I'm trying to calculate the enzyme activity for my PPO assay according to umol/mg protein (enzyme)/min. But I can't figure out how to get the mg of enzyme in my samples?
Is it derived from my sample weight pre extraction (40mg)?
I hope someone can answer. Thank you.
No (I think, its' been >50 years since I took a course in biochemistry). The figure you gave above is based on the substrate that is consumed by the enzyme. Thus, the Cmax of the enzyme varies based on its work environment (like pH and the amount of substrate). If I recall the enzyme must be crystallized 1st (whether active or inactive) in order to determine its weight by other analytical techniques.
Hi there,
Enzyme activity is expressed by mg of protein so you actually need to determine the protein content of your sample using a protein assay (Bradford, Folin, BCA...) in order to determine the protein content in the enzymatic assay.
Determining the protein content of enzymatic solution through Lowry or Bradford methods, you can apply to estimate the enzyme specific activity.
You have to calculate protein content in crude enzyme extract ( by Lowry or Bradford method) prepared from sample you extracted it is from 40 mg in your case as you mentioned.
You have to note the crude extract volume.
Calculate the protein in the total volume of crude extract. It is crude extract prepared from 40 mg sample in your case.
Can then calculate activity per mg protein.
It is the activity of PPO in crude extrac.
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