Purification of Bovine Thyroid Peroxidase*

Abstract

Trypsin-solubilized peroxidase [EC 1.11.1.7] activity from beef subcellular particles was resolved by DEAE-cellulose chromatography into 5 fractions, which contained enzymatically active components that ranged in molecular size from 73,000 to 340,000 daltons. The most active fraction (92,000 MW by gel filtration) was further purified (59,000-fold overall) by chromatography on hydroxylapatite. This highly purified peroxidase preparation had an absorbance purity ratio (A410:A280) of 0.55 and oxidized iodide (I3- formation) and guaiacol at rates of 300 and 460 .mu.mol/min per mg, respectively, which were about 3 and 1 1/2 times, respectively, greater than any previously described preparations. The enzyme was contaminated with an inactive protein of equal size. The highly purified peroxidase preparation lost its activity within a few days even when stored at -15.degree. C with iodide. Two of the other DEAE-cellulose fractions contained peroxidase components with estimated sizes (gel filtration) of 73,000, 96,000 and 98,000, which were further purified (1,600- and 15,600-fold) on hydroxylapatite. They were 1/4 to 1/40 as active as the highly purified preparation and also became increasingly labile on purification. The remaining 2 DEAE-cellulose fractions were heterogeneous mixtures of stable peroxidase components whose average molecular sizes (gel filtration) were 220,000, 300,000 and 340,000 daltons, and which were not amenable to further purification on hydroxylapatite. The ratio of guaiacol to iodide activity decreased from 3.0 in the particles to about 1.5 in the highly purified preparations. The turnover numbers of the purest peroxidase component (92,000 MW) for iodide and guaiacol were very similar to those of highly purified, commercial lacto- and horseradish peroxidases. The pH maxima for iodide oxidation were 7.4, 6.0, and 4.5 for thyroid, lacto-, and horseradish peroxidases, respectively, whereas guaiacol oxidation peaked at pH 7.0-7.8 for all 3 enzymes. On the basis of these results and the dissimilar molecular sizes reported for trypsin-solubilized thyroid peroxidase by several other investigators, it was concluded that the molecular size is primarily determined by the conditions of proteolysis.