Enzyme activity by direct monitoring of transamination rxn

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

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Hello,
I am tasked with developing a method for determining enzyme activity of 2 transaminase enzymes using LCMS. The reaction principle is simple enough, load up substrate and coenzyme with sample (serum) --> monitor for set period --> quench reaction with high organic --> quantitate amount of product made or substrate disappearance to then calculate enzyme activity.

My question is, is it possible to directly monitor the transamination products (or disappearing substrate) given that the reaction is double arrowed? Also, most reference method use 'couple reactions' and monitor color changes over period of time for quantifying enzyme activity but some of these enzymes that are 'coupled' ALSO have a double arrow reaction. How can one confidently quantify enzyme activity for reversible enzyme reactions?!

Any thoughts or help would be greatly appreciated!
I'm not sure I understand you question. Are you asking if this kind of measurement can be done given that the reaction is reversible? Enzyme activity for reversible enzymatically catalized reactions has been measured for years. As I read the question, nightmares of the chapter on the Michaelis-Menten equation came back to me from 50 year ago! (Biochem was not my favorite class.) There are numerous ways to get at this activity. One is to quench the reaction after a period of time - the quench stops the enzyme from functioning, locking the ratios of starting materials to products in place. And, yes, the reaction runs both ways until you quench it. One can measure the production of product on the fly. In some cases, one can use a spectroscopic technique and pull out a signal for starting material or product. And it is possible to react the product of the reaction with something else as long as that is a fast reaction relative tot he enzymatic reaction - but, a caution with that approach: you do not have product of the reaction present in the mix, and if the product somehow inhibits the enzyme, you may be missing a key part of what you are looking for.
2 posts Page 1 of 1

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