Reducing agents disulfide reduction

Using different reducing agents, disulfides and diselenides can be involved in the cross-coupling reaction. In situ reduction of diselenides with polymer-supported... 

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. 

Small molecules containing disulfide bonds (such as cystine-containing peptides) may be reduced and isolated simply by removing the immobilized reductant. Separation of reduced molecules from reductant is much more difficult if a soluble reducing agent is used with low-molecular-weight disulfides. 

Cleavage of disulfide bonds is done easily by incubation with a reducing agent at a level of 10-100 mM concentration. If the disulfides in the crosslinks are the only ones present in the complexed molecules, then reduction will yield unconjugated molecules—one or both of... 

Figure 8.1 Cleavage of disulfide-containing crosslinking compounds can be done using a reducing agent such as DTT. Reduction causes the conjugates to break apart into their original components with each component containing a portion of the crosslinker that terminates in a thiol group.

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

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