Disulfides reductions

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. 

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. 

Figure 1.74 Thiol-containing disulfide reductants reduce disulfide groups through a multi-step process producing a mixed disulfide intermediate.

To each ml of the antibody solution, add 6 mg of 2-mercaptoethylamine hydrochloride (final concentration is 50mM). Mix to dissolve. Alternatively, to limit the degree of disulfide reduction, add a 500-fold molar excess of 2-mercaptoethylamine over the concentration of antibody present. 

To overcome these issues, the water-soluble TCEP was synthesized and successfully used to cleave organic disulfides to sulfhydryls in water (Burns et al., 1991). The advantage of using this phosphine derivative in disulfide reduction as opposed to previous ones is its excellent stability in aqueous solution, its lack of reactivity with other common functionalities in biomolecules, and its freedom from odor. 

The use of immobilized disulfide reductants thus has the following advantages over solution phase agents ... 

Immobilized disulfide reductants can be used to reduce all types of biological disulfides without liberating product or by-product contaminants. 

Soluble components that interfere with the assay of free thiol groups are not present if immobilized disulfide reductants are used. 

Immobilized disulfide reductants easily can be regenerated and reused many times. 

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... 

Immobilized disulfide reductants may be synthesized as described in Hermanson et al. (1992) or obtained commercially (Thermo Fisher). 

Ethylenimine may be used to introduce additional sites of tryptic cleavage for protein structural studies. In this case, complete sulfhydryl modification is usually desired. Proteins are treated with ethylenimine under denaturing conditions (6-8 M guanidine hydrochloride) in the presence of a disulfide reductant to reduce any disulfide bonds before modification. Ethylenimine may be added directly to the reducing solution in excess (similar to the procedure for Aminoethyl-8 described previously) to totally modify the —SH groups formed. 

Disulfide reduction occurs over a broad pH range and in a variety of buffer environments. The reaction can be done in denaturants, chaotropic agents, detergents, and in high salt conditions. 

Figure 4.5 DTSSP can form crosslinks between two amine-containing molecules through amide linkages. The conjugates may be cleaved by disulfide reduction using DTT.

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

The following protocol for labeling proteins with 5-IAF is adapted from Gorman (1987). It is a bit unusual in that it involves reduction of disulfides with dithiothreitol (DTT) and immediate reaction with 5-IAF in excess without removal of excess reductant. The procedure can be changed to include a gel filtration step after disulfide reduction to remove excess DTT, but in any case, it should be optimized for each protein to be modified. An alternative to the use of DTT to produce sulfhydryls is thiolation with a compound that can generate free thiols upon reaction with a protein (Chapter 1, Section 4.1). 

Disulfide Reduction with Release of AMCA Label... 

The following protocol is a suggested method for labeling a protein with AMCA-HPDP. It is assumed that the presence of a sulfhydryl on the protein has been documented or created. The reaction conditions can be carried out in a variety of buffers between pH 6 and 9. Avoid the presence of extraneous sulfhydryl-containing compounds (such as disulfide reductants) that will compete in the reaction. The inclusion of EDTA in the modification buffer prevents metal-catalyzed sulfhydryl oxidation. Optimization for a particular labeling experiment should be done to obtain the best level of fluorophore incorporation. 

The homobifunctional photoreactive BASED (Chapter 4, Section 5.1) has two photoreactive phenyl azide groups, each of which contains an activating hydroxyl. Radioiodination of this crosslinker can yield one or two iodine atoms on each ring, creating an intensely radioactive compound. Crosslinks formed between two interacting molecules are reversible by disulfide reduction, thus allowing traceability of both components of the conjugate. 

Disulfide reduction and elution of captured antibody-protein complex... 

The hydrazone bond can be reduced to stabilize the linkage by the addition of sodium cyanoborohydride to a final concentration of 50mM. React for 30 minutes at room temperature with mixing. All operations with cyanoborohydride should be done in a fume hood. If the glycoprotein being modified is sensitive to disulfide reduction and potential denaturation, then this step should be avoided. 

---