Could you please clearly explain the extinction coefficient and its importance in enzymatic assays?
Should we consider it for all type of enzyme assays?
An extinction coefficient along with a particular wavelength that can be used depends greatly on the enzyme (or actually its substrate(s) and/or product(s)), so no, there is no particular wavelength and extinction coefficient that applies for all enzymes in general. These vary between cases and to find out a suitable method of following your reaction you should look into literature.
Now, if there is a wavelength that can be used to monitor (directly or indirectly) the conversion of substrate to product, knowing the extinction coefficient basically allows one to quantify the conversion i.e. how much product you actually are forming and what you ended up with. After all, the turnover of substrate to product is always in an equilibrium, so if your [S] = 5 mM, in the end [P] (which you are measuring) is actually determined by the equilibrium constant. Bare in mind that when using a marker enzyme with a certain ext. coef., one also needs to be aware of the stoichiometry of the reaction. Also, another way is to measure the disappearance of S. In this case the extinction coefficient allows you to determine the remaining amount of substrate after reaching equilibrium. But, just to answer simply in the context of Michaelis-Menten kinetics, you NEED the extinction coefficient to convert your absorbance units to concentration units if you want to be able to produce Michaelis-Menten parameters.
A typical example: an enzyme oxidizes substrate S to product P and as such reduces NAPD+ to NAPDH in the reaction. As NADP+ converts to NADPH, an increase at 340 nm is observed. The extinction coefficient of NADPH is 6.22 mM-1 cm-1, which can be used to directly quantify the formation and at the end the total product formed during the reaction, assuming the concentration of substrate S is limited in solution and NADP+ is available in excess. This is a very straightforward example as the reaction stoichiometry is simple. Now as you have your concentration series and reaction curves, you use the extinction coefficient together with the Beer-Lambert law (A = ε c l) to work out your conversion rates in quantity over time, like mM/s. Only after this you can construct you MM plot and work out the kinetic parameters.
Very well explained the importance of extinction coefficient in an enzyme assay by Arne Raasakka! Thanks, it also add to my knowledge. Well, i would like to ask,how one could determine the extinction coefficient (value) for a particular substrate or product in an enzyme assay?.
I suppose the easiest way to determine the extinction coefficient would be to measure the absorbance for a known amount of compound. Then you can calculate ε again using the Beer-Lambert law. This does have some practical aspect to take into consideration (pH, reducing/oxidising conditions etc.) if your compound can manifest as several species in solution. Also, the spectrometer should be as sensitive and well calibrated as possible and the measurement should be performed for several concentrations of the compound and in duplicates (or preferably even triplicates). And naturally, if the wavelenght of maximum absorbance is not known, one can always measure an absorbance spectrum to check it. Remember to have a good blank also and use as fresh a solution for the assayed compound as possible, especially if the compound is unstable in solution.
The determination can actually be done graphically very easily, as you remember the Beer-Lambert law is A = ε c l. Measure a series of different concentrations of your compound (be it substrate or product) in triplicates and plot them Abs vs. concentration (or actually it should be Abs vs. the product of c and l). The slope of the trendline for your points is equal to the extinction coefficient at the used wavelength.
Thanks a lot. It was a great help because I could not find such good explanation on web or books.
Actually, after putting all of knowledge about enzymatic assay together, Now I have mixed up!
I am doing the assay of PQQ-ADH by the following method:
1) Extraction of the membrane bound PQQ-ADH by french press and the ultracentrifuge
2) Addition of the enzyme to a mixture containing Pottassium Fericyanid, Ethanol and buffer and the crude enzyme.
3) Incubation at 30°C for 10 min
4) Stoping the reaction by addition of SDS-Fe ( then the blue-green color is appeared)
5) reading teh OD against a blank at 595
Since during the reaction, Fericyanid is reduced to Ferocyanid, it is possible to have a calibration curve on the basis of Ferocyanid.
Then, I calculate simply the rate by dividing the change in ODto 10 min. Therefore the specific rate can be calculated as well.
Now, what is the role of Extinction coefficient in these calculations?
Should I also take this coefficient into account?
It's true that you get the rate by dividing the OD (actually it's Abs) by time, but this only tells you how much the sample absorbance changes as a function of time. You can now use the extinction coefficient to calculate how much ferrocyanid (in concentration units) you produce as a function of time. This is relevant as absorbance is an arbitrary unit and presenting rates as M or mol per time unit is much more convenient for other scientists to understand.
Remember that you should record several time points during your measurement to see that your reaction is linear. Only that gives you the true rate. If you measure for too long the rate starts to decrease as a function of time.
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