# 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.

If

absorbance per time = Absorptivity x Concentration per time x path length

then

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.

Thanks,

Paul

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.

Great.

Many thanks,

Paul