Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure.

TLDR

The purification protocol used heat treatment, ammonium sulfate precipitation, and sequential chromatography on Affi‑Gel Blue, heparin‑Sepharose, and hydroxylapatite to isolate hHGF from patient plasma. Purified hHGF was obtained at ~209,000‑fold enrichment with 18% yield, displayed multiple bands (76–92 kDa) that stimulated hepatocyte growth proportionally to band intensity, had a half‑maximal DNA synthesis effect at 3.5 ng/ml, and was more potent than hEGF or insulin, with additive or synergistic effects when combined.

Abstract

Human hepatocyte growth factor (hHGF) has been purified approximately 209,000-fold with 18% yield from plasma of a patient with fulminant hepatic failure. The purification involves heat treatment of plasma, ammonium sulfate precipitation, and chromatography on Affi-Gel Blue, heparin-Sepharose, and hydroxylapatite. Purified hHGF shows several bands with molecular weights between 76,000 and 92,000. Each band shows growth-stimulating activity on cultured hepatocytes which is proportional to the intensity of the band. After reduction of the sample with 2-mercaptoethanol, SDS-PAGE yields two chains with molecular weights of 31,500-34,500 and 54,000-65,000. The effect of hHGF on DNA synthesis by hepatocytes is half-maximal at 3.5 ng/ml. hHGF stimulates proliferation of cultured hepatocytes more effectively than human epidermal growth factor (hEGF) or insulin, and the effect of hHGF is additive or synergistic with the maximal effects of hEGF and insulin. These results suggest that hHGF is a new growth factor which is different from hEGF.