Calculating enzyme activity - (Nov/20/2012 )
Can anyone advise on calculating enzyme activity?
I would like to calculate the activity of cathepsin G in a sample. I'd be grateful if anyone can advise how I can go from a measured OD to units.
Some information about the assay:
The assay uses N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide as a substrate.
1 unit of activity is defined as hydrolysis of 1micromol/min of the substrate (Alternatively 1nKat is 1nmol/second).
The molar extinction coefficient is 8,800 M-1 cm-1
OD was measured by a microplate reader at 410nm.
From what I can understand I should be using the Beer Lambert equation but I'm not sure if I'm using this correctly.
absorbance per time = Absorptivity x Concentration per time x path length
Concentration per time = Absorbance per time/(absorptivity x path length)
I have the following data
- Change in OD from time zero = 0.326
- Time for change = 60min
- Path length = 1cm
- Absorptivity = 8,800 (I think, taken from above)
So is this correct?
Concentration per time = 0.326/(8,800x1) = 3.7x10^-5 (I think units will be M/min)
so I have 3.7x10^-6microM/min therefore 3.7x10^-6 units
Is this correct?
I'm not sure if I've missed out step - for example, how do I account for assay volumes in this equation?
I also have standard curves of different enzyme concentrations against OD - do I need to use these?
Any help would be much appreciated.
You are on the right track; just go carefully through your units.
The first step is correct to get 3.7x10^-5 M
But this is over 60min so you have 6.17x10^-7 M/min
Then convert to uM/min
To get to enzyme units you need to remember uM is an abbreviation for umol/L so you can multiply out the assay volume to leave umol/min
Your standard curves of enzyme vs OD are important for demonstrating that you have a linear response wrt enzyme concentration.