Enzyme kinetics - (Apr/03/2008 )
I want to determine the initial rate of reaction of an enzyme and measure the increase of amount of product over time.
I hoped to get a standard curve with a steep and linear slope followed by a plateau phase.
My enzyme shows a lag phase in the beginning of the reaction and a very flat slope. And I have to wait really long to reach the max. product amount value (which I determined in assays where I used larger amounts of enzyme and a higher specific activity of radiolabelled substrate).
Should I increase the amount of enzyme or the specific activity of my radiolabelled substrate to get a steeper slope and a standard-type curve?
Thanks for your help,
chalet2
-chalet2-
QUOTE (chalet2 @ Apr 3 2008, 10:19 AM)
I want to determine the initial rate of reaction of an enzyme and measure the increase of amount of product over time.
I hoped to get a standard curve with a steep and linear slope followed by a plateau phase.
My enzyme shows a lag phase in the beginning of the reaction and a very flat slope. And I have to wait really long to reach the max. product amount value (which I determined in assays where I used larger amounts of enzyme and a higher specific activity of radiolabelled substrate).
Should I increase the amount of enzyme or the specific activity of my radiolabelled substrate to get a steeper slope and a standard-type curve?
Thanks for your help,
chalet2
I hoped to get a standard curve with a steep and linear slope followed by a plateau phase.
My enzyme shows a lag phase in the beginning of the reaction and a very flat slope. And I have to wait really long to reach the max. product amount value (which I determined in assays where I used larger amounts of enzyme and a higher specific activity of radiolabelled substrate).
Should I increase the amount of enzyme or the specific activity of my radiolabelled substrate to get a steeper slope and a standard-type curve?
Thanks for your help,
chalet2
Provided you know how much you are adding in total, and substrate is not limiting, adding more enzyme will lead to an increase in initial rates (Vmax) with no change in Km.
-shojjahd-