Inhibition Of Thyroid Peroxidase Research Articles

Effect of exogenous hydrogen peroxide on iodide transport and iodine organification in FRTL-5 rat thyroid cells

Hydrogen peroxide plays an important role in the regulation of iodination and thyroid hormone formation. In the present study, the effect of exogenous H2O2 on 125I transport and organification was investigated in FRTL-5 rat thyroid cells. Less than 20 passages after subcloning, cells in 24-well plates (6 x 10(4) cells/well) were maintained in a thyrotropin (TSH)-containing medium (6H) for 3 days. A TSH-free medium (5H) was then used for the next 7 days. A 1-h exposure to H2O2 stimulated 125I transport and 125I organification at 0.1-0.5 mmol/l H2O2 and had a toxic effect on FRTL-5 cell at 5 mmol/l. Hydrogen peroxide (0.5 mmol/l) augmented the iodide transport and iodine organification induced by TSH (333 U/l) by two- and threefold, respectively. The biphasic effect of H2O2 was blocked totally by 5-200 micrograms/l of catalase. Catalase by itself did not influence TSH-mediated 125I transport and 125I organification. Hydrogen peroxide (0.5 mmol/l) added to cells in 5H medium increased Na+K(+)-ATPase activity twofold. Ouabain (1 mmol/l), an inhibitor of Na+K(+)-ATPase, completely inhibited the twofold increase in 125I transport induced by 0.5 mmol/l H2O2 but only inhibited H2O2-induced 125I organification by 28%. Methimazole (1 mmol/l), an inhibitor of thyroid peroxidase, had no effect on H2O2-mediated 125I transport but totally blocked the fivefold rise in 125I organification induced by 0.5 mmol/l H2O2. The effect of H2O2 on intracellular cyclic adenosine monophosphate (cAMP) levels also was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective Organification of Iodide Causing Hereditary Goitrous Hypothyroidism

We present a survey of the current state of knowledge about the prevalence of the syndrome involved in defective organification of iodide, and the mechanism of iodination and coupling catalyzed by the thyroid peroxidase (TPO) enzyme. A brief summary of the recent developments in molecular cloning of TPO and regulation of TPO gene expression is also included. Methods for purification of the enzyme and details about the assessment of TPO activity in tissue are briefly explained. The classification of defective organification of iodide is primarily based on the site of the biochemical defect, being quantitative (TPO absent) or qualitative (TPO structure, localization or apoenzyme are defectives). The presence of TPO inhibitors is also briefly described. The rare possibility of an absent source of peroxide (H2O2) causing defective iodide organification is discussed. Analysis of the 118 reported cases shows that the biochemical classification covers a spectrum of abnormalities and it is likely that further molecular biology studies will increase this heterogeneity as well as refining it. Genetic studies have suggested linkage between the TPO gene polymorphisms and the iodide organification defect and can be of importance for carrier detection and prenatal diagnosis. Neonatal screening for hypothyroidism is likely to expand the number of cases available for DNA analysis and possibly the molecular diagnosis. The importance of the mutations that would affect the histidine (His) residues in the translated protein was recently documented by the finding of a deletion removing part of exon 9 and thus also deleting a proximal His residue. The resulting TPO enzyme was inactive for iodide organification and coupling reaction. It is hoped that in time we will be able to expand our knowledge of the molecular diagnosis of the inborn errors of iodide organification.

Antithyroid effects of coal-derived pollutants.

Endemic goiter in iodide-sufficient areas of the United States and Colombia has been linked to watersheds rich in coal and shale, which several reports suggest are the source of water-borne goitrogens. In this report the potential antithyroid activities of aqueous coal and shale extracts and of compounds identified in aqueous effluents from coal conversion processes were assayed in thyroid peroxidase (TPO) and thyroid slice systems. Aqueous extracts of coal and black shale were potent inhibitors of TPO or 125I organification by thyroid slices. The most abundant water-soluble compounds derived from coal are dihydroxy-phenols, thiocyanate, disulfides, and hydroxypyridines. The dihydroxyphenols resorcinol, 2-methylresorcinol, and 5-methylresorcinol (orcinol) were 26.7, 22.5, and 7.2 times more potent, respectively, than the antithyroid drug 6-propylthiouracil (PTU). Other dihydroxyphenols and thiocyanate were less potent but comparable in activity to PTU. All dihydroxypyridines and 3-hydroxypyridine produced inhibitory effects comparable to PTU. None of the disulfides inhibited TPO. The antiperoxidase effects of combinations of two dihydroxyphenols or one dihydroxyphenol and SCN were additive, whereas the effects of a combination of four dihydroxyphenols at threshold inhibitory concentrations were synergistic, resulting in net effects equivalent to or greater than the sum of the individual effects. Thus, antithyroid effects may be greatly amplified by exposure to multiple coal-derived goitrogens and could be many times that produced by any one of the contributing pollutants. These results demonstrate that potent water-borne goitrogens are derived from coal and shale and that their contamination of water supplies could pose a serious threat of thyroid disorders.

Inhibition by immunoglobulin G of synthesis of thyroid hormone in thyroid cultures from hypothyroid patients with goitrous Hashimoto's thyroiditis

Recently, thyroid microsomal antigen was identified as thyroid peroxidase, and thyroid microsomal antibody was found to inhibit thyroid peroxidase activity in vitro. We investigated the possibility that anti-microsomal antibody inhibits the iodination of tyrosine, in vivo. Immunoglobulin G with or without anti-microsomal antibody from hypothyroid patients with goitrous Hashimoto's thyroiditis inhibited thyroid hormone synthesis in cultured slices of normal human thyroid tissue. IgGs with anti-microsomal antibody inhibited 125I thyroidal uptake and thyroid hormone synthesis stimulated by TSH more than normal IgG did. However, the same results were obtained with IgGs without anti-microsomal antibody. This effect did not involve anti-microsomal antibody, anti-thyroglobulin antibody, TSH-binding inhibitor immunoglobulin, thyroid stimulation-blocking immunoglobulin, or the cAMP level of the thyroid tissue. The ratio of organic I to inorganic I with stimulation by TSH in slices incubated with IgG from hypothyroid patients with goitrous Hashimoto's thyroiditis or normal IgG was not significantly different, but was significantly higher in slices incubated with methylmercaptoimidazole. Therefore, IgG from hypothyroid patients with goitrous Hashimoto's thyroiditis mainly suppressed 125I thyroidal uptake, rather than inhibiting thyroid peroxidase activity. In addition, this IgG was present in the serum of 11 of the 12 hypothyroid patients with Hashimoto's thyroiditis studied. This IgG may be involved in the mechanism that causes hypothyroidism in some patients with goitrous Hashimoto's disease.

Mechanism of thyroid peroxidase inhibition by ethylenethiourea

Ethylenethiourea (ETU) is a thyroid carcinogen present in foods formed by degradation and metabolism of ethylenebis[dithiocarbamate] fungicides. ETU inhibits thyroid peroxidase (TPX), the enzyme that catalyzes synthesis of thyroid hormones. Inhibition of TPX-catalyzed reactions by ETU occurs only in the presence of iodide ion with concomitant oxidative metabolism to imidazoline and bisulfite ion. Inhibition ceases upon consumption of ETU with no loss of enzymatic activity and negligible covalent binding of ETU to TPX. TPX inhibition by ETU is unlike that for derivatives of imidazoline-2-thione, which cause suicide inactivation via covalent binding to the prosthetic heme group. These results demonstrate a metabolic route for detoxication of ETU in the thyroid and suggest that low-level or intermittent exposure to ETU would have minimal effects on thyroid hormone production.

Metabolism of35S- and14C-Labeled l-Methyl-2 Mercaptoimidazolein Vitroandin Vivo*

We previously described an in vitro incubation system for studying the mechanism of inhibition of thyroid peroxidase (TPO)-catalyzed iodination by the antithyroid drug 1-methyl-2-mercaptoimidazole (MMI). Inhibition of iodination in this system may be reversible or irreversible, depending on the relative concentrations of iodide and MMI and on the TPO concentration. Metabolism of the drug occurs under both conditions, and in the present investigation we used 35S- and 14C-labeled MMI together with reverse phase HPLC to examine the metabolic products associated with reversible and irreversible inhibition of iodination by MMI. Under conditions of reversible inhibition, MMI was rapidly metabolized and disappeared completely from the incubation mixture. With [35S]MMI, the earliest detectable 35S-labeled product was MMI disulfide, which reached a peak after a few minutes and then declined to undetectable levels. Coincident with the decrease in disulfide was the appearance of two 35S peaks, the major one corresponding to sulfate/sulfite, and the other to a component eluting at 7.5 min. Similar results were obtained for the disulfide and for the 7.5 min metabolite with [14C]MMI. The major 14C-labeled metabolite containing no S appeared to be 1-methylimidazole. Under conditions of irreversible inhibition, MMI disulfide was also the earliest detectable 35S-labeled metabolite. However, MMI decreased more slowly, and after reaching a nadir at about 6 min returned gradually to a level about halfway between the initial and the minimum value. The reformation of MMI appeared to involve the nonenzymatic disproportionation of MMI disulfide. Formation of the 7.5 min peak was also observed, but there was no formation of sulfate/sulfite. The difference in metabolic pattern between the reversible and irreversible conditions is primarily related to the rapid inactivation of TPO that occurs under irreversible conditions. The metabolism of [35S]MMI in thyroids of rats injected with the labeled drug resembles more closely conditions of reversible inhibition, since sulfate/sulfite is the only 35S-labeled metabolite. Neither [35S]MMI disulfide nor the 7.5 min component was detected in rat thyroids in vivo. However, it was demonstrated that these components do not survive homogenization with thyroid tissue, and failure to detect them in vivo does not exclude them as likely intermediates in intrathyroidal MMI metabolism. Based on the observations reported in this study, we present a revised scheme for the mechanism of inhibition of TPO-catalyzed iodination by MMI.

The mechanism for the inhibition of prostaglandin H synthase-catalyzed xenobiotic oxidation by methimazole. Reaction with free radical oxidation products.

Methimazole, an irreversible, mechanism-based (suicide substrate) inhibitor of thyroid peroxidase and lactoperoxidase, also inhibits the oxidation of xenobiotics by prostaglandin hydroperoxidase. The mechanism(s) by which methimazole inhibits prostaglandin H synthase-catalyzed oxidations is not conclusively known. In studies reported here, methimazole inhibited the prostaglandin H synthase-catalyzed oxidation of benzidine, phenylbutazone, and aminopyrine in a concentration-dependent manner. Methimazole poorly supported the prostaglandin H synthase-catalyzed reduction of 5-phenyl-4-pentenyl hydroperoxide to the corresponding alcohol (5-phenyl-4-pentenyl alcohol), suggesting that methimazole is not serving as a competing reducing cosubstrate for the peroxidase. Methimazole is not a mechanism-based inhibitor of prostaglandin hydroperoxidase or horseradish peroxidase since methimazole did not inhibit the peroxidase-catalyzed, benzidine-supported reduction of 5-phenyl-4-pentenyl hydroperoxide. In contrast, methimazole inhibited the reduction of 5-phenyl-4-pentenyl hydroperoxide to 5-phenyl-4-pentenyl alcohol catalyzed by lactoperoxidase, confirming that methimazole is a mechanism-based inhibitor of that enzyme and that such inhibition can be detected by our assay. Glutathione reduces the aminopyrine cation free radical, the formation of which is catalyzed by the hydroperoxidase, back to the parent compound. Methimazole produced the same effect at concentrations equimolar to those required for glutathione. These data indicate that methimazole does not inhibit xenobiotic oxidations catalyzed by prostaglandin H synthase and horseradish peroxidase through direct interaction with the enzyme, but rather inhibits accumulation of oxidation products via reduction of a free radical-derived metabolite(s).

Antithyroid effects of propylthiouracil and sulfamonomethoxine in rats and monkeys

Experiments were performed to investigate the species difference in antithyroid effects of propylthiouracil (PTU) and sulfamonomethoxine (SMM). Sprague-Dawley rats were administered 30 mg/kg of PTU, or 30 or 270 mg/kg of SMM orally for 5 weeks, while squirrel monkeys received 30 mg/kg of PTU or 270 mg/kg of SMM. In rats receiving 30 mg/kg of PTU, a decrease of both serum 3,5,3′-triiodothyronine and thyroxine concentrations and of 131I incorporation into the thyroid hormone precursors was observed, together with elevation of serum thyroid-stimulating hormone concentration, an increase in thyroid weight, and hyperplasia of the follicular epithelium of the thyroid gland. Similar changes were seen in rats receiving 270 mg/kg of SMM; however, the antithyroid effects of SMM were less severe than those of PTU. No change was produced in monkey thyroids by 5-week treatments with 30 mg/kg of PTU or with 270 mg/kg of SMM. The molar concentration of PTU required for the in vitro inhibition of thyroid peroxidase was markedly lower in rats than in monkeys. SMM showed a similar species difference in the inhibition of thyroid peroxidase in vitro, but the enzyme inhibition of SMM was weaker than that of PTU. These findings suggest that rats were more sensitive to the antithyroid effects of PTU and SMM than monkeys, and that inhibition of thyroid peroxidase may play an important role in species difference in the antithyroid effects of the two drugs.

Reversible and irreversible inhibition of thyroid peroxidase-catalyzed iodination by thioureylene drugs.

The mechanism of reversible and irreversible inhibition of thyroid peroxidase (TPO)-catalyzed iodination by thioureylene drugs was investigated using a model incubation system. The major observations may be summarized as follows. 1) TPO is inactivated by 1-methyl-2-mercaptoimidazole and propylthiouracil even in the presence of a relatively high concentration of iodide. The extent of this inactivation depends on the ratio of iodide to drug. 2) Spectral changes observed on oxidation of the drugs with the peroxidase-iodide system were very similar to those observed when the drugs were oxidized nonenzymatically with I3-. These findings support the view that oxidized iodine is an intermediate in TPO-catalyzed oxidation of the drugs. 3) Under conditions where TPO is largely inactivated, inhibition of iodination is complete and irreversible. Drug metabolism, on the other hand, occurs to a limited extent. 4) Under conditions where TPO is only partially inactivated, inhibition of iodination is transient (reversible). In this case, drug metabolism is extensive, and higher oxidation products (sulfate and sulfinic acid) are observed. Inhibition of iodination occurs only during the interval required to reduce the drug concentration to a low level. Thereafter, iodination may occur at a rate close to that observed in the absence of drug. Based on these and other observations, a scheme is presented to explain the mechanism of reversible and irreversible inhibition of iodination. In essence, the type of inhibition depends on the relative rates and extent of TPO inactivation and drug oxidation. These rates, in turn, depend primarily on the iodide to drug concentration ratio. A high ratio favors extensive drug oxidation and reversible inhibition. A low ratio favors TPO inactivation and irreversible inhibition.

Effects of phenylbutazone on thyroid iodine metabolism in vitro.

Abstract. Several alterations of thyroid function parameters have been reported in patients treated with phenylbutazone and we have studied the effect of this drug on the intrathyroidal iodine metabolism. An inhibition of the iodide transport expressed in terms of T/M ratios was observed in bovine thyroid slices incubated with high phenylbutazone concentrations. 10−3m produced 72% inhibition whereas lower concentrations showed no significant difference as compared with controls. Iodotyrosine synthesis was affected by 10−4m and 10−5m phenylbutazone. Formation of iodothyronine synthesis was markedly affected between 10−4m and 10−7m phenylbutazone concentrations. Thyroid peroxidase activity was measured by tyrosine-iodinase, triiodide and guaiacol assays. Soluble, pseudosolubilized and crude peroxidase preparations from bovine glands, as well as the soluble enzyme from human thyroids, have shown inhibition of tyrosine-iodinase activity when incubated with phenylbutazone in concentrations ranging from 10−3m to 10−8m, with a Ki of 4 × 10−6m for bovine thyroid peroxidase and of 6 × 10−6m for human soluble peroxidase. Formation of triiodide was affected between 10−3m and 10−8m phenylbutazone concentrations. Guaiacol peroxidation was scarcely affected by the action of the drug. We have concluded that phenylbutazone affects the intrathyroidal iodine metabolism through the inhibition of thyroid peroxidase in concentrations which are usually present in the sera of patients treated with this drug.

Thiourea and Cyanamide as Inhibitors of Thyroid Peroxidase: The Role of Iodide*

Thiourea, methylmercaptoimidazole, propylthiouracil, and thiouracil are all potent inhibitors of thyroid peroxidase (TPO)-catalyzed iodination. Unlike the cyclic thioureylenes, thiourea at 5 mM has no effect on guaiacol oxidation. If iodide is added to guaiacol assays containing thiourea, enzyme activity is lost. The latter observation may be explained as follows. In the presence of iodide, the iodinating species [TPO.Ioxid], oxidizes thiourea to formamidine disulfide. This product decomposes to cyanamide at neutral pH. We have shown cyanamide to be an inhibitor of the peroxidative and iodinating functions of TPO. Studies in rats demonstrate that doses of thiourea which completely inhibit in vivo protein-bound iodine formation have no irreversible effect on TPO, as measured by guaiacol peroxidation after removal of the thyroids. The major in vivo action of cyanamide is similar to that of thiourea. The data suggest that the primary in vivo and in vitro mode of action of thiourea is the reversible Ioxid-trapping mechanism. The anomalous inhibition of guaiacol peroxidation seen in the presence of thiourea plus iodide derives from the formation of formamide disulfide, followed by its nonenzymic decomposition to cyanamide.

ChemInform Abstract: SYNTHESIS AND ANTIPEROXIDASE ACTIVITY OF PROPYLTHIOURACIL DERIVATIVES AND METABOLITES

Abstract6‐n‐Propyl‐2‐thiouracil (II) läßt sich mit Methyljodid bzw. Benzylchlorid in 60‐65%iger Ausbeute in die S‐Alkyl‐Derivate (I) überführen.

Relative antithyroid effects of 2-thiouracil, 2-thiouridine and 2-thio-UMP.

SummaryThe presence of uridine phosphorylase and thymidine phosphorylase, enzymes capable of converting thiouracil to thiouridine, was demonstrated in thyroid tissue from several species. Uridine kinase which catalyzes further metabolism of thiouridine to thio-UMP was also present. The activity of UMP pyrophosphorylase which converts thiouracil to thio-UMP in a one-step reaction was insignificant.Comparisons of antithyroidal activity in intact rats and with partially purified porcine thyroid peroxidase demonstrate that thiouridine was approximately 10% as potent as thiouracil in vivo but less than 1% as active as thiouracil as an inhibitor of thyroid peroxidase. Thio-UMP was about 5% as active as thiouracil on thyroid peroxidase.These results indicate that it is highly unlikely that thiouracil conversion to a nucleoside or nucleotide is involved in its antithyroidal action.

Inhibition of thyroid iodide peroxidase by hydrazines and NSD-1055 (4-bromo-3-hydroxybenzyl-oxyamine).

Hydrazine (1 X10~M) and phenylhydrazine (1X10~M) inhibit thyroid peroxidase completely. As with horse-radish peroxidase (HRP), thyroid peroxidase is inhibited irreversibly by these compounds. NSD-1055 (4-bromo3-hydroxybenzyloxyamine), which is an inhibitor of histidine decarboxylase and other pyridoxal enzymes, also inhibits thyroid iodide peroxidase as it does horse-radish peroxidase, but NSD-1055 inhibits the peroxidase reversibly. {Endocrinology 88: 1264, 1971) A NDREJEW et al. (1) demonstrated that iso-£»niazid (INH) and hydrazine competitively inhibited horse-radish peroxidase. Later, Altschuler et al. (2) found that INH competitively inhibited peroxidase activity of dog thyroid gland homogenate. We have recently observed that, when horse-radish peroxidase is inhibited by iproniazid (isonicotyl-isopropylhydrazine) and other hydrazines, the inhibitor is bound to the nonheme portion of the enzyme irreversibly and stoichiometrically (3). Thyroid iodide peroxidase is also irreversibly inhibited in vivo and in vitro by iproniazid (4). Iproniazid has been shown to yield isopropylhydrazine, which then interacts with a carbonyl function in monoamine oxidase (5). Thyroid iodide peroxidase is also thought to interact with isopropylhydrazine moiety and thus might be inhibited by other hydrazine compounds. In this paper, we have studied the mechanism of inhibition of purified thyroid iodide peroxidase by hydrazine (H2N-NH2) and phenylhydrazine (CCH5NH-NH2). We also studied the effect of NSD-1055 (4-bromo-hydroxyphenzyloxyamine) on purified thyroid iodide peroxidase and HRP. Materials and Methods 1. Thyroid iodide peroxidase. Soluble enzyme (iodide peroxidase-tyrosine iodinase) from calf thyroid gland was prepared as described previously (5) and further purified by Sephadex G-200 column chromatography (7). The enzyme assay system contained, in 1.0 ml 0.05 ml enzyme (2.5-5 jug protein), 0.5 ml KrebsRinger phosphate buffer (pH 7.5), 11 nmoles KI, 1 /xCi m I , 1 jumole tyrosine, 1 nmo\e glucose and 0.1 rag glucose oxidase (Worthington Biochemical Received December 8, 1970. Supported in part bv USPHS Grant AM-13,377. 1 Division of Neurology, University of Colorado Medical Center, Denver, Colorado. 2 Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637. Co.). The ratio A4i0 mju/A2so m^ was 0.14. Incubation was carried out at 37 C for 20 min. Following incubation, enzymatically formed iodinated tyrosine was isolated on a cation exchange resin, and radioactivity was measured as described previously (8). Enzymatic activities are expressed as per cent incorporation of I into tyrosine. Inhibition studies by hydrazine, phenylhydrazine and NSD1055 were carried out in 2 different ways: a) Enzyme (5 mg protein) was incubated with each inhibitor (2X1O~M phenylhydrazine, 10~ hydrazine and 2 X10~M NSD-1055) in 0.5 ml of KrebsRinger buffer (0.02M, pH 7.4), at the periods of time indicated in the legend of Fig. 2; 0.1 ml of this solution was diluted 10-fold, and aliquots were used for checking residual enzyme activity. The final concentration of each reagent was 10~M phenylhydrazine, 5X10~M hydrazine and 10~M NSD-1055, respectively. Each reagent at the final concentration noted above has no effect on the enzyme activity if added to the incubation mixture directly without preincubation. b) Inhibitor was added at increasing concentrations to the standard assay mixture and inhibition of enzyme by each inhibitor was examined. 2. Horse-radish peroxidase. Horse-radish peroxidase, purified by electrophoresis, was obtained from Worthington Company (Grade A, RZ 2.8). Purity of the enzyme preparation was checked by ultracentrifugation and disc gel electrophoresis. Horse-radish peroxidase was assayed by a method described in the Worthington Biochemical Corporation Handbook. The rate of decomposition of H2O2 by peroxidase with o-dianisidine as hydrogen donor was determined by measuring the rate of color development at 460 m/n. One unit of peroxidase activity is the amount of enzyme which decomposes 1 jtimole of HaCh/min at 25 C. A 0.003% solution of H2O2 in 0.01M phosphate buffer, pH 6.0, and 1% o-dianisidine in methyl alcohol were prepared fresh daily. To 6.0 ml of the H2O2 solution was added 0.05 ml of o-dianisidine solution; one 2.9 ml aliquot of the mixture was transferred to a test cuvette, and a second aliquot to a control cuvette. At zero time, 0.1 ml of diluted enzyme was added to the test cuvette. Pre-incubation of HRP (0.33 mg/ml) with NSD-

Inhibition of thyroid iodide peroxidase [formula omitted] and [formula omitted] by iproniazid

Iproniazid (isopropyl isonicotnyl hydrazine), an irreversible inhibitor of monoamine oxidase, inhibits thyroid iodide peroxidase in vitro and in vivo . As with monoamine oxidase, the peroxidase is inhibited irreversibly by iproniazid which is bound to the enzyme covalently. Spectral changes of iodide peroxidase produced by iproniazid were similar to those with horseradish peroxidase. Inhibition is competitive with KI and of a mixed function type with tyrosine. When rats were given iproniazid 24 hours before injection of tracer amounts of 131I, 131I-incorporation into thyroid protein was inhibited 50%. Pargyline, a non-hydrazine monoamine oxidase inhibitor, did not inhibit thyroid peroxidase in vitro or in vivo .

Purification and Iodinating Activity of Hog Thyroid Peroxidase

Abstract 1. A peroxidase was purified from the particulate fraction of hog thyroid tissue by solubilization with trypsin, chromatography on diethylaminoethyl cellulose, and filtration through Biogel P-100. The final product, although not homogeneous, represented about a 400-fold purification over the starting particulate fraction. 2. The molecular weight of thyroid peroxidase was estimated by gel filtration studies. Elution volumes of proteins of known molecular weight were compared with the elution volume of the peroxidase, the latter being localized by its enzyme activity. The average molecular weight in three determinations was 64,000. 3. An absorption maximum was observed at 410 mµ, suggestive of a hemoprotein. The progressive increase in A410:A280 during the purification procedure suggested that the enzyme activity was associated with a hemoprotein. Inhibitor studies provided further support for this view. 4. When supplemented with H2O2 or, more effectively, with the H2O2-generating system, glucose-glucose oxidase, purified thyroid peroxidase readily catalyzed the iodination of protein, free tyrosine, or free monoiodotyrosine. Iodination was catalyzed over a wide range of iodide concentrations, from l10-7 m to g5 x 10-3 m. In an incubation system containing 1.2 µg of peroxidase, 0.5 mg of purified thyroglobulin, 1.0 mg of glucose, 0.83 µg of glucose oxidase, and I- varying from 50 µm to 4 mm, in 1.0 ml of 0.05 m phosphate buffer, pH 7.0, Vmax was 1.2 mµmoles of I- per min per µg of enzyme protein and Km was 2.5 x 10-4 m I-. 5. No consistent effect of pH on iodination was observed in the range, pH 5 to 7 (the only range tested). Iodination occurred about as readily at pH 5 as at pH 7. 6. During the peroxidase-catalyzed iodination of thyroglobulin or other protein, iodide was rapidly bound as 3-monoiodotyrosine, 3,5-diiodotyrosine, and thyroxine. The best yields of thyroxine were obtained with thyroglobulin as acceptor. The formation of 3',3,5-triiodothyronine was not observed. 7. The iodination of protein by thyroid peroxidase was markedly inhibited by low concentrations of antithyroid drugs. This was true for compounds of the aromatic inhibitor group—resorcinol, p-aminobenzoic acid, sulfathiazole, and others—as well as those of the thioamide group—thiourea, thiouracil, and 1-methyl-2-mercaptoimidazole. Glutathione and thiocyanate were also effective inhibitors, but perchlorate was not. Inhibition by 1-methyl-2-mercaptoimidazole and by thiocyanate was competitive with iodide under certain conditions. 8. Cyanides, and especially azide, were potent inhibitors of thyroid peroxidase, supporting the view that enzyme activity was associated with a hemoprotein. 9. At pH 5.0, iodination of thyroglobulin or of bovine serum albumin with thyroid peroxidase was inhibited at concentrations of I- greater than 1 x 10-4 m. This suggests a possible mechanism for the antithyroid action of excess iodide. However, for reasons which are not yet clear, this inhibition was not observed at pH 7.0. 10. Results obtained with purified thyroid peroxidase support the view that it is unnecessary to postulate the existence of a separate tyrosine iodinase in the thyroid gland. Moreover, the findings suggest that a peroxidase may be involved in the coupling reaction as well as in iodination of protein in the thyroid. 11. No evidence was obtained that 3,5-diiodo-4-hydroxyphenylpyruvic acid is an intermediate in thyroxine formation during the iodination of thyroglobulin with thyroid peroxidase. The results of this study favor the view that the coupling of diiodotyrosine to form thyroxine occurs within the matrix of the protein molecule.

Enzymatic Iodination of Tyrosine and Thyroglobulin with Chloroperoxidase

Abstract 1. Crystalline chloroperoxidase is effective in catalyzing the iodination of or thyroglobulin when supplemented with H2O2, or with the H2O2-generating system, glucose-glucose oxidase. The iodination reaction is very rapid at low concentrations of iodide (5 x 10-5 m), and at low concentrations of (1 x 10-4 m) or thyroglobulin (0.33 mg per ml). 2. Iodide is rapidly bound as 3-iodotyrosine, 3,5-diiodotyrosine, and thyroxine during the chloroperoxidase-catalyzed iodination. After 60 min of incubation with thyroglobulin as acceptor, chromatography of a Pronase digest showed approximately 45 to 50% of the added 131I in the form of 3,5-diiodotyrosine, about 20 to 25% as 3-iodotyrosine, and 4 to 5% as thyroxine. 3. Since iodination of thyroglobulin occurred very effectively in the presence of only a single crystalline enzyme, the results suggest that it may not be necessary to postulate the existence of a separate tyrosine iodinase in the thyroid. Moreover, the formation of appreciable thyroxine in the chloroperoxidase-catalyzed iodination of thyroglobulin suggests that a peroxidase may also be involved in the coupling reaction in the thyroid. 4. The chloroperoxidase-catalyzed iodination of and thyroglobulin is greatly accelerated by 0.1 m chloride and bromide. The mechanism of this stimulatory effect is not yet known, although several possibilities are discussed. 5. Iodination of thyroglobulin and in the presence of chloroperoxidase is readily inhibited by low concentrations of antithyroid drugs and by naturally occurring reducing agents such as cysteine, reduced glutathione, and ascorbic acid. Results obtained with these inhibitors support the view that antithyroid compounds act as competitive inhibitors in the formation of organic iodine in the thyroid. 6. Excess iodide inhibits the chloroperoxidase-catalyzed iodination of and thyroglobulin. These results suggest that the antithyroid effect of iodide, observed under certain conditions, may be partly due to inhibition of thyroid peroxidase. 7. Chloroperoxidase is very effective in catalyzing iodination of insulin and serum albumin. 3,5-Diiodotyrosine formation was at least as rapid in the case of these acceptors as it was with thyroglobulin, but thyroxine formation was not as marked. These results suggest that steric arrangement of newly formed 3,5-diiodotyrosine residues in thyroglobulin was more favorable than in the other proteins for promoting the coupling reaction. 8. No evidence was obtained that 3,5-diiodo-4-hydroxyphenylpyruvic acid (DHP) is an intermediate in the formation of thyroxine during the iodination of thyroglobulin with chloroperoxidase. However, when free was iodinated with chloroperoxidase, evidence was obtained that DHP was involved as an intermediate. To the extent that the chloroperoxidase-catalyzed iodination of thyroglobulin serves as a model for the thyroid coupling reaction, these results suggest that DHP is not an intermediate in thyroxine formation in the thyroid.