From: Jim Cole <jim_cole@merck.com>
  To  : RASMB <rasmb@bbri.eri.harvard.edu>
  Date: 13 Apr 1995 17:02:52 U

absorbance of reducing reag

                       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?