PREPARATION AND CHARACTERIZATION OF SPIN-LABELED DERIVATIVES OF EPIDERMAL GROWTH-FACTOR (EGF) FOR INVESTIGATIONS OF THE INTERACTIONS OF EGF WITH ITS RECEPTOR BY ELECTRON-PARAMAGNETIC RESONANCE SPECTROSCOPY

We prepared, purified, and characterized derivatives of epidermal growth factor (EGF) having a nitroxide spin-label attached covalently at the amino terminus. Characterization of these derivatives with regard to the positions of attachment of the spin-label was accomplished by a combination of peptide mapping, protein sequencing, and fast atom bombardment-mass spectrometry. One derivative was chosen for use in initial investigations by electron paramagnetic resonance (EPR) spectroscopy of receptor-bound EGF and its dissociation kinetics. This derivative was found to be equipotent with the native hormone in competitive binding assays, in activating the EGF receptor kinase, and in stimulating the formation of EGF receptor dimers in solubilized cell extracts. Upon binding to solubilized EGF receptor, the spin-labeled EGF derivative became immobilized, giving rise to a visually distinct slow-motion EPR spectrum. The resulting spectrum showed no detectable dipolar interaction between nitroxides, indicating that the nitroxide moieties of spin-labels reacted at the amino termini of receptor-bound spin-labeled EGF molecules are separated by a distance of at least 16 angstrom. An EPR study of the kinetics of dissociation of spin-labeled EGF in the presence of excess unlabeled EGF revealed a rapid component with a k(off) almost-equal-to 2 x 10(-2) s-1 and a less well resolved slow component.