Chromatographic methods to study protein-protein interactions

Proteins and enzymes are now generally thought to be organized within the c ell to form clusters in a dynamic and versatile way, and heterologous prote in-protein interactions are believed to be involved in virtually all cellul ar events. Therefore we need appropriate tools to detect and study such int eractions. Chromatographic techniques prove to be well suited for this kind of investigation. Real complexes formed between proteins can be studied by classic gel filtration. When enzymes are studied, active enzyme gel chroma tography is a useful alternative. A variant of classic gel filtration is ge l filtration equilibrium analysis, which is similar to equilibrium dialysis . When the association formed is only dynamic and equilibrates very rapidly , either the Hummel-Dryer method of equilibrium gel filtration or large-zon e equilibrium filtration sometimes allows the interactions to be analyzed, both qualitatively and quantitatively. Very often, however, interactions be tween enzymes and proteins can only be evidenced in vitro in media that mim ic the intracellular situation. Immobilized proteins are excellent tools fo r this type of research. Several examples are indeed known where the immobi lization of an enzyme on a solid support does not affect its real propertie s, but rather changes its environment in such a way that the diffusion beco mes limiting. Affinity chromatography using immobilized proteins allows the analysis of heterologous protein-protein interactions, both qualitatively and quantitatively. A useful alternative appears to be affinity electrophor esis. The latter technique, however, is exclusively qualitative. All these techniques are described and illustrated with examples taken from the liter ature. (C) 1999 Academic Press.