Citrate synthase standard for CS assay - (May/21/2013 )

Hi,

I recently performed a citrate synthase assay on rat skeletal muscle. I decided to use a commerical citrate synthase as a control for the experiment. The CS control states that 1 unit of it will form 1 umol of citrate from OAA and Acetyl co A. When analysing my data I came across a problem, I am unsure how to determine if the assay worked correctly using this control. I calculated the results to umol/min using 1 unit of citrate synthase. But I got results as low as 0.3 umol/min. If I take into account the mg/ml concentration of the citrate synthase and calculate the data the same as my samples I get ~130 umol/min/mg using 1 unit of citrate synthase.

Has anyone else done this assay? And if so do they know how to do the calculation?
I except you ment to get something around 1, if it is a bit off then you can use this to normalize you data as you know the reaction is slightly off. But I feel like I must be doing something totally wrong for mine to be this off, or I am not taking all factors into account.

Any help would be greatly appreciated!

Units for enzymes are usually defined by the amount of substrate per time. for many the unit of time is an hour - so perhaps this is your problem?

are you controlling temperature?

mixing thoroughly?

how are you assaying (kit or homebrew)?