Application of Chemical Conjugation to Solid-State Chemistry
Insolubilized biomolecules such as proteins and nucleic acids have found increasing uses in medicine, clinical analysis, afŠnity chromatography, and synthetic chemistry applications.1-10 Latex particles coated with antibodies, for example, have become standard in rapid diagnostic tests.9 These immobilized proteins have also provided valuable information about basic protein-protein interactions.11 In addition to enzymes, many different types of proteins, including antibodies, protein antigens, enzyme inhibitors, protein toxins, and peptide hormones, have been attached to insoluble carriers and have been shown to be biologically active. Quite often, immobilization of an enzyme actually results in an increased stability of the protein.2-4
Various approaches have been used to insolubilize proteins.2-6 These methods involve either noncovalent adsorption, encapsulation, and entrapment, or covalent linkage. In this chapter, only covalent immobilization will be considered in detail, particularly the use of chemical cross-linkers. Readers interested in other methods of protein insolubilization are encouraged to consult the numerous books and reviews3,12,13 that address this area. Covalent attachment of a protein to a solid support involves the formation of a covalent bond between a functional group on the protein and a reactive group on the surface of the solid phase. The methods of such covalent bonding are as varied as organic chemistry itself. During the last few decades, thousands of proteins have been immobilized, and hundreds of matrices have become available. In immobilization of proteins to solid supports, several factors must be considered in order to retain the optimum activity of the proteins. The proteins must be attached in such an orientation that their active sites or binding domains are accessible to the surrounding milieu and not buried in or blocked by the matrix or other components on the matrix surface. During the coupling reaction, the protein must be in its active state. Royer and Uy14 have demonstrated, for example, that, in some cases, enzymes can be immobilized in an active conformation in the presence of their substrates. Another factor that must be considered in the covalent attachment of proteins is the possibility of chemically altering the protein in such a way that its reactivity is reduced. Since reactive groups on the proteins are involved in the chemical bonding, it is possible that groups associated with the active site or binding site of a protein could be involved in the reaction. In addition, chemical cross-linking could take part intramolecularly causing conformational changes in the protein, thereby reducing the attachment efŠciency. New developments in the techniques of immobilization of proteins involving emulsiŠcation in the presence of cross-linkers, cross-linked enzyme crystals, cross-linked enzyme aggregates, and carrier-free enzyme particles may resolve some of these problems. In this chapter, the chemistry of protein insolubilization will be discussed.