I have a question about the calculation of the enzymatic activity from the absorbance. In particular I have a series of curves corresponding to the different concentrations of my samples. The slope of these curves (variation of absorbance per time unit) corresponds to the velocity of the reaction..is that right? If I diluted my samples, do I need to multiply the value of the slope by the dilution factor?Is that right?I don't have the standard curve
Thanks
The slope of your absorbance vs time plot - or its linear part - gives you the rate of change of absorbance over time (s-1). Use the molar absorption coefficient (L mol-1 cm-1) at the given wavelength and the optical pathlenght of your cuvette (cm) to convert this into a concentration change (mol L-1 s-1). Using the volume of your experiment (mL) you can then calculate the enzymatic velocity (mol/s = kat).
If you dilute your enzyme by adding, say, 1 µL of enzyme solution to 4 µl buffer, then you have a 1 / (1+4) = 1/5 dilution. The above experiment gives you the concentration of enzyme in the diluted sample, to get the concentration of the stock you have to divide by the dilution, that is, multiply by 5 (the reciprocal).
Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry by Irwin H. Segel (1976) is still a very relevant book for the beginner. You should be able to get a used copy at bargain price.
Hi Engelbert,
thank you so much for your reply. The experiment was carried out in 96 well plates. So, how do I get the optical pathlenght then? And how do I get the molar absorption coefficient at a given wavelength?
In addition,
Couldn't I multiply the rate of change of absorbance over time by the dilution factor directly? I mean, without converting into concentration change.. I don't know how to get the pathlength of the 96 well plates and the molar coefficient..
The molar absorbtion coefficient (epsilon) you usually get from the literature. But you can determine it yourself: Measure the absorbance of a solution with known substrate concentration at the wavelength you want to use in the assay. That is usually the wavelength where absorbance has a maximum. Then use the rule of three to calculate what the absorbance at 1 M would be under the same conditions.
You need epsilon to calculate concentration (and ultimately amount) of substrate from the measured absorbance, using Lambert-Beer's law. Without it you could only measure relative values for enzymatic activity (x times that of a reference solution).
If you are using 96-well plates, you have to use a constant volume in all wells, so that the hight is the same in all wells (since they all have the same radius).
If you measure absorption spectra of the same substance at different concentrations, you often will notice a wavelength where absorption is independent of concentration (all curves go through the same point). This is called the isosbestic point, you should use it for your reference wavelength. Since substrate concentration has no effect on absorbance at this wavelength any change in absorbance must be due to scratches in the plastic or similar happenstance.
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