How can I transform the graph from concentration vs. absorbance to concentration vs. velocity in my research on human liver cytosol using spectroscopy to estimate the Km and Vmax values? I have absorption data at different concentrations and would like to determine the velocity values corresponding to each concentration for the purpose of creating a concentration vs. velocity graph.
If you can use Lambert-Beer for the concentration-absorbance relationship (which I hope for you, otherwise...nasty), you calculate the reaction rate (that's more common than velocity)
r=dc/dt=d[ln(I/I0)/(alpha*d)]/dt
Both the intensity-concentration recalculation as well as the derivative can be calculated in a simple Excel spreadsheet.
Another way to state the method is: Measure the rate of the reaction by finding the slope during the initial, linear part of the absorbance versus time plot. To do this, you must measure the absorbance at several times during the reaction.
Convert absorbance to concentration using the Beer-Lambert Law, as stated by Jürgen Weippert .
Adam B Shapiro Jürgen Weippert thanks a lot! If the path length for a cuvette is 1 cm in lambert-beers law, what is the equivalent path length that should be used when using a 96-well microplate?
I have never used microplates, but Agilent seems to provide a guide on it:
https://www.agilent.com/cs/library/applications/protein-analysis-5994-2770EN-agilent.pdf
The path length in a 96-well plate is determined by the volume of the sample. Some plate readers can do a path length correction for you. You can also try to measure the path length from one of the edge wells using a ruler, but it is only approximate. The measurement should be made at the center of the well, since there may be a meniscus.
My usual approach is to measure the extinction coefficient of the substance by making a set of solutions of different concentrations in the same buffer and volume that is used in the assay, then making an absorbance versus concentration plot. The slope of the plot is the extinction coefficient for that specific condition. Since the volume is the same for the assay, you can ignore the path length component of the extinction coefficient. This method depends, of course, on the availability of the substance to be measured, which is usually the product of the reaction. If you don't have it, then you can sometimes make it in situ by running the reaction with the enzyme to completion.
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