Mechanism of Iodide-dependent Catalatic Activity of Thyroid Peroxidase and Lactoperoxidase*

Mechanisms that have been proposed for peroxidase- catalyzed iodination require the utilization of 1 mol of HzO, for organic binding of 1 mol of iodide. When we measured the stoichiometry of this reaction using thyroid peroxidase or lactoperoxidase at pH 7.0, we con- sistently obtained a ratio less than 1.0. This was shown to be attributable to catalase-like activity of these en- zymes, resulting in unproductive cleavage of HzO,. This catalatic activity was completely iodide-depend- ent. To elucidate the mechanism iodide-dependent activity, the effects of various agents were investigated. The protein concentration in the clear supernatant was determined the expression, Other Methods and Materials-The procedure for measuring HZ0, together with other information relating to methods and materials, is presented in the Miniprint. mixtures contained 100 p~ H& 50 p~ CBZ, and either 0, 25, or 100 pM I- in 0.067 M POI, pH 7.0. A shows effect of varying concentrations of CBZ on H202 degradation. The reaction was initiated with thyroid peroxidase (1.3 pg/ml) at 37 "C, and aliquots of the solution were removed at intervals for measurement of H20, concentration. B shows effect of 50 pM CBZ on 0, evolution after addition of thyroid peroxidase. The incubation


Mechanism of Iodide-dependent Catalatic Activity of Thyroid
Mechanisms that have been proposed for peroxidasecatalyzed iodination require the utilization of 1 mol of HzO, for organic binding of 1 mol of iodide. When we measured the stoichiometry of this reaction using thyroid peroxidase or lactoperoxidase at pH 7.0, we consistently obtained a ratio less than 1.0. This was shown to be attributable to catalase-like activity of these enzymes, resulting in unproductive cleavage of HzO,. This catalatic activity was completely iodide-dependent.
To elucidate the mechanism of the iodide-dependent catalatic activity, the effects of various agents were investigated. The major observations may be summarized as follows: 1) The catalatic activity was inhibited in the presence of an iodine acceptor such as tyrosine.
2) The pseudohalide, SCN-, could not replace Ias a promoter of catalatic activity. 3) The inhibitory effects of the thioureylene drugs, methimazole and carbimazole, on the iodide-dependent catalatic activity were very similar to those reported previously for thyroid peroxidase-catalyzed iodination. 4) High concentrations of 1inhibited the catalatic activity of thyroid peroxidase and lactoperoxidase in a manner similar to that described previously for peroxidase-catalyzed iodination.
On the basis of these observations and other findings, we have proposed a scheme which offers a possible explanation for iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase. Compound I of the peroxidases is represented as EO, and oxidation of Iby EO is postulated to form enzyme-bound hypoiodite, represented in our scheme as [EOII-. We suggest that the latter can react with Hz02 in a catalaselike reaction, with evolution of 0 2 . We postulate further that the same form of oxidized iodine is also involved in iodination of tyrosine, oxidation of thioureylene drugs, and oxidation of I-, and that inhibition of catalatic activity by these agents occurs through competition with HzO, for oxidized iodine.
In the course of experiments in which we measured the stoichiometry between H20, utilization and organic iodine formation in thyroid peroxidase-and lactoperoxidase-catalyzed iodination, we observed nonproductive disappearance of TO whom all correspondence should be addressed.
H202. This raised the possibility that these enzymes possess catalatic activity. Evidence for iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase was recently presented in a brief report from this laboratory (I).
In the present communication we describe the stoichiometry experiments that led us to investigate the catalatic activity of thyroid peroxidase and lactoperoxidase. These are described in the Miniprint. ' The main text of the paper presents further studies on the mechanism of the iodide-dependent catalatic activity of these peroxidases. A scheme is proposed to explain this activity and to show its relationship to various peroxidative functions of these enzymes.

MATERIALS AND METHODS
Measurement of 0, Evolution-Oxygen evolution was measured polarographically with a Clark-type oxygen electrode. The waterjacketed cell and the oxygen electrode were purchased from Gilson Medical Electronics, Middleton, WI. The input and output electronic components were built in our Bioengineering Department from schematic diagrams kindly provided by Gilson. Stirring was accomplished with a TRI-R model MS-7 micro-submersible stirrer (TRI-R Instruments, Rockville Center, NY), and recording was performed on a Gilford model 242 recorder (Gilford Instruments, Oberlin, OH).
Incubations were performed in 0.067 M phosphate, pH 7.0, a t 24 "C. All of the components of the incubation mixture, minus the enzyme, were added to the reaction cell in a total volume of 1.85 ml. After the cell was sealed with the glass, capillary bore stopper, the baseline was allowed to stabilize (1-2 min). The reaction was started by adding thyroid peroxidase, lactoperoxidase, or catalase in a small volume (<lo pl) through the capillary bore of the stopper. A Drummond microdigital dispenser (Drummond Scientific Co, Broomall, PA) was very convenient for this purpose. Instrument response was determined in each experiment by addition of catalase (1 pg/ml) to a solution containing 100 p M H202 (equivalent to 50 p M 02). The linearity of the response was established by adding small aliquots of Hz02 in 25 W M increments to 1 pg/ml of catalase in the reaction chamber. Electrode membranes were replaced daily.
When incubations were performed with lactoperoxidase, it was necessary to wash the cell extensively to remove residual enzyme, presumably adsorbed to the membrane or to the glass wall of the cell. This was accomplished by rinsing the reaction cell successively with several rinses each of deionized water, 0.7 M NH40H, 95% ethanol, and finally deionized water again. Rinsing with water alone sufficed to remove residual thyroid peroxidase or catalase from the reaction cell after these had been used in the incubation system.
Catalase-Bovine liver catalase (2 X crystallized, suspension in water containing 0.1% thymol, 30,000-40,000 units/mg) was purchased from Sigma (catalog No. C-100). An aliquot of the well mixed suspension was diluted 200-fold with 0.067 M phosphate, pH 7.0, warmed at 37 "C for 5 min, and centrifuged a t 2500 rpm for 10 m~n . The protein concentration in the clear supernatant was determined from the expression, milligrams of protein/ml = AZRO X 0.667 (29).
Other Methods and Materials-The procedure for measuring HZ0, together with other information relating to methods and materials, is presented in the Miniprint.

Effect of Iodide Concentration on Degradation of H202 by
Thyroid Peroxidase and Lactoperoxidase- Fig. 7 shows that the disappearance of 100 p~ Hz02 from an incubation mixture containing thyroid peroxidase or lactoperoxidase was highly dependent on the I-concentration. Very little, if any, H,02 was degraded in the complete absence of I-. However, as little as 1 pM Ihad a definite stimulatory effect, especially with thyroid peroxidase. Increasing concentrations of 1resulted in progressive increases both in the rate and the extent of H20z disappearance. At the highest Iconcentration (100 p~) all of the H202 was degraded within 1 min in the presence of lactoperoxidase, and more than 90% was degraded in a similar period in the presence of thyroid peroxidase. Low concentrations of I-were more effective with latter, but high concentrations of I-were more effective with the former. Presumably, therefore, (K& is greater for thyroid peroxidase but V,,,,, is greater for lactoperoxidase. Only a small fraction of the H202 that disappeared from the incubation mixture could be accounted for by oxidation of Ito 12. This was most apparent in the samples containing 1 p~ I-. Under these conditions 25 ~L M H202 was degraded by thyroid peroxidase and 15 p~ H202 by lactoperoxidase. In samples containing 100 PM Ionly about 5 pM 1, was formed (data not shown), compared to the 90-100 PM H202 that disappeared from the reaction mixture. Utilization of H202 for Ioxidation, therefore, could not account for the disappearance of H20, observed in Fig. 7 . Rather, it appeared that thyroid peroxidase and lactoperoxidase display catalatic activity in the presence of iodide. Further evidence for this was obtained in experiments measuring 0 2 evolution, described in the following section.
0, Evolution from H202 Catalyzed by Thyroid Peroxidase, Lactoperoxidase, and Catalase: Effect of Iodide and Iodine Acceptor- Fig. 8 shows the results of experiments in which O2 evolution was measured in incubation mixtures containing HZ02 and thyroid peroxidase, lactoperoxidase, or catalase. Fig. 8a shows results obtained with thyroid peroxidase in the absence of iodide. Under these conditions very little evolution of OS was detected. However, in the presence of 10 p~ 1- (Fig.  ab), a rapid and marked evolution of 0, was observed. Addition of catalase after the reaction had plateaued showed no further evolution of 02, indicating that all the H202 had been degraded. Fig. 8c shows results obtained when the incubation mixture included an iodine acceptor (150 p~ tyrosine). Under these conditions Oz evolution was greatly reduced, both in rate and in extent. Only about 8 nmol/ml of O2 were evolved, compared to about 50 nmol/ml observed in the absence of tyrosine (Fig, 86). The much reduced 0, evolution cannot be attributed to lack of H202, since addition of catalase after the reaction had plateaued resulted in release of an additional 36 nmoi of 0 2 . It seems more likely that utilization of the iodide €or tyrosine iodination was responsible for the greatly reduced catalatic activity, since in other experiments (results not shown) it was observed that under the conditions of Fig. 8c almost all the iodide was utilized for tyrosine iodination within 1 min after the initiation of the reaction. Fig. 8, d, e, and f, shoxvs results of analogous experiments performed with lactoperoxidase. As in the case of thyroid peroxidase, very little 0, was evolved in the absence of I- (Fig. 8 4 . However, in the presence of 25 GM I-, O2 evolution was very evident (Fig. 8e). The reaction was slower with lactoperoxidase than with thyroid peroxidase, in agreement with the results shown in Fig. 7. When tyrosine was present in the incubation mixture (Fig. 8f), O2 evolution was greatly diminished, although not quite to the low level observed with thyroid peroxidase (Fig. 8c). The somewhat higher level obtained with lactoperoxidase is probably attributable to the higher iodide concentration that was used with this enzyme.
Results obtained with catalase are shown in Fig. 8   H,O,-Thiocyanate anion (SCN-) is classified as a pseudohalide and bears many resemblances to iodide in its chemical behavior. It was of interest in the present study, therefore, to determine whether thiocyanate, like iodide, stimulates catalatic degradation of H,02 by thyroid peroxidase and lactoperoxidase. Experiments were also performed with perchlorate (C104-), a potent inhibitor of iodide transport in the thyroid gland (30). Fig. 9A shows the disappearance of H202 from incubation mixtures containing Hz02, thyroid peroxidase, and either SCNor clo4-. No effect on HZ02 degradation was observed with Clod-. On the other hand, a progressive increase in both the rate and extent of H202 degradation was observed with SCN-as its concentration was raised from 0 to 250 p~. Although the results with SCN-resemble those observed with iodide, SCN-was much weaker than I-in promoting H20, degradation. As seen in Fig. 9A, 100 p~ SCN-was less effective than 10 pM I-. It seemed possible, therefore, that the mechanism of H202 degradation observed with SCN-is different from that seen with I-.
That this is indeed the case was demonstrated in experiments measuring 0, evolution. As shown in Fig. 9B, no catalatic activity of thyroid peroxidase was detected in an incubation mixture containing 250 pM SCN-(Curue a), in contrast to the results obtained in the presence of 100 p~ I-(Curue b). Thus, the disappearance of HZ02 observed with 250 p~ SCN-in Fig. 9A cannot be attributed to catalatic activity. Since SCN-has been reported (31) to be oxidized by lactoperoxidase + H202, it seemed likely that the observed disappearance of H,02 could be explained by thyroid peroxidasecatalyzed oxidation of SCN-.
Oxidation of SCNby H20, in the presence of thyroid peroxidase was measured both spectrophotometrically and by examination of the reaction products of "S-labeled SCN-(results not shown). By following the absorbance (AZ~R) of SCN-on addition of successive increments of H202, we observed that 2.7 mol of HZ02 were required for complete oxidation of 1  Effect of Methimazole on Iodide-dependent Catalatic Actiuity of Thyroid Peroxidase-In a previous study in this laboratory (32) we showed that the thioureylene drug, "I,, inhibits thyroid peroxidase-catalyzed iodination reversibly or irreversibly. The type of inhibition is determined largely by the ratio of iodide to drug. When this ratio is high, enzyme-catalyzed oxidation of the drug is favored and inhibition of iodination is reversible. However, at lower iodide to drug ratios, thyroid peroxidase is rapidly inactivated, and inhibition of iodination is irreversible. It was of interest to examine the effect of MMI on the iodide-dependent catalatic activity of thyroid peroxidase.
Results obtained with MMI are illustrated in Fig. 10. Effects of 10, 25, and 50 p~ MMI were determined on H20, disappearance ( all the H,02 disappeared from the reaction mixture. The rate was slower with 25 p~ than with 10 p~ MMI. Both drug concentrations gave slower rates than the control sample containing no MMI. At the highest MMI concentration (50 p~) , both the rate and the extent of the HZ02 disappearance were markedly inhibited. Under these conditions only about 25% of the H202 disappeared from the reaction mixture.
Investigation of the effect of MMI on the catalatic activity of thyroid peroxidase, illustrated in Fig. 10B, provided information about the mechanism of the H20a disappearance depicted in Fig. 1OA. The observation that no O2 was evolved in the presence of 50 pM MMI (Curue d ) indicates that the disappearance of about 25% of the H20, from the reaction mixture ( Fig. 1OA) cannot be attributed to catalatic activity of thyroid peroxidase. It appeared likely, therefore, that H2OZ was utilized to oxidize MMI. This was shown to be the case by performing the reaction in a cuvette and following the peak absorbance of MMI in the recording spectrophotometer (results not shown). The decrease in A,,,, corresponded to the disappearance of approximately 40 nmol/ml of MMI. Assuming that the sole oxidation product was the disulfide of MMI, this reaction would consume 20 nmol/ml of H202. Within the limits of the experimental error of the measurements, this could readily explain the observed disappearance of H202 from the reaction mixture. Parallel experiments in which enzyme activity was followed by guaiacol   (32), demonstrated that the thyroid peroxidase was completely inactivated during the course of the MMI oxidation and that failure to oxidize the MMI to higher oxidation products could be attributed to enzyme inactivation. In contrast to the results with 50 PM MMI, there was extensive catalatic activity of thyroid peroxidase in the presence of 25 p~ MMI (Fig. 1OB, Curve c ) . Under the latter conditions the 0, evolved on addition of the enzyme was equivalent to 75 ~L M H202, showing that the disappearance of H,O, in the presence of 25 p~ MMI in Fig. LOA was largely attributabIe to catalatic activity of thyroid peroxidase. The remainder of the H202 (25 p~) was presumbly utilized for oxidation of the 25 p~ MMI in the reaction mixture. The stoichiometry of the reaction (1 mol of H2O2/mol of MMI) indicates that the MMI was oxidized beyond the disulfide stage. As shown in our previous study (32), under conditions where MMI was oxidized to higher oxidation products, inac-tivation of thyroid peroxidase was not a major factor in limiting enzyme activity.
Results obtained with 10 PM MMI were comparable to those observed with 25 ~L M MMI. When the reaction mixture contained 10 ~L M MMI, the 0, evolved on addition of thyroid peroxidase was equivalent to 89 p~ H202 (Fig. 10B, Curve b). The remainder of the H202 was presumably utilized for oxidation of the 10 p~ MMI in the incubation mixture.
Correlation between the Effect of M M I on O2 Evolution and on Iodination-It may be seen in Fig. 10B that there was a time lag between the addition of thyroid peroxidase and the start of 0, evolution in samples containing 10 and 25 pM MMI. We reported (32) a similar (though longer) lag in previous studies in which we investigated the inhibitory effects of MMI on thyroid peroxidase-catalyzed iodination in reaction mixtures containing glucose-glucose oxidase as the source of HzO,. It was of interest in this connection to determine whether, under the conditions of the present ex-periment, we would observe a lag in iodination comparable to that seen in Fig. 10B. Accordingly, experiments were performed under the conditions used in Fig. 10B, except that an iodine acceptor (bovine serum albumin) was added to the reaction mixture. The results are shown in Fig. 11. A lag in iodination was observed, which compared very well with that seen in the oxygraph experiments. With 25 IM MMI, iodination was completely inhibited for 30 s, agreeing very closely with the lag in catalatic activity shown in Fig. 10B. With 10 PM MMI, iodination was greatly inhibited at 15 s, corresponding closely to the approximately 10-s lag observed for catalatic activity. Both iodination and catalatic activity were irreversibly inhibited by 50 PM MMI, presumably because the thyroid peroxidase was inactivated under these conditions (32).

Effect of Carbimazole on H 2 0 p Degradation and Catalatic
Actioity-Carbimazole is a derivative of MMI, which, unlike MMI, does not readily inactivate thyroid peroxidase. As described in a previous communication (32), it acts as a purely reversible inhibitor of thyroid peroxidase-catalyzed iodination. It was of interest, therefore, to compare its effects with those of MMI described in the preceding section. Iodination studies performed as in Fig. 11 indicated that 50 KM CBZ inhibited iodination more than 95% (results not shown). In this case the result resembled that obtained with MMI (Fig. 11). However, the mechanism of inhibition of iodination was different. CBZ inhibited iodination competitively by utilizing the H202 for its own oxidation. However, MMI inhibited iodination primarily through inactivation of thyroid peroxidase.
Effect of Excess Ion Catalatic Effect of Thyroid Peroxidase-High concentrations of iodide inhibit thyroid peroxidase-catalyzed iodination (6,8), presumably by utilizing the Ifor I, formation rather than for iodination. It was of interest, therefore, to test the effect of increased concentrations of I-on catalatic activity. Experiments were performed both with thyroid peroxidase and with lactoperoxidase, and the results are shown in Fig. 13. Catalatic activity of both enzymes was progressively inhibited as the concentration of I-was raised from 30 pM to 10 mM. A slight inhibitory effect was observed at 100 PM I-, the standard concentration used in most of our studies. The inhibitory effect of I-was greater for lactoperoxidase than for thyroid peroxidase. This correlates with observations made in this laboratory indicating that lactoperoxidase is much more active than thyroid peroxidase in oxidizing I-to Ip (data not shown). As the concentration of Iwas increased in the incubation mixtures, a progressive increase in the formation of 13was observed, evident by its characteristic color. This suggested that oxidation of I-to I, was a competing reaction and that the inhibition of catalatic activity observed in Fig. 13 involves competition between H202 and Ifor some common intermediate. The reaction was initiated at 24 "C with either 1.3 pg/ml of thyroid peroxidase or 1.0 pg/ml of lactoperoxidase and was continued until all the H202 was utilized.

DISCUSSION
The mechanism of thyroid peroxidase-and lactoperoxidasecatalyzed iodination of tyrosine and tyrosyl residues in protein has been discussed by various investigators. Mechanisms involving an iodine free radical (2,8,9), iodinium ion, (13-15, 34, 35), or hypoiodite (1, 10) have been proposed. According to all these schemes it would be expected that 1 mol of Hz02 is required for organic binding of 1 mol of iodide. In the present study we examined the stoichiometric relationship between organic iodine formation and H20, utilization in iodination reactions catalyzed by thyroid peroxidase and lactoperoxidase. Iodination of tyrosine, bovine serum albumin, or low iodine thyroglobulin was carried out under conditions where H,O, was limiting. The H202 was either generated with glucose-glucose oxidase or added as a bolus. With generated H,O, the ratio, moles of iodide bound per mol of H202 utilized, although close to unity, was always less than 1.0 (generally about 0.9). Similar ratios were observed with low levels of directly added H,02 (5-10 p~) , but at higher concentrations of H,O, (100 FM) the ratio declined to less than 0.5. Despite the low ratio, it was observed that all the H20, disappeared from the reaction mixture, indicating that H202 was utilized in some reaction other than iodination. This raised the possibility that thyroid peroxidase and lactoperoxidase possess catalase-like activity, resulting in unproductive cleavage of H,O,. Catalatic activity was established by demonstrating that O2 is readily evolved from H,O, by thyroid peroxidase and lactoperoxidase. This activity, however, was completely iodidedependent. The pseudohalide, thiocyanate, could not replace iodide, although thiocyanate itself was readily oxidized by H,O, in the presence of the peroxidases. The observation of the iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase explains our inability to obtain a ratio of 1.0 for moles of I bound per mol of H 2 0 n utilized in the peroxidase-catalyzed iodination reactions.
The distinction between peroxidases and catalase is generally based on the manner in which they utilize H202. Catalase catalyzes the rapid degradation of H202 to form oxygen.
Peroxidases catalyze the utilization of H20z for the oxidation of a donor compound. Thomas et al. (36) showed that chloroperoxidase, a mold enzyme, displays significant catalatic activity and that it bridges some of the classical differences between enzymes of the peroxidase and catalase type. The catalatic activity of chloroperoxidase was observed both in the presence and in the absence of C1-. The pH optimum for the halide-independent reaction (4.5) was greater than that for the halide-dependent reaction (2.75). Subsequent studies by Manthey and Hager (37) demonstrated that bromoperoxidase, isolated from a marine alga, also catalyzed the evolution of O2 from H202. Significant activity was observed at pH 6.8 in the absence of halide, but the activity was greatly enhanced in the presence of 100 mM Br-. In the present study we have shown that thyroid peroxidase and lactoperoxidase display significant catalatic activity. This activity, in contrast to that of chloroperoxidase and bromoperoxidase, is completely halide-dependent. A possible catalatic activity of thyroid peroxidase was briefly reported by Ohtaki et al. (13, 14), but no mention was made of an iodide requirement.
To elucidate the mechanism of the iodide-dependent catalatic activity, we have made use of previously reported (32, 38) inhibitors of thyroid peroxidase-and lactoperoxidasecatalyzed iodination. Following is a summary of observations made in this and in our previous study (I), which led to the development of a general scheme showing how the iodidedependent catalatic activity may relate to the various peroxidative functions of these enzymes (see below).
1 ) Thyroid peroxidase and lactoperoxidase exhibit marked catalatic activity in the presence of iodide, based both on measurements of H,O, disappearance and O2 evolution. In the absence of iodide, the enzymes catalyze neither H20, disappearance nor 0, evolution.
2) The presence of only 1 ~L M Iin an incubation mixture containing 100 p M H,02 and 1.3 pg/ml of thyroid peroxidase resulted in the degradation of 25 PM H20z (Fig. 7). Clearly, therefore, oxidation of Ito I, could not stoichiometrically account for the disappearance of the H202, and it appeared the Imust be catalytically involved in the enzymatic reaction leading to H20, disappearance. In samples containing 100 FM Iit was possible to measure I2 formed in the reaction, and again it was evident that oxidation of I-to Is was much too small to account for the measured disappearance of H202.
Oxidation of iodide to iodate was negligible, as shown in our previous study (1).
3) The pseudohalogen, SCN", cannot replace Ias a promoter of catalatic activity, even though SCN-is readily oxidized by H20, f thyroid peroxidase or lactoperoxidase. As shown in Fig. 9, H20, was degraded in the presence of SCN-, but no evolution of O2 accompanied the reaction. In this case H202 was utilized solely for the oxidation of the anion, in contrast to the catalatic cleavage of H20, that occurred in the presence of I-. Another anion, ClOj-, which competes with 1for a transport system in the thyroid, also had no stimulatory effect on the catalatic activity of thyroid peroxidase and lactoperoxidase (Fig. 9). Unlike SCN-, C104-is not oxidized by the peroxidase system.

4)
The thioureylene drug, MMI, was shown in previous studies in this laboratory to act both as a reversible and irreversible inhibitor of thyroid peroxidase-and lactoperoxidase-catalyzed iodination, and a scheme was proposed to explain its mechanism of action (32). In the present study we examined the effects of MMI on the catalatic activity of thyroid peroxidase, and we observed that this reaction may also be inhibited reversibly or irreversibly by MMI, depending on the concentration of drug (Fig. 10). Moreover, in the presence of concentrations of MMI leading to reversible inhibition, a time lag was observed which corresponded closely in extent to the time lag seen in iodination experiments performed under similar conditions (Figs. 10 and 11).
5) The thioureylene drug, CBZ, is a derivative of MMI, but, unlike MMI, it does not readily inactivate thyroid peroxidase. For this reason, as shown in a previous study (32), it acts only as a reversible inhibitor of thyroid peroxidase-catalyzed iodination. In the present study we have shown that CBZ inhibits the catalatic activity of thyroid peroxidase, and that in this case also, the inhibition is reversible (Fig. 12). Thus, the inhibitory effects of CBZ on iodination and on catalatic activity suggest that this drug acts as a competitive inhibitor in both reactions. 6) High concentrations of Iwere shown to inhibit the catalatic activity of thyroid peroxidase and lactoperoxidase (Fig. 13). A similar inhibition of thyroid peroxidase-catalyzed iodination by excess Iwas reported previously (6,8), and in both instances oxidation of Ito 1, appeared to be correlated with the degree of inhibition. 7) Conversion of thyroid peroxidase and lactoperoxidase to a less active or inactive form (Compound 111) by excess H202 has been described by other investigators (13, 39), and it was necessary to consider the possibility that the iodide dependence of the catalatic activity of these peroxidases might reflect a protective action of iodide against inactivation by H202. We examined this possibility in our previous study (l), and we observed that although thyroid peroxidase, and to a lesser extent, lactoperoxidase, were significantly inactivated on addition of 100 PM H202, the inactivation was not prevented by iodide. Thus, the role of iodide in promoting catalatic activity cannot be attributed to a protective effect on the enzymes. Discussion of a possible mechanism for iodide-dependent catalatic activity must also be based on an understanding of the mechanism of thyroid peroxidase-and lactoperoxidasecatalyzed iodination. The complete details of the iodination mechanism have not been elucidated, but the most recent evidence favors a 2-electron rather than a free radical mechanism (13-15). In their extensive studies on the mechanism of chloroperoxidase-catalyzed chlorination, Hager and coworkers (40,41) postulated that the chlorinating intermediate may be represented as an enzyme-bound halogenium ion (Cl+), or as a hypohalite ion. Morrison and Schonbaum (10) proposed a scheme for peroxidase-catalyzed iodination involving an enzyme-hypoiodous acid complex. The postulation of a hypoiodite intermediate in the iodination reaction offers a possible explanation for iodide-dependent catalatic activity observed in the present study.
Of special interest in this connection is the following reaction, reported by Liebhafsky (42): This reaction provides a chemical basis for O2 evolution, the essential element of catalatic activity.
In Fig. 14 we propose a scheme to explain iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase and to show the relationship of this reaction to various peroxidative functions of these enzymes. A discussion of the various reactions in Fig. 14 is provided in the following comments. This would imply that there may be two different forms of EO. If this proposal is confirmed, the scheme in Fig. 14 would require some revision, but this would not affect the major conclusions of the present study. Although for the sake of simplicity the scheme shows this inactivation to occur with Compound I, Compound I1 may also be involved in these reactions.

At low Concentrations of
H20z. the Patlo, moles I boundlmoler HZ02 added, was s l i g h t l y lover than 1.0, resembling the r a t i o s observed when H202 was generated with glucose-glucose oxidase.
Honever. t h e r a t i o f e l l Off s i g n l f l c a n t l y as the H2O2 concentratlon was increased, declining to values below 0.5 a t 100 WM H202.
uK HzDz YI P added to the incubation mixture.