Solubilization of peroxidase from porcine thyroids and characterization by photoaffinity labelling.
Peroxidase was solubilized without proteolysis from porcine thyroid particulate fraction with the nonionic detergent, 1-O-n-octyl-beta-D-glucopyranoside. The enzyme was able to catalyze the oxidation of guaiacol and the iodination of bovine serum albumin (33 atoms of iodine per molecule protein). Binding studies performed with the partially purified enzyme indicated that the substrates thyroxine (T4) and tyrosine compete for the same binding site on the enzyme. Dissociation constants of 0.9 nM and 0.5 nM were found for T4 and tyrosine, respectively. After photoaffinity labelling with underivatized 125I-labelled T4, gel chromatography on Sephacryl S-1000 revealed a relative molecular weight of about 100 000 for the solubilized enzyme. The peroxidase activity and haem-absorbance peak coeluted from the Sephacryl S-1000 column. SDS-polyacrylamide gel electrophoresis under reducing conditions indicated two major radiolabelled polypeptides, Mr 83 000 and Mr 42 600, as well as a smaller peak at Mr 15 400. The 15 400 molecular weight species is probably not part of the peroxidase complex, since it could partially be removed by Sephadex G-25 prechromatography . Further analyses confirmed that the partially purified enzyme is a haemoprotein absorbing maximally at 412 nm. The Soret band is shifted to 423 nm by reducing agents and the haem-cyanide complex has a maximum absorbance at 416 nm.
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000032:analysis; D006420:Hemeproteins / Q000378:metabolism; D007453:Iodide Peroxidase / Q000302:isolation & purification* / Q000378:metabolism; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D010544:Peroxidases / Q000302:isolation & purification*; D012995:Solubility; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology*; D013974:Thyroxine / Q000378:metabolism
D000345:Affinity Labels; D006420:Hemeproteins; D007457:Iodine Radioisotopes; D006418:Heme; D010544:Peroxidases; D007453:Iodide Peroxidase; D013974:Thyroxine