DTT and BME are reducing agents used for
the chemical reduction of disulfide bonds. They are commonly added to SDS-PAGE
sample buffers and are often used interchangeably. While both DTT and BME are
used to achieve the same purpose in SDS-PAGE, they exhibit different chemical
properties.
BME
This is very volatile and readily
evaporates from solution. Because of its volatility and toxicity, solutions of
BME are often handled in a fume cupboard. The disadvantage of this is that
frequent usage will increase the rate of evaporation, leading to a decrease in
the concentration of a solution of BME over time.
The issue with this is that the chemical
reduction of disulfide bonds within proteins and peptides is an equilibrium
reaction where bonds are continually breaking and re-forming. Accordingly,
excess BME is required to drive the reaction forward to completion.
Reciprocally, insufficient quantities of BME in a given reaction will not
adequately reduce all protein disulfide bonds with some bonds undergoing
reoxidation.
DTT
This is volatile but not to the extent as BME.
Unlike BME, the chemical reaction in reducing disulfide bond linkages within
proteins and peptides is not an equilibrium reaction. A disulfide reduction
reaction using DTT leads to an irreversible change in the DTT molecule where its straight chain structure is altered to a ring structure. Accordingly, use of
DTT will avoid issues of disulfide bond reoxidisation. However, DTT is unstable
in solution and must be made fresh each time.
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