Hello,
I am having trouble in preparing some standard solutions for my enzyme of interest to check its enzymatic activity.
In the product sheet it says it contains > 50000 units/mg of solid and the molecular weight is written as mol wt 108 kDa (two 54 kDa subunits). I want to do molarity calculations and I can do it using molecular weight information but, because I don't really know the meaning of unit, I am hesitating to do further experiments.
The unit definition is given as: One unit will cause an increase in A234 of 0.001 per min at pH 9.0 at 25 deg C when Linoleic Acid is the substrate in 3.0 ml volume (1 cm light path) which seems to be related with the enzyme capability to react with the substrate but I am not sure..
Thank you,
Hi there,
You are right : unit is related to the catalytic activity of the enzyme. But obviously you are only interested in the molarity. So provided your solid is pure you are able to convert weight into mole. In your case 1g corresponds to 1/108000 mol. So for instance if you prepare a solution of 1g / L it will be 1/108000 M = 9.26 µM.
You should check if your enzyme is prepared in any excipient. Normally when they are lyophilized it is so. I recommend that you determine the protein content so that you can determine the specific activity. You can use the Bradford reagent.
If you know the sequence (or at leat amino acid composition) of your enzyme, you can also determine its concentration from absorbance at 280 nm, from the known molar absorption coefficients of Tyr, Trp and cystine:
@ARTICLE{Per-86,
title = {Protein Volumes and Hydration Effects},
author = {S.J. Perkins},
journal = {J. Biochem.},
year = {1986},
pages = {169-180},
volume = {157},
doi = {10.1111/j.1432-1033.1986.tb09653.x},
language = {eng},
}
@ARTICLE{Gil-89,
title = {Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data},
author = {S.C. Gill and P.H. von Hippel},
journal = {Anal. Biochem.},
year = {1989},
number = {2},
pages = {319-326},
volume = {182},
doi = {10.1016/0003-2697(89)90602-7},
language = {eng},
}
Hi
I agree with ll answer but you must checking activity before do any thing
good luck
Dominique Liger Thank you for the explanation. It follows the rule of Molar concentration equals Mass concentation/Molar weight.
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