Purifying and Concentrating ATP

Bruce D. Ray bray at iupui.edu
Tue Jul 22 13:46:49 EST 2003

In article
<Pine.LNX.4.44.0307211935530.29859-100000 at cardinal5.Stanford.EDU>,
Bhadresh Rami <brami at stanford.edu> wrote:

> Hello Folks,
>         I am trying to purify ATP (commercially supplied - Sigma/
> Calbiochem) from its hydrolysis product ADP (even 3-5% ADP contamination
> is not acceptable for my experiments).
>         Running a KCl gradient on an ion-exchange column separates ADP
> from ATP.  However, the ATP gets very diluted coming off the column.
> I cannot increase the stock ATP that I load on the column, because the ADP
> and ATP peaks are closely spaced and at high ATP concentrations, the two
> peaks begin to merge.
>         Moreover, the ATP elutes at about 50-100 mM KCl.
>         My main problem is to remove the KCl from the ATP solution without
> enhancing ATP hydrolysis in any manner AND also concentrate the ATP soln.
> to obtain a greater working concentration (although the latter is not
> absolutely essential).
>         changing pH conditions, temperature, lyophilization, etc., could
> promote hydrolysis of ATP to give ADP, which defeats the purpose of my
> purification procedure.
>         either I am missing something very obvious, or it is indeed
> difficult to do this!
>         Any useful and tested insights on how to purify and concentrate
> ATP without promoting hydrolysis will be of great help.

I've synthesized and purified stable isotope labeled ATP's
for a number of years.  I've never detected any hydrolysis
product either in Sigma's A7699 or in Sigma's A2383 ATP.
In contrast, I've always seen some in various isotope
vendors' products.  What grade are you using?

That said, I do have some comments on purification of ATP.

1. Do not use KCl.  Use a pH 8.5 ammonium bicarbonate buffer
   instead.  As a general rule, use a volatile buffer when
   doing a substrate purification such as this.  I start with
   the column equilibrated to 10 mM buffer and begin elution
   with 50 mM buffer.  With DEAE cellulose, if the resin is
   chloride free, ATP elution will not occur until after 200
   mM buffer.

2. Lyophilize to remove buffer and water with the flask maintained
   at -20 C. You can do this by wrapping the lyophilizer flask with
   tubing through which you run an antifreeze solution cooled by a
   refrigerated recirculator.  With sufficient time, usually about
   three days, almost all of the volatile buffer will evaporate.
   One can speed the process by lyophilizing just to dryness,
   then reconstituting with methanol and rotavaporating down
   several times.

3. Apparently divalent and trivalent metal ions catalyze hydrolysis.
   Therefore, chelex all solutions before use to remove them.

4. I've used both DEAE cellulose and Q-sepharose and find that
   either works.  In my experience, the chromatography must be
   done at 4 C to avoid on column hydrolysis.  If one is purifying
   GTP, this is even more of a problem, but that's only to be

I hope this helps.

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