Homobifunctional Sulfhydryl-Reactive Cross-linkers

Many varieties of homobifunctional, sulfhydryl-reactive cross-linkers have been synthesized and described in the literature. Some have been based on bis-mercurial salts (Edsall et al., 1954 Edelhoch et al., 1953 Kay and Edsall, 1956 Singer et al., 1960 Mandy et al., 1961). Such mercurial reactive groups also have been used in reversible covalent chromatography applications to purify sulfhydryl-containing pro- 

The differences within these families of reagents generally relate to the length of the spacer or bridging portion of the molecule. Occasionally, the bridging portion itself is designed to be cleavable by one of a number of methods (Chapter 7). The great majority of homobifunctional, sulfhydryl-reactive cross-linkers mentioned in the literature are not readily available from commercial sources and would have to be synthesized to make use of them. The ones listed in this section are obtainable from Pierce Chemical. 

DPDPB is insoluble in aqueous solutions and should be initially dissolved in an organic solvent prior to addition of a small aliquot to a buffered reaction medium. Preparation of a stock solution in DMSO at a concentration of 25 mM DPDPB works well. The addition of an aliquot of this stock solution to the conjugation reaction should not result in more than about 10% organic solvent by volume in the buffered mixture or protein precipitation may occur. 

DPDPB has two absorbance maxima, one peak at 237 nm (e = 1.2 x lO M icm i) and another at 287 nm (e = 8.8 X 10 M icm i) (Zecherle, 1990 Traut et ah, 1989). Reduction of the pyridyldithio groups causes a shift in the absorbance characteristics of the molecule, such that the peak at 237 nm is shifted to 272 nm and the peak at 287 nm is shifted to 343 nm. This absorbance shift correlates to the release of the pyridine-2-thione groups. 

DPDPB can be used to conjugate reduced antibody molecules to p-D-galactosidase using essentially the same protocol as that described by O Sullivan et al. (1979). 

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