Dithiothreitol, a New Protective Reagent for SH Groups^*

Abstract

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...

Enhancing effect of pre-treatment of cells with misonidazole in hypoxia on their response to melphalan in air

“…n which Under these conditions significant potennt with tiation is still apparent 5h after exposure hypoxic to MISO. (Cleland, 1964) and in our experiments does not protect against the cytotoxicity of melphalan alone. This contrasts with the protective effect of several other thiols against the cytotoxicity of alkylating agents (Conners, 1966).…”

Chemical Reactivity and Spectroscopy of the Thiol Ester-Linked p-Coumaric Acid Chromophore in the Photoactive Yellow Protein from Ectothiorhodospira halophila

“…Hydrolysis of this bond by performic acid (at low pH) yields doubly protonated p-coumaric acid with an absorbance band at approximately 310 nm ( Figure 6A). Both DTT and performic acid are classical reactants for the disruption of disulfide bridges (Cleland, 1964;Sanger, 1949). Our results show that these reactants also disrupt thiol ester linkages.…”

The chemical reactions of the haemagglutinins and neuraminidases of different strains of influenza viruses: II. Effects of reagents modifying the higher order structure of the protein molecule

“…The results, however, tended to be irregular, except in the case of mercuric chloride, these irregularities being attributed to reversibility of the reaction with many of the chemical reagents used. More recent work using dithiothreitol (Cleland, 1964) which reacts completely and irreversibly with the disulphide bond have shown that the disulphide bonds in the virus proteins are inaccessible to attack except under conditions permitting some disruption of hydrogen bonds. A study has therefore been made of the effects on the virus properties produced by reagents acting on non-covalent bonds alone and in combination with dithiothreitol.…”