From: Tom Laue <tml@hopper.unh.edu>
  To  : Jim Cole <jim_cole@merck.com>
  Date: Fri, 14 Apr 1995 07:51:03 -0400

Re: absorbance of reducing reag

Dear Jim-
The formation of disulfide bonds in proteins requires that an oxidant be
present. For typical protein solutions, it is molecular oxygen that provides
the oxidizing potential. Therefore, if you degas a solution and add only
tiny amounts of DTT or BME, then store in an oxygen-free environment,
disulfide bond formation is usually immeasureably slow.

What we have done (successfully) is to use a buffer that is clear at 230
(PO4 is good, though divalent metal ions require that the PO4 be kept low ~5
mM and cool, Tris is OK, but only at low, 10 mM or so, concentrations, use
Na+ not K+ salts, if possible) including 1-2 mM of fresh DTT. The freshness
is critical, for it is the oxidized form of DTT and BME that absorbs in this
region of the spectrum. You can distill BME or DTT (watch out!) or buy it
fresh, in small amounts and keep it cold, dark and tightly sealed against
air. Flush with argon or nitrogen before capping and sealing with parafilm
before storing the left over DTT or BME.

With the centrifuge cells, flush the sectors with argon (preferred because
it is so dense) or nitrogen just prior to filling. Add your solutions and
seal up. One caveat is that the centerpiece materials contain dissolved
oxygen (particularly Kel-F) which will leach out during the run. You can
minimize this problem by vacuum-treating the centerpieces prior to storage,
then storing them in an argon-filled, sealed container (we have found capped
urine specimen jars to work very well) until used.

These precautions take longer to write out than to observe, and they will
let you work with BME/DTT-containing solutions. 

One last note- you may find that you have a significant (~0.1, even)
baseline offset, either positive or negative, when you scan. This is a
consequence of the differential rate of oxidation of the BME/DTT in the
solvent and solution sectors. It is not a big problem and not too
unexpected. Use of dc/dt for sedimentatin velocity or NONLIN for
sedimentation equilibrium will take this into account.

Best wishes and good luck!
Tom Laue

>                       Subject:                               Time:4:40 PM
>  OFFICE MEMO          absorbance of reducing reagents        Date:4/13/95
>When using the XL-A centrifuge I have been  frustrated by the UV absorbance of
> reagents such as DTT or 2-mercaptoethanol which are  commonly used to keep
>proteins sulfhydryls in the reduced state. At the concentrations usually used
>in biochemical buffers, ~ 1 mM, these reagents prevent one from taking
>advantage of the intense protein absorbance at ~ 230 nm. So far,  my solution
>has been to either omit these reagents or to use 2-mercaptoethanol at a
>concentration of 0.1 mM, which has an O.D. of only 0.02 /cm  at 230 nm.
>However, many proteins are susceptible to oxidation over the long timescale of
>equilibrium experiments. Does anyone know if submillimolar concentrations of
>sulfhydryl reagents are sufficient to keep protein samples reduced in a sealed
>cell over several days. Also, does anyone know of other reducing agents for
>protein sulfhydryls which lack intense absorbance at 230 nm? 
>
>
>