Two forms of murine epidermal growth factor: Rapid separation by using reverse-phase HPLC

Epidermal growth factor (EGF) has been isolated from acid extracts of C57BL6/J mouse submaxillary glands by using hydrophobic chromatography. High yields of EGF in large amounts (10 mg) can be isolated reliably from the acid extract of the glands in less than 4 hr. The reverse-phase HPLC techniques used to purify the EGF initially yielded what appeared to be a single homogeneous EGF molecule. However, ion pairing reagents (e.g., heptafluorobutyric acid) altered the chromatographic properties, revealing two distinct species: EGF-α and EGF-β. The apparent molecular weights, isoelectric points, and antigenic properties of EGF-α and EGF-β were identical, and both forms stimulated a mitogenic response in 3T3 cells. Analysis of different preparations of purified EGF (commercial and experimental) indicated the presence of EGF-α and EGF-β in constant proportion. Previous EGF binding studies must have used mixtures of ¹²⁵I-labeled EGF-α and ¹²⁵I-labeled EGF-β. The two molecules appear to compete for an identical receptor on the cell surface.