finding EC-50 value against proliferation. While doing MTT assay I have seen EC-50 values of a compound decreases when cell count is less.

It's a matter of finding a good protocol to follow up. For example (standard NCI anticancer SRB assay, IC50 is called GI50), you calculate IC50 from dose-response curve by the interpolation of end-poits (percent of growth relative to the control) obtained from gradually increasing/reducing tested compound concentration (usually by the power of 10 - 0,01->0,1->1->10->100 mcM). Next, finding a half maximal inhibitory concentration gives you IC50. Typically, like you already know, colony of 10^6 cells of most of human cancer cell lines grows within 24-48 h, depending on the tested compound potency you incubate it in the above mentioned concentrations - for anticancer assays IC50 falling

Hi Kamil Brozewic Thank you very much for clearing my doubt. Kindly help me to find a solution for finding IC-50 values for enzymatic inhibition. I am working with an enzyme inhibitor and for determination of IC-50 I took 220mcM of enzymes + 1000mcM substrate + varying concentration of inhibitor A. I got an IC-50 value of 570mcM for the inhibitor A. Kinetic studies shows that it is competitive inhibitor (Vmax remained same and Km of the substrate increased). What do you think about the concentrations of each components i took for this assay? I got IC-50 value 571mcM does that mean the inhibitor is weak? ( I am confused because IC-50 values of inhibitor depends on enzyme concentration and substrate concentration ). Do i need to do comparative study with other known inhibitors?

It's always some kind of relativity you should consider planning your research. Find a good, well known (this will increase your further article chances;) so called 'active comparator' and review your results (determine ic50 using your protocol). The absolute values mean nothing if you could not compare them with something - unfortunately for us, this always means more job for us to do in the lab, because of 'the consistency and comprehensiveness' of the results obtained if you plan publishing them in the future... From my own practice (not only mine) the best approach is plannig and doing research taking into account publishability of the results. Good luck!

As you mentioned, I am trying to publish the same and writing is almost done . In the manuscript i have mentioned the IC-50 value along with the concentration of enzyme, substrate and reaction volume so that any body can repeat and see if the data is valid. But I could not compare my IC-50 value with any other known inhibitor. Hope this wont create any problem, if it does, I am trying to get some known inhibitors.

Thank You very much for you suggestions

The short answer to your enzyme inhibitor IC50 question is yes, the enzyme concentration does affect the measured IC50 of its inhibitor, especially if the inhibitor is of the competitive inhibitor variety.

Yes, it depends. IC50 is not a true affinity parameter. It is defined as the inhibitor concentration required for 50% inhibition or saturation of the enzyme. Therefore, no matter how high the affinity is, it will have a lower limit value: half of the enzyme concentration. Usually is larger than this lower limit value if the affinity is not high enough. Then, if you get two inhibition curves, one obtained with 10 nM enzyme and the other with 100 nM enzyme, you will probably get different IC50 values.

I do'nt think IC50 should get influenced with conc of enxyme you take for the assay.The only requirement is that you should ensure that your enzyme activity should be in linear range and substrate should not be a limiting in the assay

@Rajeev, IC50 does depend on enzyme concentration. IC50 is an operational parameter defined as the concentration of inhibitor (ligand) required for achieving 50% inhibition (binding saturation) of the enzyme (receptor). Therefore, independent of the binding affinity, there is a lower limit for IC50: 50% of enzyme (receptor). Let's say you have a very potent inhibitor (e.g. with an inhibition constant of 1 pM). If you perform an inhibition assay with 20 nM enzyme concentration, your IC50 will be 10 nM; but if you perform an inhibition assay with 200 nM enzyme concentration, your IC50 will be 100 nM. The reasoning is that, in order to inhibit 50% of the enzyme you must add, at least, the same amount (concentration) of inhibitor.

@Rajeev, as far as my understanding of assays, enzyme assays and ligand binding assays are not similar in nature. In enzyme assays usually IC50 is independent of concentration. But if you consider simple receptor ligand binding assay IC50 is dependent of receptor concentration. So, selecting the appropriate receptor concentration is very important. you can follow the following guideline for developing your assay. I personally found it very handy..

http://www.ncbi.nlm.nih.gov/books/NBK91992/

@Ankur, enzyme assays and ligand binding assays are more similar than you consider (enzyme activity requires substrate binding and enzyme inhibition requires inhibitor binding, the same way receptor activity requires ligand binding). In both cases, IC50 is dependent on the concentration of enzyme/receptor. As I said before, IC50 is an operational parameter defined as the concentration of inhibitor/ligand needed for achieving 50% inhibition/binding saturation. No matter the actual value of the affinity, IC50 has a lower limit value: 50% of the enzyme/receptor concentration. If the affinity is very high, IC50 is equal to 50% of the enzyme/receptor concentration. If the affinity is low, IC50 is larger than 50% of the enzyme/receptor concentration. Of course, if the affinity is low (e.g. dissociation or inhibition constant of 1 mM) and you use different concentrations of enzyme/receptor in the nM or microM range, then the IC50 may seem concentration independent, because in these cases IC50 will be approximately equal to 1 mM.

@Adrian Velazquez-Campoy, thanks for the explanation. Could you please give me a reference that demonstrates your argument? I observed a difference between our assay (7 nM) and a published one (2 nM). Our argument is also the effect of concentration (Substrate concentration 50 nM-theirs and 1 uM-ours), and other experimental conditions (I don't think 2 vs 7 nM is that much a big deal but I need to convince the editor). 

IC50 is always larger than the inhibition constant Ki,app.

The mass conservation applied to the inhibitor gives:

[I]t = [I]b + [I]f

where [I]t is the total concentration of inhibitor, [I]b is the concentration of inhibitor bound to enzyme (or EI complex), and [I]f is the concentration of free inhibitor. By definition, IC50 is the "total" concentration of inhibitor required to reach 50% inhibition (or 50% enzyme saturation fraction), and Ki,app is the free concentration of inhibitor required to reach 50% inhibition (or 50% enzyme saturation fraction). Therefore:

IC50 = E0/2 + Ki,app

where E0 is the total concentration of enzyme.

Consequently, IC50 depends on enzyme concentration. Moreover, because Ki,app may depend on substrate concentration, IC50 will depend on enzyme and substrate concentrations. Obviously, IC50 is always larger than half enzyme concentration, and, no matter how strong is the enzyme-inhibitor interaction (that is, how small is Ki,app), IC50 has a lower limit value: E0/2 (50% of total enzyme concentration).

The point is the distinction between "total" and "free" concentration. For example, if Ki,app is 50 nM, the Ki,app is 50 nM, that is, when the free concentration of inhibitor is 50 nM, the enzyme is 50% saturated (50% inhibitoed) or the enzymatic rate is Vmax/2. But, obviously the total concentration of inhibitor required to reach 50 nM free inhibitor is larger than 50 nM.

If you look up in Google "IC50 depends on enzyme concentration" you will find:

www.biokin.com/slides/1305-arqule.pdf

where Petr Kuzmic explains nicely and throrougly the difference between IC50 and Ki,app, and the drawbacks surrounding IC50. There you will find appropriate references.

Finally, the dependece of Ki,app on concentration of substrate and Km depend on the inhibitory mechanism (competitive, uncompetitive...):

Ki,app = Ki x f([S],Km...)

Thus, overall IC50 is an operational parameter to describe the effective strength of the enzyme-inhibitor concentration, but it depends on enzyme and substrate concentration; it will not be useful to rank potent inhibitors according to potency if their K,app is much lower tan E0/2. On the contrary, Ki,app only depends on substrate concentration and it may be used to estimate the intrinsic Ki.

Ok. You mean the lower the concentration,the higher will be the IC50.

No, the contrary.

From what I wrote before:

IC50 = E0/2 + Ki,app, for an enzyme and

EC50 = R0/2 + Kd,app, for a receptor.

Thus, the larger the concentration of enzyme (E0) or receptor (R0), the larger the IC50 or EC50.

The logical and detailed argument by Adrian also explains why people tend to neglect enzyme concentration: when it is well below the apparent Ki (E0

Robert A. Copeland, Wiley-VCH, New York, 2000, ISBN: 0-471-35929-7

good luck

Please tell, How to quantify amount of free drug in solution a solution containing enzyme-substrate-drug complex.

How to quantify amount of free drug in a solution containing enzyme-substrate-drug complex.

If you consider the initial situation, with no significant substrate processing and assuming just a single substrate binding site and a single drug binding site in the enzyme, you can solve the ternary equilibrium:

[E]t = [E] + [ES] + [ED] + [ESD]

[S]t = [S] + [ES] + [ESD]

[D]t = [D] + [ED] + [ESD]

where each complex involves a dissociation constant:

[ES] = [E] [S] / KS

[ED] = [E] [D] / KD

[ESD] = [E] [S] [D] / KS KD k

where k is the cooperativity interaction constant for the ternary complex (k=0 if the ternary complex is not possible, that is, if S nad D are competitive; k1 if the binding of substrate and drug show positive cooperativity).

Once you know [E]t, [S]t and [D]t, and entering values for KS, KD, and k, solving the previous system will provide you [E], [S] and [D] (you are looking for [D]).

When substrate starts being processed, its concentration will diminish and the equilibrium state will be modified, but a new species, the product P, will appear, which might enter into the equilibrium (then, quaternary) if the product P can bind to the enzyme.

Of course, the ternary equilibrium can be substituted by a quasi-simple approximation in which a binary equilibrium is considered:

[E]t = [E] + [ED]

[D]t = [D] + [ED]

where each complex involves a dissociation constant:

[ED] = [E] [D] / KD,eff

where KD,eff is the effective dissociation constant for the ED complex, which contains the influence of substrate binding on the E-D equilibrium. For example, if S and D are competitive:

KD,eff = KD (1 + KS [S])

and if S and D behave cooperatively:

KD,eff = KD (1 + KS [S]) / (1 + KS k [S])

Again, once you know [E]t and [D]t, and entering values for KD,eff (or KS, KD, and k), solving the previous system will provide you [E] and [D] (you are looking for [D]).