I have already conducted 6 different substrate concentration and 6 different enzyme concentration. I obtained the absorbance value converted to concentration using A = e. c.l (beer's law). I got the concentration in mMol. After this I am not sure how to proceed?How to use this value in lineweaver burk plot to calculate km? Finally I wonder do I need to conduct 5 substrate concentration for every single concentration of enzyme? Then calculate km and take average of it?
Any one can provide me protocol.
The value of km serves to tell how much affinity the enzyme has for the substrate. In the enzymology it is said that the lower the value of km, the greater the affinity of the enzyme for the substrate.
Soon you need the lowest concentration of enzyme in which you can dose the enzymatic activity and saturate it with different concentrations of substrate.
Now that you already have the absorbances in mMol, also convert the substrate concentrations you used in mMol. Convert the substrate concentrations to 1/[Substrate]mMol and place on the X axis. On the Y axis, the absorbances as 1/abs mMol. Draw a line with the points you got on the chart. The point of the line crossing the X-axis is the value of km (x = -1/km). Remember that the value of km must be between the minimum and maximum amount of substrate concentration you have used.
As for the repetitions, as soon as you can achieve enzyme saturation, repeat the protocol for another 3 to 4 times. If you get similar results it means that your result is reproducible, so you will calculate the standard deviation of the absorbances and the result of the km will already be the average of the values.
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