General Approaches for Chemical Cross-Linking
Considering the diversity of cross-linking reagents as well as their varying degrees of selectivity and speciŠcity and their different chemical reaction mechanisms, and also the vast number of biomolecular systems in which they may be used, the technical procedures of conjugation and crosslinking are clearly highly diverse. The reaction protocols depend on which reagents are used and for what purpose.1 In Chapter 4, we discussed the choice of these reagents based on their chemistry. In that regard, it is obvious that the right reagent is necessary for the right chemical reaction. The other criterion of choice of these reagents is based on the biological systems to be studied and the exact purpose.2,3 For analysis of subunit organizations in a stable multimeric complex system, for example, various homobifunctional, heterobifunctional, or multifunctional reagents can be used. In this case, analysis of subunit interactions is relatively straightforward. Typically, a puriŠed complex is incubated with one or several different cross-linkers to form covalent bonds with amino acid side chains, such as the ε-amino groups of lysines, the sulfhydryl groups of cysteines, or the carboxyl side chains of aspartate and glutamate. For investigations of weak or transient subunit interactions, where the entities associate only under certain physiological conditions such as ligand-receptor interactions, or transient association of subunits for regulatory or enzymatic purposes, a different set of cross-linkers would be used. These may include photoactivatable cross-linkers, cleavable reagents, or oxidation cross-linking reagents. For detection of conformational changes of proteins, different reagents would be employed. Furthermore, the biological systems to be studied also dictate which cross-linkers should be used. Different reagents would be used for studying nucleic acids, protein-nucleic acid interactions, membrane-bound systems, glycoproteins, etc. The choice of reagent would be based on the speciŠc properties of the target macromolecules. Each of these approaches requires a different reaction protocol. It is therefore not possible to give a standard description of reagents and reaction conditions for general applications. However, a broad classiŠcation of reaction types may be useful for understanding the applications of these reagents. It is possible to generalize the reaction procedures for common functional groups. One must be aware, though, that in some cases different protocols may give rise to different products or ratios of products, and undesired side reactions may occur. One should always be open to the possibility that conditions may be varied in order to achieve the desired results. Thus, if a procedure is not established, trial and error may be necessary to attain the cross-linking objectives. In the following sections, established reaction procedures are provided as a guide for well-known cross-linkers.