Reducing dithiothreitol

The cellular effects of Ang II are mediated by distinct high-affinity cell surface receptors. Two subtypes can be discriminated by specific nonpeptide antagonists. Additionally, exposure to the sulfhydryl-reducing dithiothreitol (DTT) inactivates Ang II Type 1 receptors (ATiRs), whereas Ang II Type 2 receptors (AT2RS) remain... 

The efficiency of various regeneration systems was investigated for reactivation of reduced chymotrypsinogen (Orsini et a/., 1975). The redox system of 0.5 mM reduced dithiothreitol and 5 mM oxidized dithiothreitol was the most effective, allowing a renaturation yield of 45-50% at pH 8.5 (see Table 5.9). 

The four disulfide bonds of nine homologous short neurotoxins were cleaved by reduced dithiothreitol. The kinetics of refolding, induced by air reoxidation or in the presence of thiol-disulfide exchange reagents, indicated differences in refolding rates. Three toxins refolded 4-10 times more slowly than the six others. It was proposed from consideration of the sequence that a single additional amino acid insertion is responsible for these differences (Menez et al, 1980). These data indicate the role of noncovalent interactions in the formation of disulfide bridges. 

Chemical Properties. The most significant chemical property of L-ascorbic acid is its reversible oxidation to dehydro-L-ascorbic acid. Dehydro-L-ascorbic acid has been prepared by uv irradiation and by oxidation with air and charcoal, halogens, ferric chloride, hydrogen peroxide, 2,6-dichlorophenolindophenol, neutral potassium permanganate, selenium oxide, and many other compounds. Dehydro-L-ascorbic acid has been reduced to L-ascorbic acid by hydrogen iodide, hydrogen sulfide, 1,4-dithiothreitol (l,4-dimercapto-2,3-butanediol), and the like (33). 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

There are other substrates for the E. coli Met(0) peptide reductase, one of which is Met(0)-a-l-PI. The native protein is the major serum elastase inhibitor that functions by forming a binary complex with elastase which inhibits its activity. Met(0)-a-l-PI, on the other hand, which can be formed by treatment of the protein with TV-chlorosuccinimide, cannot form a complex with elastase and therefore is not able to inhibit elastase activity117,118. Table 6 shows, however, that when Met(0)-a-l-PI is incubated in the presence of Met(0)-peptide reductase and dithiothreitol the protein regains its ability to form a complex with elastase and inhibit elastase activity119. Similar to results found with Met(0)-L12 reduced thioredoxin could replace the dithiothreitol as reductant in the enzymatic reaction. 

Rodrlguez-Garrido B, MC Arbestain, MC Monterroso, F Macfas (2004) Reductive dechlorination of a, 3,5, and y-hexachlorocyclohexane isomers by hydroxocobalamin in the presence of either dithiothreitol or titanium(III) citrate as reducing agents. Environ Sci Technol 38 5046-5052. 

Lodish, H. F., and Kong, N. (1993). The secretory pathway is normal in dithiothreitol-treated cells, but disulfide-bonded proteins are reduced and reversibly retained in the endoplasmic reticulum. J. Biol. Chem. 268, 20598-20605. 

Dithiothreitol (DTT) and dithioerythritol (DTE) are the trans and cis isomers of the compound 2,3-dihydroxy-1,4-dithiolbutane. The reducing potential of these versatile reagents was first described by Cleland in 1964. Due to their low redox potential (—0.33 V) they are able to reduce virtually all accessible biological disulfides and maintain free thiols in solution despite the presence of oxygen. The compounds are fully water-soluble with very little of the offensive odor of the 2-mercaptoethanol they were meant to replace. Since Cleland s original report, literally thousands of references have cited the use of mainly DTT for the reduction of cystine and other forms of disulfides. 

---