Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor

The kinetics, binding equilibria and stoichiometry of the interaction betwe en epidermal growth factor and the soluble extracellular domain of the epid ermal growth factor receptor (sEGFR), produced in CHO cells using a bioreac tor, have been studied by three methods: analytical ultracentrifugation, bi osensor analysis using surface plasmon resonance detection (BIAcore 2000) a nd fluorescence anisotropy. These studies were performed with an sEGFR prep aration purified in the absence of detergent using a mild two step chromato graphic procedure employing anion exchange and size exclusion HPLC, The flu orescence anisotropy and analytical ultracentrifugation data indicated a I : I molar binding ratio between EGF and the sEGFR, Analytical ultracentrifu gation further indicated that the complex comprised 2EGF:2sEGFR, consistent with the model proposed recently by Lemmon er al. (1997), Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction do es not adequately describe the EGF : sEGFR interaction and that more comple x interaction mechanisms are operative. Furthermore, analysis of solution b inding data using either fluorescence anisotropy or the biosensor, to deter mine directly the concentration of free sEGFR in solution competition exper iments, yielded Scatchard plots which were biphasic and Hill coefficients o f less than unity. Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a K-D of 2-20 nM and th e other with a K-D of 400-550nM.