Unexpected observation of concentration-dependent dissociation rates for antibody-antigen complexes and other macromolecular complexes in competition experiments.
The dissociation rate constant of bimolecular complexes between macromolecules (k(off)) is often measured in solution by competition experiments and is generally expected to follow first-order kinetics.When measuring k(off) constants by competition for three complexes of high-affinity recombinant antibody fragments with the cognate antigen and for one calmodulin/peptide complex, a surprising dependence between apparent dissociation rate and concentration of competitor (antigen or calmodulin-binding peptide) was observed. Our results may be characteristic for macromolecules consisting of two domains (such as single-chain Fv fragments) and may reflect a transient opening of the two domains which are involved in the binding reaction, and which are connected by a polypeptide linker.
Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETHZ, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
Jörg
J
Scheuermann
Francesca
F
Viti
Dario
D
Neri
D000906:Antibodies / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000276:immunology; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D018448:Models, Immunological; D009113:Muramidase / Q000276:immunology; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000941:Antigens; D002147:Calmodulin; D005353:Fibronectins; D008024:Ligands; D046911:Macromolecular Substances; D010455:Peptides; D009113:Muramidase