Because of its low redox potential (-0.33 volts at pH 7), dithiothreitol (and its isomer, dithioerythritol) is capable of maintaining monothiols completely in the reduced state and of reducing disulfides quantitatively. Since this compound is a highly water-soluble solid with little odor and little tendency to be oxidized directly by air, it should prove much superior to the thiols now used as protective reagents for sulfhydryl groups.Thiol groups such as those of coenzyme A and of some enzymes are readily oxidized in air to disulfides.To maintain these groups in the reduced state, another thiol such as cysteine, glutathione, mercaptoethanol, 2,3-dimercaptopropanol, or thioglycolate is often added so that interchange takes place according to reactions (1) and (2):However, the equilibrium constants of these reactions are near unity, so that a sizable excess of the second thiol must be used. It occurred to this author that if reaction (2) were intramolecular and RSSR were a sterically favorable cyclic disulfide, there would be two products produced from one reactant, so that the equilibrium should be displaced to the right, particularly in dilute solutions. It appeared that a 1,4-dithiolbutane structure would produce the most sterically favorable cyclic disulfide, and that addition of hydroxy groups on the middle carbons should make the compound water soluble and reduce the stench of the thiol groups. Dithiothreitol (DTT) 1 and dithioerythritol (DTE), the threo and erythro isomers of 2,3-dihydroxy-1,4dithiolbutane, were therefore prepared as described by Evans et al. (1949) and found to have the desired properties. Reaction with a disulfide takes place according to reactions (3) and (4), and is complete in several minutes a t p H 8. *Supported in part by a grant (HE-05095) from the National Institutes of Health, U. S. Public Health Service. 1 Abbreviations used in this work: DTT, dithiothreitol; DTE, dithioerythritol; oxidized DTT, cyclic disulfide of DTT (trans-4,5-dihydroxy-o-dithiane); oxidized DTE, cyclic disulfide of DTE (cis-4,5-dihydroxy-o-dithiane). R-SS-R + HS--CH*(CHOH)nCH2---SH RSH + R-SS-CHp(CHOH)zCH2--SH (3) S , S-R S (4 1 / C H ! ' S SH J I I CHOH CH2 CHOHCH2 'C/ HOH 'CdOH I + RSH CH?Attempts were made to determine the over-all equilibrium constant for reactions (3) and (4) by following the reduction of cystine by DTT or DTE, which can be conveniently measured because the thiol groups of D T T and DTE give only 4% as much color as cysteine in the nitroprusside assay of Grunert and Phillips (1951). Within experimental error, reaction between cystine and D T T or DTE went t o completion, even when concentrations of the cyclic oxidized form of DTT or DTE (prepared by ferricyanide oxidation of DTT or DTE) ten times those of DTT or DTE were added.The actual redox potential of D T T was measured by equilibrating the DTT-oxidized DTT system with the DPN +-DPNH system in the presence of lipoamide and dihydrolipoic dehydrogenase, and measuring the amount of DPNH at equilibrium at 340 mp (making suitable corre...