Characterization of human thyroid peroxidase purified by monoclonal antibody-assisted chromatography.
A murine monoclonal antibody (mAb) to human thyroid peroxidase (TPO) was produced and used for purification of the enzyme. This report describes studies done to characterize the mAb and the purified human TPO. The mAb bound to human TPO at a molar ratio of 1:2 or 1:1 and to human thyroid microsomes with a dissociation constant of 1.2 nM. The mAb had no effect on TPO activity to catalyze guaiacol oxidation. As immunoglobulin G, the mAb consisted of a light chain (kappa) of 26 kilodaltons (kDa) and a heavy chain (gamma 1) of 53 kDa; its pI value was 5.9. Using an mAb immunoaffinity column, 85% of TPO activity was recovered from an ammonium sulfate precipitate of a human thyroid microsomal preparation solubilized with deoxycholate. The purified TPO had a specific activity of 164 guaiacol U/mg protein and an A413 nm/A280 nm ratio of 0.26. The enzyme showed bands only at 107 and 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TPO was very stable at alkaline pH, and its ability to catalyze iodide oxidation and tyrosine iodination was increased to the same extent as its ability to catalyze guaiacol oxidation. For comparison with a conventional method, the immunoaffinity-purified TPO was trypsinized and rechromatographed on the immunoaffinity column. The trypsinized enzyme had a specific activity of 336 U/mg and an A413 nm/A280nm ratio of 0.65. The absorption spectrum of the enzyme suggested that the prosthetic group of human TPO is protoheme IX. These results indicate the value of the immunoaffinity procedure for human TPO purification. The major advantages of this purification procedure are not only high yield and speed, but also recovery of intact enzyme.
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007527:Isoenzymes / Q000302:isolation & purification*; D051379:Mice; D009195:Peroxidase; D010544:Peroxidases / Q000302:isolation & purification*; D013961:Thyroid Gland / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010544:Peroxidases; D009195:Peroxidase