An Affinity‐Column Procedure Using Poly(l‐proline) for the Purification of Prolyl Hydroxylase

Abstract

An affinity column procedure is reported for purifying prolyl hydroxylase. The procedure is based on the affinity of the enzyme for its competitive polypeptide inhibitor, and involves affinity chromatography in a column containing poly(l-proline) of molecular weight 30000 linked to agarose, and the elution of the enzyme from the column with poly(l-proline) of molecular weight 5700. The enzyme is finally separated from this polyproline by gel filtration. The procedure was employed for purifying prolyl hydroxylase from an ammonium sulphate fraction of chick embryo extract. The recovery of enzyme activity varied in ten enzyme preparations from 50 to 82%, and the purified preparations synthesized from 59.3 to 91.5 pmol hydroxyproline per mg enzyme per h at 37°C with a saturating concentration of (Pro-Pro-Gly)₅ as substrate. The enzyme was pure when examined by polyacrylamide gel electrophoresis as a native protein or in the presence of sodium dodecyl sulphate, and the amino acid composition of the enzyme agreed with that reported previously with only minor exceptions. The molecular weight of the enzyme measured by equilibrium in an analytical ultracentrifuge was 240000, indicating that the enzyme had been isolated in the tetramer form.