Affinity chromatography of thyroid peroxidase using tyrosine coupled to Agarose.
A selective adsorbent for thyroid peroxidase was prepared by attaching tyrosine, a possible substrate of peroxidase, to agarose beads. When partially purified calf thyroid peroxidase was passed through a column containing this adsorbent, the peroxidase activity present was bound to the agarose. The binding of thyroid peroxidase on the adsorbent was inhibited by tyrosine and iodotyrosines. Quantitative elution was readily achieved by modifying the pH of eluting buffers. The peroxidase eluted at pH 8.5 or pH 9.8 was slowly inactivated. During this inactivation, enzyme activity assayed by triiodide formation was not affected, while peroxidase activity assayed by guaiacol oxidation and tyrosine iodination were slowly reduced. Enzyme activity was protected by elution under a partially anaerobic state. Iodide and guaiacol did not interfere with the adsorption of thyroid peroxidase by tyrosine residues on agarose. These data indicate the following characteristics of thyroid peroxidase. 1. Tyrosine and iodotyrosines are the substrates of thyroid peroxidase. 2. Thyroid peroxidase has specific active site(s) for tyrosine and iodide which are independent of each other. 3. The active site(s) for tyrosine and iodotyrosines are common. 4. The active site(s) for tyrosine and guaiacol are similar but are not identical. 5. Thyroid peroxidase is able to bind tyrosine before it is activated to "Complex I" by hydrogen peroxide.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D007453:Iodide Peroxidase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D010544:Peroxidases / Q000302:isolation & purification*; D012685:Sepharose; D013961:Thyroid Gland / Q000201:enzymology*; D014443:Tyrosine
D014443:Tyrosine; D012685:Sepharose; D010544:Peroxidases; D007453:Iodide Peroxidase