ABSTRACT
Before we can discuss the chemical cross-linking and conjugation of proteins, we must understand some basic protein chemistry, as the cross-linking reagents depend on the reactivities of the constituents of proteins. In most cases, the biological activities of the individual proteins in the conjugated products have to be preserved. This condition dictates that those amino acids involved in the biological functions must be conserved and only those residues not involved in the biological activities be modiŠed. In addition, the three-dimensional (3D) structure of a protein should remain as invariant as possible during the process of chemical modiŠcation. Disturbances of protein structures and properties may occur with reagents that change the charge, size, and other characteristics of the modiŠed amino acid residues. For example, rat liver glycine methyltransferase is completely inactivated on introduction of a large and anionic 2-nitro-5-thiobenzoate, while a smaller and neutral cyano group has no effect.1 Similar results have been observed for the modiŠcation of cysteine residues of many proteins.2 Thus, only those amino acid residues that are not situated at the active centers or settings critical to the integrity of the tertiary structures of proteins may be targets for chemical cross-linking. Such amino acids are ideally located on the surface of the molecule. It follows, therefore, that the identity of the reactive functional groups on the exterior of a protein is often the most important factor controlling the protein’s reactivity toward cross-linking reagents. By knowing which functional groups are located at the protein-solvent interface, one may modify the protein without sacriŠcing its biological activity. However, this strategy is not always as straightforward as one would like. Proteins vary in their 3D structures as well as their surface compositions. A particular amino acid may occur both buried in a protein’s interior and exposed on the protein’s surface. This duality may or may not be true in another protein. In addition, the chemical properties of an amino acid side chain may be in¦uenced by the nearby residues with which it interacts. In fact, such differences in reactivity may be used to evaluate the microenvironment of the residue.3,4 On the other hand, some studies looking at the modulation of biological activities on interaction with other proteins may speciŠ- cally involve the inhibition of its biological activity. One end of a bifunctional cross-linker would react with the active site and the other end would attach to the nearby modulating protein. The reactivity of the active site residues and residues on neighboring proteins need to be well deŠned for the modifying agents to work as planned. In order to understand the principles that govern the reactivity of a protein toward chemical reagents, it is necessary to consider the general properties of the amino acid side chains. With the advent of genomic projects, protein-nucleic acid interactions have become increasingly crucial to our understanding of gene regulation and expression. Chemical modiŠcations and cross-linking of protein-nucleic acids have provided valuable information in many biological systems. It is therefore important to review the chemical reactivities of nucleic acids toward chemical reagents as well.
