Bifunctional Cross-linking Agents

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

SASD (Chapter 5, Section 3.2) behaves in a similar manner, except it contains an amine-reactive end that can be coupled to proteins and other molecules. Its photoreactive end can be iodinated using any of the radioiodination reagents discussed previously. Just as in the case of APDP, SASD cross-links can be cleaved by a disulfide reductant to transfer the radioactive component to a second molecule. 

Finally, the small amine-reactive and photoreactive cross-linker NHS-ASA (Chapter 5, Section 3.1) can be iodinated to provide a noncleavable radioactive conjugate. 

There are many other compounds that have been investigated for their use in indirect radiolabeling of proteins. For an excellent overview of these chemical reactions, see Wilbur (1992). 

The technology of bioconjugation has affected every conceivable discipline in the life sciences. The application of the available chemical reactions and reagent systems for creating novel complexes with unique activities has made possible the assay of minute quantities of substances, the in vivo targeting of molecules, and the modulation of specific biological processes. Modified or conjugated molecules also have been used for purification, detection, or location of specific substances, and in the treatment of disease. 

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Birnbaumer, M.E., Schrader, W.T., and O Malley, B.W. (1979) Chemical cross-linking of chick oviduct progesterone-receptor subunits using a reversible bifunctional cross-linking agent. Biochem. /. 181, 201-213. 

The neutral hydrophilic polyacrylamides are entirely gels formed by copolymerization of acrylamide (CH2=CH—CONH2) with the bifunctional cross-linking agent, NA -methylene-bis-acrylamide (CH2=CH—CONHCH2 NHCO—CH=CH2) (Eigure 3.4b). 

PBQ is a bifunctional cross-linking agent (Section 11.2.3.1.2) for tissue constituents and is, therefore, used at lower concentrations (0.4-0.5% in PBS). Fixation for 30 min at room temperature has been particularly successful for the visualization of neuropeptides and other small molecules (lessen et al., 1980 lessen, 1983). PBQ reacts not only with proteins but also with sugars. 

For preparing whole ricin IT, the intact toxin is normally derivatized with a bifunctional cross-linking agent before being mixed with antibody prepared for cross-linking by partial reduction of disulphide bonds [183]. 

Obviously, multiple sites for attachment of even stronger bonds (i.e., polar and especially primary valence bonds) are even more important to substan-tivity [22], Multiple covalent attachment sites could conceivably occur via in situ polymerization reactions and with bifunctional cross-linking agents. However, for pragmatic reasons, this is not nearly as important as multiple ionic attachments to hair (e.g., with cationic polymers). 

Polyacrylamide is a synthetic polymer derived from acrylamide monomer introduced as a support matrix for electrophoresis. Polyacrylamide gels result from polymerization of acrylamide with a suitable bifunctional cross-linking agent, most commonly, A/,A/ -methylenebisacrylamide or bisacrylamide. 

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