Quantification of Bacteria Urease Activity??? Berthelot method. - (Jul/25/2012 )
I'm hoping someone may be able to help me.
My lab is a microbiology lab and there is no one here with Biochem experience, so I'd really appreciate it if someone could read this and let me know if I'm going in the right direction.
I am trying to quantify the urease activity of my bacterial cells using the Berthelot method and I want to express urease activity as uM urea hydrolysed / min-1/ mg-1 protein.
-Produced a standard curve ranging from 0-500uM of Ammonia.
-Pellet of bacterial cells contains 0.6mg of protein - cells are lysed in 200ul of lysis buffer and 1,000ul of Reagent A is added to the sample and incubated for 30mins;
-a 90ul aliquot of the sample is removed and mixed with 1,050ul of Reagent B; Absorbance measured.
Urease activity expressed as uM of urea hydrolysed/min-1/mg-1 of protein
0.6mg protein in pellet
0.6mg of protein in 200ul
0.6mg of protein in 1,200ul
0.045mg of protein in 90ul aliquot
If OD570 is 0.03, extrapolation from standard curve; 60uM ammonia; representative of the 90ul sample.
As urease activity is expressed as uM of urea hydrolysed/min-1/mg-1 of protein
Total of 30mins; 60uM/30min= 2uM of urea hydrolysed/min/ 0.045mg of protein
1mg of protein= 1000ug protein. I had 0.045mg protein in my 90ul aliquot.
1000/0.045=22.22 therefore 44.44uM of urea hydrolysed/min/mg of protein.
Does this make sence?
Thank you for reading this and I'd appreciate any comments.
i can't say anything about the assay method because i'm not familiar with it but the calculation works out for specific activity (i think your units are umoles/min/mg protein, not uM/min/mg).
if you want to quantify then you multiply the specific activity by the total protein.