Radiommunoassay for human and chick prolyl hydroxylases.
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 usingpoly(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.
D000328:Adult; D000818:Animals; D001665:Binding Sites; D002642:Chick Embryo; D002645:Chickens; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D005333:Fetus; D006801:Humans; D046911:Macromolecular Substances; D008832:Microchemistry; D008875:Middle Aged; D009928:Organ Specificity; D011247:Pregnancy; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis*; D011485:Protein Binding; D011863:Radioimmunoassay / Q000379:methods; D012867:Skin / Q000201:enzymology
D046911:Macromolecular Substances; D011393:Procollagen-Proline Dioxygenase; D004229:Dithiothreitol