[Sis290025]

A student purified cox-2. The crude extract contained 12 g of protein and had 23,000 units of cox-2 activity in 800 mL. The final purification using an affinity column resulted in 3 mL that was 2,300 fold pure. What was the activity of 5 microL of the final purification?

Under my assumption, enzyme units will be represented by micromol/sec/mL for each purification step. Purification is reflected through specific activity. Therefore,

Crude extract’s specific activity = micromol/sec/mg protein = (23,000 micromol/sec/mL * 800 mL)/(12,000 mg) = 1533.33 micromol/sec/mg

Fold of purification = final purification’s specific activity / crude extract’s specific activity


final purification’s specific activity = Fold of purification * crude extract’s specific activity = 2300*1533.33 micromol/sec/mg = 3.53*10^6 micromol/sec/mg

This part is where I am confused over as total enzyme activity does not include mg of protein.

Is this setup permissible?

12 g / x = 800 mL / 3 mL, where x = final purification’s total mass of protein in g??

If above is true, then:

X = .045 g = 45 mg

3.53*10^6 micromol/sec/mg * 45 mg = 1.587*10^8 micromol/sec

Activity in 3 mL = (1.587*10^8 micromol/sec)/(3 mL) = 5.29*10^7 micromol/sec/mL

The total enzyme activity in 5 microL of final purification is: 5.29*10^7 micromol/sec/mL * (5*10^-3 mL) = 2.65*10^5 micromol/sec.

I don’t know if I have my unit definitions correct, so please tell if my setup is correct. If my approach is wrong, then guide me.

Thank you.

[aHerraez]

1. Enzyme units are not per volume unit. Enzyme activity is not the same as concentration of enzyme activity. In other way: enzyme activity (enzyme "units") is not a concentration, it is an amount.

So your first calculation is not correct.

Apart from that, why do you assume per mL? It would have been per L.

2.

12 g / x = 800 mL / 3 mL, where x = final purification’s total mass of protein in g??

This makes no sense. You are not picking 3 mL out of the 800 mL of solution. There are concentrations or dilutions going on during the purification.