The Protein Man's Blog | A Discussion of Protein Research

How can the Vmax of an enzyme reaction be calculated from the colorimetric SAM assay?

Posted by The Protein Man on Jan 29, 2014 1:56:00 PM
The Protein Man

To calculate the Vmax and Km of the reaction you will need to run various concentrations of the methyltransferase to be tested and calculate the activity at each concentration.

The following are example numbers and should not be used with the assay. The optimal starting amount of enzyme will need to be determined empirically

An Example: The following concentrations of a methyltransferase were used in the SAM510 assay and the activites were calculated as per the protocol.

[Methyltransferase] (mM)

Activity

0.1

0.04855

0.125

0.05664

0.167

0.06827

0.25

0.08395

0.5

0.11227

1

0.133

 Convert both columns of data in the above table to the inverse. i.e Divide 1 by each number in the table above.

1/[Methyltransferase] (mM)

1/Activity

10

20.59836979

8

17.65574554

6

14.64772963

4

11.91231024

2

8.907403153

1

7.518796578

 Plot the above as a chart and determine the linear equation for a linear trendline:

 Vmax Determination

 

1/Vmax is the y-intercept. This is 6.0373 in the above example. So Vmax = 1/6.0373 = 0.1656 µmol/min/ml

 
The x intercept is -1/km. So in the above equation. 0=1.4527(-1/Km)+6.0373. So Km=-1.4525/-6.0337 or 4.5812mM

Topics: Apoptosis Assays, Assay Development (ELISA)

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