Purification and characterization of hypoxia-inducible factor

Guang L. Wang, Gregg L. Semenza

Research output: Contribution to journalArticle

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding protein that activates erythropoietin (Epo) gene transcription in Hep3B cells subjected to hypoxia or cobalt chloride treatment. HIF-1 DNA binding activity is also induced by hypoxia or cobalt in non-Epo-producing cells, suggesting a general role for HIF-1 in hypoxia signal transduction and transcriptional regulation. Here we report the biochemical purification of HIF-1 from Epo-producing Hep3B cells and non-Epo-producing HeLa S3 cells. HIF-1 protein was purified 11,250-fold by DEAE ion-exchange and DNA affinity chromatography. Analysis of HIF-1 isolated from a preparative gel shift assay revealed four polypeptides. Peptide mapping of these HIF-1 components demonstrated that 91-, 93-, and 94-kDa polypeptides had similar tryptic maps, whereas the 120-kDa polypeptide had a distinct profile. Glycerol gradient sedimentation analysis suggested that HIF-1 exists predominantly in a heterodimeric form and to a lesser extent as a heterotetramer. Partially purified HIF-1 bound specifically to the wild-type HIF-1 binding site from the EPO enhancer but not to a mutant sequence that lacks hypoxia-inducible enhancer activity. UV cross-linking analysis with purified HIF-1 indicated that both subunits of HIF-1 contact DNA directly. We conclude that in both cobalt chloride-treated HeLa cells and hypoxic Hep3B cells HIF-1 is composed of two different subunits: 120-kDa HIF-1a and 91-94-kDa HIF-1β.

Original languageEnglish (US)
Pages (from-to)1230-1237
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number3
DOIs
StatePublished - Jan 20 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Purification and characterization of hypoxia-inducible factor'. Together they form a unique fingerprint.

  • Cite this