Purification and characterization of epidermal growth factor (beta-urogastrone) and epidermal growth factor fragments from large volumes of human urine.
We purified human epidermal growth factor (hEGF) and hEGF fragments from a benzoic acid precipitate of the materials not adsorbed to silica gel in 330 liters of human urine by a relatively brief, simple, and efficient method employing sequential batch absorption to and stepwise elution from CM-cellulose and DEAE-cellulose, Bio-Gel P-10 chromatography in 50 mM HCl, and three reverse-phase HPLC steps for final resolution and purification of hEGF components. Recovery of hEGF was 29%. Eight apparently homogeneous hEGF components were recovered, each of which had similar activities in a homologous hEGF radioimmunoassay and an EGF radioreceptor assay using human placental membranes. Amino acid composition analysis indicated that there were four pairs of components that represented intact 53-amino acid hEGF, hEGF-(1-52), hEGF-(1-51), and hEGF-(1-50); intact hEGF accounted for one-third of the total materials recovered. Automated Edman degradation of each component for at least 10 cycles revealed a single amino acid sequence identical to that proposed for human beta-urogastrone. Similar immunoreactive hEGF components were observed in similar proportions in freshly voided urine, indicating that they were not artifacts of the purification process. Thus, multiple forms of fully biologically active hEGF (i.e., beta-urogastrone) can be relatively easily and efficiently purified from large volumes of human urine.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004815:Epidermal Growth Factor / Q000302:isolation & purification / Q000652:urine*; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification / Q000652:urine*; D011863:Radioimmunoassay; D011869:Radioligand Assay; D012997:Solvents
D000596:Amino Acids; D010446:Peptide Fragments; D012997:Solvents; D004815:Epidermal Growth Factor