Chromatographic methods to study protein-protein interactions.
Proteins and enzymes are now generally thought to be organized within the cell to form clusters in a dynamic and versatile way, and heterologous protein-protein interactions are believed to be involved in virtually all cellular events. Therefore we need appropriate tools to detect and study such interactions. 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 chromatography is a useful alternative. A variant of classic gel filtration is gel 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-zone equilibrium filtration sometimes allows the interactions to be analyzed, both qualitatively and quantitatively. Very often, however, interactions between enzymes and proteins can only be evidenced in vitro in media that mimic the intracellular situation. Immobilized proteins are excellent tools for this type of research. Several examples are indeed known where the immobilization of an enzyme on a solid support does not affect its real properties, but rather changes its environment in such a way that the diffusion becomes 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 electrophoresis. The latter technique, however, is exclusively qualitative. All these techniques are described and illustrated with examples taken from the literature.
Beeckmans
S
S
Laboratory of Protein Chemistry, Vrije Universiteit Brussel, Paardenstraat 65, Sint-Genesius-Rode, B-1640, Belgium. sbeeckma@vub.ac.be
D000327:Adsorption; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity
D011506:Proteins; D007521:Isocitrate Dehydrogenase; D007655:Ketoglutarate Dehydrogenase Complex