Basic question about PMSF

Pow Joshi via methods%40net.bio.net (by pow.joshi At gmail.com)
Fri Dec 15 15:50:01 EST 2006


On 12/14/06, Dennis At oat.bio.indiana.edu <Dennis At oat.bio.indiana.edu> wrote:
> On 2006-12-14 17:42:43 -0500, Dennis said:
>
> > On 2006-12-13 12:03:47 -0500, "Breslauer" <breslauer1981 At gmail.com> said:
> >
> >> Listen,
> >> is it normal that when adding PMSF (250mM solution in MeOH) to cell
> >> free extract from E.coli (broked by sonication and spinned 12000g for
> >> 30min) to the final concentration of 0,5mM buffer becomes cloudly and
> >> st become to precipitate (probably protein?).
> >>
> >> Thans for any clue
> >>
> >> Julio
> >
> > You are alkylating at least some protein and increasing its
> > hydrophobicity, so a precipitate is not surprising.
> >
> > Why on earth are you adding PMSF to a protein extract?  The common use
> > of PMSF is to add it to a water solution to alkylate and inactivate
> > RNAses.
>
> OOps, I was thinking of TPCK.  Yes, PMSF would be useful to inhibit
> proteases, but it is a rather nondiscriminate alkylator and could cause
> precipitation.

the mechanism of action of PMSF is, apparently,  by suphonylation of
the active site serine-OH, of the serine protease,  rather than
alkylation or arylation or acylation..... the suphonyl group, I think
is -SO3 (-) rather than -CH3 (methyl) or -C6H5 (phenyl) ......
hope that hlps to clarify some issues....

pow

P.S: it's been a while since I studied reaction mechanisms, so whoever
is from core chemistry here, please feel free to add details, and I
would also appreciate correcting my mistakes, if I have made any.




> >
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