Sulfhydryl-Reactive Derivatives

Figure 5.9 SIAX can be used to modify amine-containing molecules to produce sulfhydryl-reactive derivatives. Subsequent reaction with a thiol compound produces a thioether linkage.

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form a sulfhydryl-reactive derivative. Toxin molecules containing disulfide-linked A-B chains may be reduced with DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with the reduced A chain to effect the final conjugate by disulfide bond formation.

Figure 21.10 Cystamine may be used to make immunotoxin conjugates by a disulfide interchange reaction. Modification of antibody molecules using an EDC-mediated reaction creates a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol can be coupled to the cystamine-modified antibody to form disulfide crosslinks.

Reduction of the pyridyl disulfide end after SPDP modification releases the pyridine-2-thione leaving group and generates a terminal—SH group. This procedure allows sulfhydryl-reactive derivatives such as maleimide-activated enzymes (Chapter 26, Section 2.3) to be conjugated with DNA probes for use in hybridization assays (Malcolm and Nicolas, 1984). 

Conjugates of avidin or streptavidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with SPDP to create a sulfhydryl-reactive derivative, followed by modification of avidin or streptavidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of avidin or streptavidin can be found in Section... 

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