b-Mercaptoethanol has the simple structure HO—CH2—CH2—SH, abbreviated here as HOEtSH, and its thiol group is of typical reactivity and ionizes with a pKa of about 9.6. It is one of the most extensively used reagents for protecting biological materials, especially proteins, from oxidation. It has the advantage of being a stable, colorless liquid that is readily miscible with water. Its thiol group will react with oxidants and heavy metals, thereby protecting biological materials, and it can also be used to keep the thiol groups of proteins and other macromolecules in the reduced form. It is frequently used to reduce disulfide bonds in biological materials, especially in preparation for SDS-PAGE.
Mercaptoethanol has limitations in this respect that should be recognized. Its thiol group is not a very potent reductant, very similar to that of glutathione. The equilibrium constant for thiol-disulfide exchange between these two molecules, has been measured with glutathione and will be about the same for mercaptoethanol; values in the region of less than 5 x 10" J have been measured for the overall reaction of Eq. 3 (1). Therefore, a large excess of mercaptoethanol will be required to reduce stable protein disulfide bonds.
A further problem is that mercaptoethanol becomes air-oxidized readily, especially at alkaline pH values, and the disulfide form is a potent oxidant (Eq. 2 and 3). If only 1% of a 10 mM solution of mercaptoethanol becomes oxidized, it will be unable to reduce stable disulfide bonds like those previously described. In the presence of significant amounts of the disulfide form of mercaptoethanol, mixed disulfides of the mercaptoethanol with protein thiol groups will be significant. The reagents dithiothreitol and dithioerythritol are much more powerful in reducing disulfide bonds because they form a stable, intramolecular disulfide bond; the disulfide bond of HOEtSSEtOH can be considered to be intermolecular because two molecules are produced when the disulfide bond is reduced. A solution in which 99% of dithiothreitol has become oxidized will be no less potent at reducing protein disulfide bonds than the above solution of mercaptoethanol that was only 1% oxidized.