Enzyme Activity - (Jul/18/2008 )
I have 4 enzymes to work on, and plan to overexpress and purify each of them, then assay their activity using published methods. However, none of the published methods explain how to calculate the activity from the raw data (change in absorbance over time), and none give extinction coefficients.
The equation I used to use was for reduction of ferricytochrome c, which has an extinction coefficient of 17.3 mM-1, so the equation was 1/vol protein used X change in absorbance/17.3. this gave me results in nmol product/min.
My question is this:
1. how do I find an extinction coefficient for each of my reactions?
and 2. if I can't find one, how do I calculate enzyme activity?
If anyone can answer this, or point me in the direction of a straightforward tutorial, that would be great!
Thanks.
The equation I used to use was for reduction of ferricytochrome c, which has an extinction coefficient of 17.3 mM-1, so the equation was 1/vol protein used X change in absorbance/17.3. this gave me results in nmol product/min.
My question is this:
1. how do I find an extinction coefficient for each of my reactions?
and 2. if I can't find one, how do I calculate enzyme activity?
If anyone can answer this, or point me in the direction of a straightforward tutorial, that would be great!
Thanks.
I think you can use a similar commercial enzyme as an internal marker, and based on that reduction in Abs to measure your enzyme activity.
The equation I used to use was for reduction of ferricytochrome c, which has an extinction coefficient of 17.3 mM-1, so the equation was 1/vol protein used X change in absorbance/17.3. this gave me results in nmol product/min.
My question is this:
1. how do I find an extinction coefficient for each of my reactions?
and 2. if I can't find one, how do I calculate enzyme activity?
If anyone can answer this, or point me in the direction of a straightforward tutorial, that would be great!
Thanks.
a reliable source for extinction coefficient are manufacturer´s data sheets or Merck Index;