I have Purchased AchE from Sigma Aldrich. I want to make stock solution of This Enzyme. But the unit is not defined for me. Just it has wrote 200 unit. and i dont know its gram or other unit. I want to make it in 1 ppm and other stock solution concentration. I would be grateful explain it completely.
The amount of enzyme ina preparation/sample can be given as enzyme units (U).
1 U corresponds to an activity of 1 µmol/min.
Peter
Hi, the enzyme unit (U or IU) is a unit for the amount of a particular enzyme. 1 U or IU is defined as the enzyme quantity that liberates 1 microgram or micromole of product per ml of the reaction mixture per minute (usually at 25 °C).
I believe there is something like 200 unit/mg or the similar on the label. If there is not, you can get the product information sheet and specification sheet from Sigma Aldrich online. There you can find the unit and percentage of the specific ingredient.
Hi Shariat. I absolutely hate working with enzyme units because they are dependent on the assay being used, the purity of the enzyme preparation, how the assay is performed, etc. etc.
But Peter is right. 1 U corresponds to 1 uM substrate conversion/min for enzymes.
But then you also have units for heparin, protamine, vWF, cofactors, etc. I wish that there was a measurement that all biochemists could understand, namely molarity and weight/volume.
Thank you dear friends,
your answers were very helpful.
i want to prepare AchE stock solution.
how can i make 200m units of Ache.T he Enzyme is soluble in Water and tris buffer too. I have a little amount of Ache in soild form ( less than 1mg)
That is a classic problem of solution preparation. What you need to know of your stock is the concentration. Two hundred mU is a quantity. If you take your package and dissolve it in 1 mL of buffer (check the buffer needed for best stability, it is usually indicated on Sigma catalogue) you would have a 200 U/ml (=200mM/µl) preparation, 1 µl would contain 200 mU. To know how much protein you have per µl you need to know the total weight of your package and its protein content (usually indicated as %). The ratio of activity to protein should indicate the maximum specific activity. Any enzyme preparation has a maximum specific activity, representing the ration Vmax/[protein] (usually in µmol min-1 mg prot.-1, equal to unitis/mg prot). When the preparation has contaminant other than your protein, the ratio decreases. When the protein is pure, the ratio reaches a constant value characteristic of a protein and a function of the catalytic rate constant (kCAT). Knowing the specific activity tells you about enzyme's purity in the preparation.
Your preparation may or may not be pure, but It should indicate the Biological source of the protein (which organism it comes from, it should be in the catalogue, at least). Go to Brenda (http://www.brenda-enzymes.org/) and find out the reported specific activity of your protein. (i.e. Units/mg of pure protein). That should tell you how many mg of your enzyme are present in the package.
You should study this book:
https://www.amazon.com/Biochemical-Calculations-Mathematical-Problems-Biochemistry/dp/0471774219
It is basic knowledge you should have in this line of work.
You may also find this IUBMB definitions' page useful:
http://www.iupac.org/publications/pac/2001/pdf/7306x0927.pdf
Best wishes,
Rogelio
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