Radioimmunoassay for Human and Chick Prolyl Hydroxylases

Abstract

A radioimmunoassay is reported for measuring prolyl hydroxylase. The assay is based on the displacement of radioactively-labelled prolyl hydroxylase from its antibody by the non-labelled enzyme, and on the subsequent precipitation of the enzyme-antibody complex by a cellulose-bound second antibody. Pure prolyl hydroxylase was isolated from foetal human or chick embryo tissues by an affinity column procedure using poly(l-proline). The enzyme was labelled with tritium using a technique of reductive alkylation with formaldehyde and sodium [³H]borohydride. No conversion of the enzyme tetramer to its monomers was found to take place during the tritiation reaction. Experiments on the dissociation of the non-labelled enzyme indicated that the degree of displacement of the labelled enzyme was similar regardless of whether the non-labelled enzyme was in the tetramer form or in that of the subunit monomers. The sensitivity of the radioimmunoassay is of the order of 5-10 ng immunoreactive prolyl hydroxylase. The concentrations of the immunoreactive prolyl hydroxylase assayed with the present method in human serum and skin and in several chick embryo tissues are reported.