Microisolation and Sequence Analysis of Human Epidermal Growth Factor

Abstract

Human epidermal growth factor (EGF) has been characterized by a combination of microisolation, -amino acid analysis and -sequencing. The purification from milk was monitored with a human placental membrane radioreceptor assay using murine EGF as a competitive ligand and was achieved exclusively by the use of reverse phase liquid chromatography (RPLC). The sequential use of preparative, semipreparative and analytical RPLC on octylsilica supports with solvent systems of different solute selectivity such as pyridine formate, triethylammonnium phosphate or perluorocarboxylic acids in the presence of n-propanol or acetonitrile allowed the purification to homogeneity with five consecutive runs, The molecular weight, amino acid composition and NH2-terminal sequence of human EGF were determined. Gas phase micro-sequencing of residues 1 through 17 revealed the following sequence:ASN-SER-ASP-SER-GLU-X-PRO-LEU-SER-HIS-ASP-GLY-TYR-X-LEU-X-ASP which is identical with the NH2-terminius of urogastrone from human urine. Although human epidermal growth factor has been unequivocally identified in human milk and shown to be identical with urogastrone from human urine, the high resolution techniques employed have also revealed the presence of EGF-related molecules which await further characterization. It is possible that EGF and the EGF-related growth factors possess important regulatory functions in normal growth of the human breast during pregnancy and lactation as well as in abnormal growth during mammary tumor formation and progression.