help, buffer solution

michelle michelle at twister.tamu.edu
Sun Sep 8 11:40:36 EST 1996


Luis Lopes da Fonseca <llfonseca at mail.telepac.pt> wrote:
>I have been asked the following questions, that i am not able to
>answer.
>
>How would you select a buffer solution, if you wanted to:
>
>a) purify an enzime
>b) characterize kineticly an enzime associated 
>   with the glicolitic pathway
>c) determine the enzimatic activity of a NAD dependent dehydrogenase
>
>and why (justify)?
>
>I thank all the help.
>
>Luis
>llfonseca at mail.telepac.pt
>Lisbon, Portugal

First, you need to know what pH the enzyme is most stable.  Once 
this is determined you should look for a buffer with its pKa at that
pH. 

In order to characterize an enzyme from the glycolitic pathway, 
you must first have an assay to determine the activity of the enzyme.
So you should know the substrates and products for the reaction 
catalyzed by the enzyme.  Is this reaction able to be followed 
optically?  For example the reduction of NADH can be followed at 
340 nm.  If the reaction can not be followed you should use other
enzymes to couple the reaction you are interested in to a reaction that 
can be followed optically.  

To Determine the activity of an NADH dependent dehydrogenase, you just
need to have an assay mix that contains buffer, NADH, and the substrates 
for the reaction.  Start the reacion by adding the dehydrogenase and
monitor the reduction of NADH at 340nm.

I hopes this helps.

Michelle
michelle at twister.tamu.edu



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From: Luis Lopes da Fonseca <llfonseca at mail.telepac.pt>
Reply-To: llfonseca at mail.telepac.pt
Newsgroups: bionet.molbio.methds-reagnts
Subject: help, buffer solution
Date: Thu, 05 Sep 1996 23:05:51 -0700
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Message-ID: <322FBF3F.63D7 at mail.telepac.pt>

I have been asked the following questions, that i am not able to
answer.

How would you select a buffer solution, if you wanted to:

a) purify an enzime
b) characterize kineticly an enzime associated 
   with the glicolitic pathway
c) determine the enzimatic activity of a NAD dependent dehydrogenase

and why (justify)?

I thank all the help.

Luis
llfonseca at mail.telepac.pt
Lisbon, Portugal


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