Macroscale high-performance liquid chromatographic separation and instrumental identification of components of diethylaminoethyl murine epidermal growth factor

Abstract

Murine epidermal growth factor (m-EGF), a polypeptide produced as a chromatographically homogeneous peak on diethylaminoethyl (DEAE) cellulose by the method of Savage and co-workers, and characterised as a single compound, has been shown by ourselves and several other groups to be a mixture. The present contribution extends our previously reported work and discuss the separation of this material, termed DEAE—m-EGF, into its components by preparative ion-pair reversed-phase high-performance liquid chromatography (RP-HPLC) on C 18 μBondapak in quantities up to 50 mg per run. Isocratic elution was used and the mobile phase was acetonitrile—water (26:74, v/v, 0.04 M in triethylamine acetate); pH was 5.6, temperature 40°C, and detection was by ultraviolet absorption at 254 nm, and (for some runs) by differential refractometry. Seven significant peaks, four major, three minor, were detected. Of the major peaks, two designated α- and β-EGF, constituted 70% of the total mass and were the most important to our work. Each of the eluted peaks was recovered by lyophilisation, and this product checked for homogeneity by ion-pair RP-HPLC on a C 18 μBondapak analytical column, with utraviolet detection as before. All recovered peaks were found to be homogeneous by this criterion. These chromatographically homogeneous compounds were investigated by modern physicochemical instrumentation to determine their structure. The molecular weight of each of the species was determined by fast atom bombardment mass spectrometry. High-field proton magnetic resonance at 270 MHz provided structural and conformational information. Polarimetry and ultraviolet absorption were also used to characterise the compound. α-EGF, for example, had a molecular weight of 6040 corresponding to the 53 amino acid residue peptide previously designated EGF; β-EGF had a molecular weight of 5930. This molecular weight differential of 110 suggested the hypothesis that β-EGF was a 52 residue peptide corresponding to α-EGF minus the terminal asparagine at position 1. Proton magnetic resonance difference spectroscopy (β spectrum subtracted from α) provided powerful confirmatory evidence for this hypothesis. All materials recovered from RP-HPLC were tested in the sheep and found to retain their biological activity.