Purifying and Concentrating ATP
nobody at elnino.com
Tue Jul 22 12:16:38 EST 2003
"Bhadresh Rami" <brami at stanford.edu> wrote in message
news:Pine.LNX.4.44.0307211935530.29859-100000 at cardinal5.Stanford.EDU...
> 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.
> Best wishes
> Bhadresh Rami
May I ask you what is the downstream application the purified ATP is going
to be used in? If it is some sort of enzymatic reaction and ADP in the
sample is your concern, then you could use an ATP regeneration system to
remove convert ADP to ATP. One good example is creatine phosphokinase and
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