[Nescafe]

Hello,

Does anyone know the formula from Michaelis menten that allows for IC50 calculation? I am confused by the following statement made by authors of a paper I just read.

IC50 assay: C50 values were derived by a sigmoidal dose-response (variable slope) curve using GraphPad Prism software. A substrate concentration equivalent to the Km value for each enzyme was used for IC50 determinations. The reported data are average of at least three independent experiments.

Thanks,

Nescafe.

[Babcock_Hall]

I am not sure what you are asking.  It sounds to me that the workers set the concentration of substrate equal to its K_m.  Then they found IC50 for an inhibitor under the condition of constant substrate concentration.  If I am not mistaken, the IC₅₀ would not be equal to the K_i of the inhibitor.  I used to have a nice paper which discussed the various measures of inhibition
EDT
1.   The IC50 Concept Revisited.
Caldwell GW, Yan Z, Lang W, Masucci JA.
Curr Top Med Chem. 2012 Jun 1;12(11):1282-90.
PMID: 22571790 [PubMed - in process]

2.   Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.
Cheng Y, Prusoff WH.
Biochem Pharmacol. 1973 Dec 1;22(23):3099-108. No abstract available.
PMID: 4202581 [PubMed - indexed for MEDLINE]

3.   Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes.
Brandt RB, Laux JE, Yates SW.
Biochem Med Metab Biol. 1987 Jun;37(3):344-9.
PMID: 3606895 [PubMed - indexed for MEDLINE]

[Nescafe]

I am not sure what you are asking.  It sounds to me that the workers set the concentration of substrate equal to its K_m.  Then they found IC50 for an inhibitor under the condition of constant substrate concentration.  If I am not mistaken, the IC₅₀ would not be equal to the K_i of the inhibitor.  I used to have a nice paper which discussed the various measures of inhibition
EDT
1.   The IC50 Concept Revisited.
Caldwell GW, Yan Z, Lang W, Masucci JA.
Curr Top Med Chem. 2012 Jun 1;12(11):1282-90.
PMID: 22571790 [PubMed - in process]

2.   Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.
Cheng Y, Prusoff WH.
Biochem Pharmacol. 1973 Dec 1;22(23):3099-108. No abstract available.
PMID: 4202581 [PubMed - indexed for MEDLINE]

3.   Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes.
Brandt RB, Laux JE, Yates SW.
Biochem Med Metab Biol. 1987 Jun;37(3):344-9.
PMID: 3606895 [PubMed - indexed for MEDLINE]

Thanks for the response Babcock. Does this  mean that I have to run a separate experiment measuring enzyme velocity at a constant substrate concentration to determine IC50 of my inhibitor?  I usually measure absorbance over time at various substrate concentration and calculate enzyme velocity (Abs/min). Then plot the michaelis menten v v.s. substrate graph. But for IC50 determination of an inhibitor, do I have to run a separate experiment at a constant substrate concentration (= Km) and various inhibitor concentration? or, as I originally thought,all I have to do is rerun the initial experiments I did with just substrate but this time in presence of varying inhibitor concentration and use the following formula to calculate IC50.

vi = v0/ 1 + /IC50

Thanks in advance,

Nescafe.

[kriggy]

I think you want to measure constant concentration of enzyme, known (maybe excess) amount of substrate and known different amounts of inhibitor. Or at least thats what I didnt measure enzyme activity, rather enzymme mimic activity.
Basicaly what I did:
a) measure the absorbance of solutions with different amounts of inhibitor (A)
b) measure the absorbacne of solution without inhibitor (B)
c) calculate % of inhibition %inh = A/B * 100
d) plot the %inh vs -log contentration of inhibitor and from the line equation I calculated the concentration where the %inh is 50.

Note that my reaction followed 1st order kinetics so the plot was rather easy but enzyme follow different kinetics so the plot might be more difficult.
hope it helps

[Nescafe]

I think you want to measure constant concentration of enzyme, known (maybe excess) amount of substrate and known different amounts of inhibitor. Or at least thats what I didnt measure enzyme activity, rather enzymme mimic activity.
Basicaly what I did:
a) measure the absorbance of solutions with different amounts of inhibitor (A)
b) measure the absorbacne of solution without inhibitor (B)
c) calculate % of inhibition %inh = A/B * 100
d) plot the %inh vs -log contentration of inhibitor and from the line equation I calculated the concentration where the %inh is 50.

Note that my reaction followed 1st order kinetics so the plot was rather easy but enzyme follow different kinetics so the plot might be more difficult.
hope it helps

Hmmm that makes sense.

Thanks!

Nescafe.

[Babcock_Hall]

The enzyme concentration need not be constant when one performs enzyme assays.  In fact, it is often convenient to alter the enzyme concentration so that the reaction does not become too fast or too slow.  However, one must normalize the rates for changes in enzyme concentration.  99% of the time, the rate is is a linear function of [E], and in fact observing this behavior is a key control experiment.  It is far more common to measure initial rates (in which less than, say, 5% of the substrate is consumed) in enzyme kinetics than it is to use integrated rate equations.  It seems to me that the substrate concentration was constant and equal to K_m in the work you described.  I am pressed for time, but here is one more reference:
Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data.  Cortés A1, Cascante M, Cárdenas ML, Cornish-Bowden A.Biochem. J. (2001) 357 (263–268)

Cornish-Bowden is well known in the field of enzyme kinetics.

[Yggdrasil]

The enzyme concentration need not be constant when one performs enzyme assays.  In fact, it is often convenient to alter the enzyme concentration so that the reaction does not become too fast or too slow.  However, one must normalize the rates for changes in enzyme concentration.  99% of the time, the rate is is a linear function of [E], and in fact observing this behavior is a key control experiment.  It is far more common to measure initial rates (in which less than, say, 5% of the substrate is consumed) in enzyme kinetics than it is to use integrated rate equations.  It seems to me that the substrate concentration was constant and equal to K_m in the work you described.  I am pressed for time, but here is one more reference:
Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data.  Cortés A1, Cascante M, Cárdenas ML, Cornish-Bowden A.Biochem. J. (2001) 357 (263–268)

Cornish-Bowden is well known in the field of enzyme kinetics.

This is true to some extent, but you have to keep in mind that as you increase the enzyme concentration, you can get to a point where enzyme concentration becomes saturating and you are no longer looking at at steady state kinetics (where you're measuring the rate of multiple turnovers of the enzyme) , but at the pre-steady state kinetics (where you're looking at a single turnover per enzyme).  In general, if the [S ] >> [E] then you're in the steady state regime and when [E] >> [S ], you're in the pre-steady state regime.

[Babcock_Hall]

Yes, but I had in mind somewhat less extreme variation in enzyme concentration.