Use of Disulfide Reductants

One of the most convenient ways of generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. 

In some cases, mild reducing conditions can free one or more —SH groups for conjugation or modification purposes. The creation of free sulfhydryls in this manner allows for site-directed modification at a limited number of locations within the protein molecule. 

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. 

As with all reductants, DTT and DTE will reduce disulfides only if they are accessible. The three-dimensional structure of a protein molecule often contains disulfides buried deep in the inner structure of the polypeptide chains. A protein retaining its native conformation is frequently protected from complete reduction. In the absence of denaturants such as urea, guanidine or SDS, DTT is not capable of reducing all available disulfides within some proteins 

In a comparative study of disulfide reducing agents, it was determined that use of the relatively strong reductants DTT and TCEP required only 3.25 and 2.75 mole equivalents per mole equivalent of antibody molecule to achieve the reduction of two interchain disulfide bonds between the heavy chains of a monoclonal IgG (Sun et al., 2005). This limited reduction strategy retains intact bispecific antibody molecules while providing discrete sites for conjugation to thiols. 

The presence of two sulfhydryl groups in DTT and DTE, however, allows the formation of a favored cyclic disulfide during the course of target protein reduction (Fig. 67). This drives the equilibrium toward the reduction of target disulfides. Therefore, complete reduction is possible with much lower concentrations of DTT or DTE than when using monothiol systems. 

Complete Reduction of Disulfides in Protein Molecules Using DTT Protocol 

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Yang, J, Chen, H, Vlahov, I. R, Cheng, J. X. and Low, P. S. (2006a). Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging. Proc. Natl. Acad. Sci. USA 103, 13872-13877. 

Immobilized dihydrolipoamide (thioctic acid) (Gorecki and Patchornick, 1973 Gorecki and Patchornick, 1975) and immobilized N-acetyl-homocysteine thiolactone (Eldjarn and Jellum, 1963 Jellum, 1964) are the two most commonly used immobilized disulfide reductants. In addition, immobilized TCEP provides a reducing matrix that is free of thiols (Thermo Fisher). Such immobilized reductants successfully can be used to reduce many types of biological disulfides, including small molecules like oxidized glutathione and bovine insulin. They... 

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

Before proceeding to describe the mechanistic features of disulfide reduction, it is useful to first describe the effect of substituents on the stability of thiyl radicals and disulfide radical anions as well as the consequences, from a theoretical viewpoint, of the unpaired electron on both the bond energy and the S—S bond length of disulfides. 

Another special application is given by the use of Cl relaxation to follow the rate of disulfide reduction in proteins [476], This technique involves inter alia the addition of dithiothreitol to effect... 

Diethanolamine (DEA) has replaced MEA as the most widely used amine solvent. High load DEA technologies, such as that developed by Elf Aquitaine, permit the use of high (up to 40 wt % DEA) concentration solutions. The Elf Aquitaine—DEA process allows lower cinculation rates, and has consequent reductions ia capital and utility expenses. DEA tends to be more resistant to degradation by carbonyl sulfide and carbon disulfide than MEA. DEA is, however, susceptible to degradation by carbon dioxide. 

Tetrabutylammonium iodide in trifluoroacetic anhydride is an effective reducing reagent [dS] This system can be used for direct reduction of arenesulfonic acids to the corresponding thiols or disulfides m moderate yields under mild conditions (equation 18) Alkanesulfonic acids are reduced by this system to disulfides with 30-57% yields [dfi]... 

If refined products, such as gasoline, diesel, jet fuel, or kerosene, are transported in a pipeline, where otherwise sour hydrocarbon fluids are transported, there may be an undesired enrichment of sulfur in the refined products. This can be avoided if the oxygen level of the transportant is maintained at below 20 ppm [570]. The dissolved oxygen level in the hydrocarbon product is controlled by reducing the amount of air injection employed in mercaptan or disulfide reduction or by the use of oxygen scavengers prior to the introduction of the refined hydrocarbon product into the pipeline. 

When unamalgamated zinc dust is used, a considerable proportion of yellow insoluble product is often noted at this point. Since the disulfide has been isolated as an intermediate in a similar reduction of m-chlorosulfonylbenzoic acid,4 it is probable that this material is a mixture of disulfides of varying molecular weight. 

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