General questions for enzyme assay - (Aug/27/2015 )

Hello BioForum!

  I have confirmed that my purified enzyme is active by a very rough assay.  I performed a very rough enzyme assay, for the sole purpose of testing whether or not my renatured protein is active or not.  The good news is that the substrate was shown to be degraded by spectrophotometry and that my His-tagged renatured protein is active.  I must now perform a time-course enzyme assay of this enzyme to properly measure the activity of this enzyme.  I have a few questions because I require TWO enzymes to complete the reaction.  I am not sure how to conduct an assay when two enzymes are required for the reaction to be 100% completed.

  I hope that someone may lend advice when there are two enzymes involved.  Should I measure the rate for one enzyme and separately measure for the second enzyme?  Should I test them simultaneously, and how should I measure that? THe thing is that two enzymes may be required to fully complete the reaction and for the reaction to be measured.

  Any insights, link, or sources would be wildly appreciated for enzyme assay.

Thank you, I will post updates.

here is an example of a two enzyme coupled assay:

pyruvate kinase

here is an example of a three enzyme coupled assay:

aspartate aminotransferase

in general, the enzymes other than the enzyme of interest are enough that the enzyme of interest is the limiting factor for the reaction rate.

the reactions of the coupled enzymes should be well established so that you only need to know that they are active and their concentration.

Thanks mdfenko, I really appreciate it.  Can't wait to get into lab tomorrow, hopefully everything will run smoothly.