The intracellular environment is reducing. I know there are no disulfide
bridged intracellular proteins in E. coli. In enzyme purification, if some
of the reduced proteins start to form disulfide bridges, the net charge of
the proteins will change, and you will have perhaps doubled the number of
protein bands to get rid off. Without a low concentration of reducing
agents, one has a gradually changing protein composition that can be a
nightmare if ion-exchange steps are the mainstays of a purification scheme.
It is debatable which is the better reducing agent to use. That depends on
your protein of interest, whether it is more stable in the presence of, or
the absence of, an reducing agent. Many enzymes die in reducing
environments, perhaps of missing their natural partners.
The preference of MSH (BME) versus DTT is one of style as much as
chemistry once you know your protein of interest is stable in both. The
reason is that MSH, cheaper, has a half life of about two weeks in open
air. If one works slowly, the mixture of reduced and oxidized thios
provides a -ss- relay environment that is much more oxidative than having
just the oxidized MSH by itself. I only use MSH under an argon atmosphere,
and if I can finish all purification steps in 4 to 7 days. Obviously, if
one has a protein that requires over 10,000 fold purification, the
multistep purification scheme may suffer from the choice of using MSH. In
general, half of to 1 mM DTT is a good choice to keep your extract
composition stable. The redox potential of DTT is much lower than MSH, as
much as about 5 logs (please check this out on the CalBiochem's free
booklet on Clevland's Reagent DTT, I am home today and cannot verify this
for you). DTT oxidation leads to cyclization, and it will stay that way
instead of forming -SS- relays. There is a trans-isomer of DTT called DTE
dithioerytheotol. It cannot cyclize, and is not a popular choice. In a
mixture with MSH, the DTT will keep the MSH reduced. DTT comes in several
grades. You need a good enzyme grade so as not to bring in metals, etc,
that can catalyze oxidation.
After purifications is completed, whether your protein likes to stay in
the presence of reducing agents (hence gradually oxidative) environment,
under different storage conditions, is another issue. That has to be tested.
A retired protein chemist Alfred Zweidler taught me that both DTT and MSH
are too reducing for some proteins. In that situation, his preference is to
use thioglycol. Thioglycol is able to keep reduced proteins reduced, and
yet will not reduce existing disulfide bridges, such as when extracellular
proteins are present. He said that this fact is not well known. I have not
yet tested thioglycol in my purification schemes, and would only pass this
information on as a hypothesis.
Hope this helps!
Tony
At 02:58 PM 1/22/99 -0800, Deb McMillen wrote:
>Hi, all,
>A chemistry question
>
>I have someone who runs their protein preps in low concentration of BME
>and DTT (with basically a PBS recipe at pH 7)--is their some
>advantage/disadvantage to doing this. Does BME or DTT work on the
>other?
>Deb McMillen
>Institute of Molecular Biology
>University of Oregon
>Eugene OR 97403
>
>
>
>
Anthony T. Yeung, Ph.D.
Director, Fannie E.Rippel Biotechnology Facility
Fox Chase Cancer Center
7701 Burholme Avenue, Philadelphia, PA 19111
215-728-2488 voice
215-728-3647 fax
AT_Yeung@fccc.edu email
http://www.fccc.edu.research