Disulfide Reagents

Unfortunately, 2,2 -dipyridyl disulfide is relatively insoluble in aqueous buffers. The use of this compound to modify molecules usually involves prior dissolution in an organic solvent 

4 -dipyridyl disulfide can be used in aqueous solutions, but it has been found that modification of proteins with this reagent yields rapid disulfide bond formation. Only when 2-iminothiolane is used in tandem with 4,4 -dipyridyl disulfide can 4-dithiopyridyl groups be introduced into proteins (King et al., 1978) (Section 4.1, this chapter). This is due to disulfide interchange reactions predominating without the addition of 2-iminothiolane. 

For one-step methods, the use of Ellman s reagent (previous section) to yield a similar reversible sulfhydryl blocking group is probably a better choice with protein molecules. 

Unfortunately, 2,2 -dipyridyl disulfide is relatively insoluble in aqueous buffers. The use of this compound to modify molecules usually involves prior dissolution in an organic solvent such as acetone and then performing the blocking reaction in an aqueous/organic mixture. Many proteins will not tolerate high concentrations of organic solvents without precipitation. 

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Ruan, Q. Chen, Y. Kong, X. Hua, Y. Comparative studies on sulffiydryl determination of soy protein using two aromatic disulfide reagents and two fluorescent reagents. J. Agric. Food Chem. 2013, 61, 2661-2668. 

It involves the ionized form of the thiol and therefore is pH dependent. The reoxidation of sulfhydryl in proteins can be monitored either with a linear disulfide reagent and proceeds as follows ... 

Fig. 5.14. Effect of oxidized DTT on renaturation of reduced BPTI at pH 8.5 (courtesy of Creighton, 1974a) concentrations of oxidized DTT are indicated (in mM) in the graph. The renaturation proceeds more rapidly in the presence of disulfide reagent. In each case, the reaction follows a pseudo-first-order kinetics with respect to reduced BPTI, except at higher concentrations of disulfide reagent when the reaction becomes biphasic (see the curve for 30 mM DTT).

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