Affinity labeling and photoaffinity labeling

Affinity labeling is a technique for labeling the binding (or active) site of proteins by virtue of a ligand analog to which a chemically reactive or photoreac-tive group has been attached. The latter can form a covalent bond with a suitably oriented amino acid in the binding (active) site. Such a labeling reagent (R-L) would consist of l) a biologically active substance (R) capable of forming a reversible complex with a given protein (P), and 2) a properly positioned, chemically or photochemically reactive leaving group (L) (Figure 1). Upon incubation, the affinity label interacts with its protein counterpart, resulting in the formation of an irreversible protein-ligand complex. This process is illustrated in Figure 1 and described in the following formula:

Figure 1 Affinity labeling.

The formation of the initial reversible complex (P- • ■R-J.) increases the local concentration of the reagent at the active site relative to its concentration in solution. This ensures that the labeling reaction will take place at the binding site and not elsewhere. Following the formation of the reversible complex (P- ■ -R—L), a functional group(s) on the protein adjacent to the active site reacts chemically with the affinity label, resulting in a covalent bond to form P-R. The affinity-labeled protein is either totally or partially inactivated by virtue of covalent bond formation.

The experimental criteria for a good affinity label are stipulated as follows: 1) its reaction with a given protein should result in a concomitant inactivation of the reversible binding activity; 2) it should not significantly label unrelated proteins; 3) specific ligands should protect against the affinity labeling; 4) it should facilitate the localization of the covalently bound ligand at the active site of the protein.

In planning an affinity labeling reagent, one should ensure that the reactive group (/„) is relatively small and does not interfere significantly with the protein-ligand interaction. One should also consider the availability of radioactively labeled precursors and the facility of synthesis and stability. Finally, the labeled residue should remain stable to degradative techniques in order to permit its identification.

The first demonstration of affinity labeling by deliberate design was accomplished in 1961. Although affinity labeling was originally developed for investigations of purified enzymes, it has also added significantly to the study of the structure of active sites in other systems - for example, antibodies, ribosomes and membrane receptors. Affinity labeling of active transport carriers and hormone receptors has been used to identify and isolate these systems.

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