Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme.

Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A₄₁₀/A₂₈₀ of 0.54, and its specific activity based on the guaiacol assay (794 μmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of Asp] plus Glu] to Lys] plus Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves α helix or β structure.