Dear Mike
BME readily autooxidizes (need for metal traces and dissolved O2). Is
yours a metalloprotein? Is your buffer contaminated with metals? Those
situations would make the case a bit different. If not, unless your
BME stock is already oxidized, blocking of the accesible thiols with
BME should take some time. If you treat your protein for 40 min with
fresh BME you should not observe thiol blocking. If you let the
preparation to stay for several days, even at 4-6 °C you may observe
the blocking that you may be observing.
If you want to prevent Cys blocking you can also change to DTT (it is
a dithiol, does not readily form mixed disulfides) and use it with
caution (for thiol reduction it is advisable to use stoichiometric DTT
(with respect to the number of Cys you need to reduce) and 10 fold
excess of BME, look for their redox potentials). Take care of not
"over-reducing" your protein if internal disulfide bonds are expected.
Once reduced I suggest you to remove any reducing agent and store the
protein at -80 °C.
External Cys can be easily oxidized, they are highly expossed to
metals and oxidants (H2O2, BME disulfides, etc). Diffusion is for sure
much faster than SS bond formation, although some cys react at almost
diffusion-controlled rates with oxidants (is yours a thiol'dependen t
peroxidase?) You can take a look at the following reference
(advertising):
2011. Factors Affecting Protein Thiol Reactivity and Specificity in
Peroxide Reduction. Chem Res Toxicol.
Metals can contaminate bad quality materials (water, salts, buffers,
etc), take care of that too. If you need to control the redox state of
your protein you should use DTNB (Ellman´s reagent), or DTDPy, to
measure accesible reduced thiol groups.
Good luck!
Horacio
Quoting Kendall Nettles <[email protected]>:
We see BME adducts in all of our estrogen receptor structures,
though we don't always put them in the models. Sometimes we only see
one or two atoms of the adduct, and in others it is completely
ordered. We only see it on the solvent accessible cysteines. We do
it on purpose. We used to treat the protein with iodoacetic acid to
generate uniform modification of the cysteines, but then we realized
we could get then same homogeneity with 20-50mM BME.
Kendall Nettles
On Apr 15, 2011, at 4:09 PM, "Michael Thompson" <[email protected]> wrote:
Hi All,
I was wondering if anyone knew whether or not it is possible for
reducing agents with thiol groups, such as DTT or
beta-mercaptoethanol (BME), to form covalent S-S bonds with Cys
residues, particularly solvent-exposed Cys? I have some puzzling
biochemical results, and in the absence of a structure (thus far),
I was wondering if this might be something to try to control for. I
have never heard of this happening (or seen a structure where
there was density for this type of adduct), but I can't really
think of a good reason for why this wouldn't happen. Especially
for something like BME, where the molecule is very much like the
Cys sidechain and seems to me like it should have similar
reactivity. The only thing I can think of is if there is a kinetic
effect taking place. Perhaps the rate of diffusion of these small
molecules is much faster that the formation of the S-S bond?
Does anyone know whether or not this is possible, and why it does
or does not happen?
Thanks,
Mike
--
Michael C. Thompson
Graduate Student
Biochemistry & Molecular Biology Division
Department of Chemistry & Biochemistry
University of California, Los Angeles
[email protected]
