[Partial purification of hog thyroid peroxidase using detergent treatment and its spectral changes induced by hydrogen peroxide (author's transl)].
Thyroid peroxidase (TPO) was partially purified from hog thyroid microsomes after solubilization by means of deoxycholate treatment followed by ammonium sulfate farctionation and affinity chromatography with Con A Sepharose. The absorption spectra of the preparation showed the maxima at around 410 nm for oxidized form, 422 nm for dithionite-reduced form and 422 nm for CO complex of reduced form. The cyanide difference spectrum showed a peak at 431 nm and a trough at 403 nm. This preparation was contaminated with little cytochrome b5 and it was shown that the TPO preparation was able to be used for the following spectrophotometric experiments. The addition of H2O2 to the TPO preparation induced the characteristic change in the difference spectrum with a peak at 430 nm and a trough at 407 nm, which was gradually disappeared in a few minutes, but at the high concentration of H2O2 (35 muM) the trough at 411 nm was observed after decomposition of H2O2, accompanying loss of peroxidase activity. This deepening of the trough caused the heme degradation which was dependent with the concentration of H2O2 added and to less extent at the low concentration of H2O2 (3.5 MUM). Since the difference spectrum produced by the addition of small amount of H2O2 disappeared rapidly after the addition of KI or ascorbate and resembled the spectral change due to the formation of Compound II in the reaction of other perioxidases, it was concluded that the difference spectrum with a peak at 430 nm and a trough at 407 nm observed after the addition of H2O2 was ascribable to the formation of Compound II of TPO. Although Compound I was not observed under the experimental conditions used, the results were accounted for the presence of I bound to TPO or other endogeneous reducing agents. We tentatively concluded that Compound I and Compound II are formed in the reaction of TPO with H2O2 as well as in that of horseradish peroxidase.
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008861:Microsomes / Q000201:enzymology; D010544:Peroxidases / Q000302:isolation & purification*; D013053:Spectrophotometry; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology*
D003902:Detergents; D006861:Hydrogen Peroxide; D010544:Peroxidases