PMSF dire straits

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Jan 31 11:53:23 EST 2000


pathos wrote:

> In article <86tjvs$81$1 at bgtnsc03.worldnet.att.net>, "simm"
> <simm at worldnet.att.net> wrote:
>
> > I am currently running experiments requiring the use of PMSF. I have to
> > prepare fresh
> > solutions each time which as you may know takes up time and wastes a lot of
> > the
> > reagent. Is there a way to prepare a solution which is stable in storage?
> > are there other similar protease inhibitors? I am specifically interested in
> > stability of PMSF in methanol,  acetone and isopropanol over time. I have
> > heard some colleagues store such solutions for up to a month, but I need
> > confirmation from the scientific body.
>
> The problem with PMSF is not the stability in ethanol or methanol which is
> years, it is the stability after you place it in agueous media (like lysis
> buffer).  The half life now is about 20 minutes.  For my purposes, that
> stinks.

In fact, the stability of PMSF depends both on temperature and pH of your
solution. It may be as short as seconds.
HOWEVER, stability of PMSF is hardly ever a concern because its mode of action.
It covalently modifies the active center of many (serine) proteases, and makes
them proteolytically inactive. This modification is fast, and once it happened it
is irreversible, so you really donut have to care about breakdown of the excess
PMSF afterwards. Remember to add PMSF to lysis buffers directly before use, to
instantly inactivate proteases that are released from digestive organelles. For
protein purification, it is a good idea to treat buffers with PMSF, but the
timing is not critical because of the irreversible mode of action. AND be careful
when handling PMSF, it is really terribly toxic.


Frank







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