AK('HIVW OF AIO('HEMISTKY AND BIOPHYSICS Vol. 287, No. 2, rJune, pp. 288-296, 1991

Peroxidase-Catalyzed Bromination of Tyrosine, Thyroglobulin, and Bovine Serum Albumin: Compar is0 n of Thyroid Peroxidase and Lactoperoxidase’ Alvin Taurog’ and Martha Department

Received

of Pharmacology,

December

L. Dorris

University

7, 1990, and in revised

of Texas Southwestern

form

January

by NIDDK Grant 03612. should be addressed.

Center,

Dallas,

Texas

75235-9041

29, 1991

A recent paper (Buchherger, W., 1988, J. Chromatogr. 432, 57) on lactoperoxidase-catalyzed bromination of tyrosine and thyroglobulin stated, without evidence, that thyroid peroxidase (TPO) is able to use bromide as a substrate. This was in disagreement with unpublished experiments previously performed in this laboratory, and we undertook, therefore, to examine this subject further. Highly purified porcine TPO was compared with lactoperoxidase (LPO) and chloroperoxidase (CPO) for ability to catalyze bromination of tyrosine, thyroglobulin, and bovine serum albumin (BSA). The incubation mixture contained 50-100 nM peroxidase, IO-500 pM S2Br~, tyrosine (150 PM), thyroglobulin (0.3 or 1 PM), or BSA (7.5 FM), and a source of H202. The latter was either generated by glucose (1 mg/ml)-glucose oxidase (0.5 or 1 Kg/ml), or added initially as a bolus (100 PM). With TPO, formation of organically bound *‘Br was undetectable under all conditions in the pH range 5.4-7.0. Lactoperoxidase and CPO, on the other hand, displayed considerable brominating activity. Lactoperoxidase was much more active at pH 5.4 than at pH 7.0 and was more active with BSA as acceptor than with tyrosine or thyroglobulin. The distribution of “Br among the various amino acids in LPO-brominated thyroglobulin and BSA was determined by HPLC. As expected, monobromotyrosine and dibromotyrosine together comprised the greatest part of the bound “‘Br. However, a surprisingly high percentage (ZO-25%) was present as monobromohistidine. Evidence was also obtained for the presence of a small percentage of the bound s2Br as tetrabromothyronine. Peroxidasecatalyzed bromination probably depends on the oxidation of Br to Br+ by the Compound I form of the enzyme. Since oxidation of Bra to Br’ requires a stronger oxidant than oxidation of I to I+, our results suggest that Com-

’ This work was supported ’ To whom correspondence

Medical

pound I of LPO and of CPO has a higher oxidation potential than Compound I of TPO. In viva experiments with rats on a low iodine diet injected with 82Br~ showed that even under conditions of high stimulation by thyrotropic hormone, there is negligible formation of organic bromine in the thyroid. Measurements of thyroid:serum concentration ratios for 82Br~ in similar rats provided no evidence that Bra is a substrate for the iodide transport system of the thyroid. I~> ISSI Academic PFM, IW.

Haloperoxidases can be grouped according to their ability to oxidize specific halide ions in the presence of Hz02 (1). Oxidation of the halide, which is mediated by the heme prosthetic group, is necessary for covalent binding to carbon. The standard oxidation-reduction potentials of the halides, including the pseudohalide CNS, are as follows:

21~ H 2CNS tf 2Br~ tf 2Cll 2Hz0 -

I2 + 2e (CNS)2 + 2e Brz + 2e Cl, + 2e HzOZ + 2H+ + 2e

E” (volts) -0.54 -0.77 -1.07 -1.36 -1.77

I is the most readily oxidizable of all the halides, whereas Cll requires a more powerful oxidant for its oxidation. Although HzOz itself, in the presence of Ht, is a sufficiently powerful oxidant to oxidize all the halogens, the oxidant in peroxidase-catalyzed halogenation is not H,O, itself but rather the reaction product of the peroxidase with Hz02, known as Compound I. A haloperoxidase having an oxidation potential sufficient to oxidize chloride (e.g., chloroperoxidase and myeloperoxidase) is also able to oxidize bromide and iodide. Haloperoxidases capable of oxidizing bromide (e.g., lac-

‘88 Copyright CC:1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

PEROXIDASE-CATALYZED

BROMINATION

toperoxidase and bromoperoxidases) are also able to oxidize iodide. It is well known that thyroid peroxidase (TP0)3 catalyzes oxidation of iodide and iodination of tyrosine and tyrosyl residues in protein (2). It also catalyzes oxidation of the pseudohalide, CNS (3). Horseradish peroxidase is stated to catalyze oxidation of iodide only (4). It has been reported (5) that thyroid peroxidase may catalyze bromide oxidation, although no data were presented to support this contention. A continuing interest in this laboratory in the properties of thyroid peroxidase led us to examine this question in further detail. We have used radioactive bromide (s’Br-) to determine whether thyroid peroxidase catalyzes bromination of tyrosine, thyroglobulin, and bovine serum albumin. For comparison, experiments were also performed with lactoperoxidase (LPO) and with chloroperoxidase (CPO), which are known to act as bromoperoxidases. We have also performed experiments with rats to test whether bromide is a substrate for the iodide-transport system of the thyroid. MATERIALS

AND

METHODS

“Bromide. This was purchased from DuPont-New England Nuclear Corp. The half-life of s*Br is 35.3 h, thus greatly limiting the number of experiments that can be performed with a single shipment. Experiments were performed with four separate shipments during the course of this study. The radioactivity varied from 1.08 to 4.43 mCi/ml and the bromide concentration from 0.58 to 3.8 mg/ml. Porcine TPO was prepared as previously deThyroid peroxidase. scribed (6), with minor modifications as later described for Preparation XII (7). The TPO used in this study was Preparation XIII (A,,,,/A,,,

= 0.48). Lactoperoxidase. Two different preparations were used, one from Pharmacia (A,,,/A,,, = 0.90) and one from Sigma (A,,,/A,, = 0.88). The material was provided as a lyophilized solid. This was dissolved in 67 mM phosphate, pH 7.0, at 1 mg/ml and dialyzed against a large volume of the same bufIer. For comparison of TPO and LPO on an equimolar basis, the Soret absorbance of the heme prosthetic group (412 nm for LPO, 413 nm for TPO) was used. A value of 114 for the millimolar extinction coefficient of LPO was reported by Morrison and Bayse (8). This value was used in the present study for both LPO and TPO.

Chloroperoxidase. This was purchased from Sigma. It was dialyzed against 0.1 N acetate buffer, pH 4.8, before use. The enzyme concentration was based on a millimolar extinction coefficient of 75.2 at 403 nm (9). The major purpose of this study was to comIncubation procedure. pare TPO with LPO for ability to catalyze bromination of tyrosine, thyroglobulin, and BSA. A typical incubation mixture contained peroxidase, %“Br , tyrosine, thyroglobulin, or BSA, and a source of H,O,. The latter was either generated by glucose-glucose oxidase or added initially as a bolus. Concentrations of individual components were as follows: peroxidase, 50-100 nM; bromide, lo-500 PM; tyrosine, 150 PM; thyroglobulin 0.3 or 1 PM; BSA, 7.5 PM; glucose, 1 mg/ml; glucose oxidase, 0.5 or 1 ag/ml; and H,O,, 100 PM. The incubations were performed at

” Abbreviations used: TPO, thyroid peroxidase; LPO, lactoperoxidase; CPO, chloroperoxidase; Tg, thyroglobulin; MMI, 1.methyl-2.mercaptoimidazole; PTU, 6-propyl-2.thiouracil; BSA, bovine serum albumin; LID, low iodine diet.

OF TYROSINE

AND

PROTEIN

289

37°C in 67 mM phosphate buffer at pH 7.0, 6.0, or 5.4, and in the case of chloroperoxidase in 0.1 N acetate buffer at pH 4.8. The reaction was started with glucose oxidase or H,O,, and it was stopped by the addition of 1.methyl-2-mercaptoimidazole (final concentration, 5 mM) and cooling of the reaction tubes in an ice slurry. Details of individual experiments are provided with the results. A few experiments were also performed involving TPO- and LPOcatalyzed iodination of BSA. In this case the incubation mixture contained [‘“‘Iliodide and lower concentrations of peroxidase than those used in the 82Br experiments.

Animals. Male Sprague-Dawley rats weighing 120-150 g were obtained from Simonsen laboratories. Control rats received regular rat chow. Iodine-deficient rats were prepared by the feeding of a Remington low iodine diet as previously described (10). For measurement of thyroid iodide transport, the rats were injected first with 2 mg of propylthiouracil (PTU) to block organic iodine formation and after 30 min with 0.1 pmol ‘“‘I-iodide. Blood and thyroid were taken 1 h after the injection of iR’I, and the thyroid:serum ““I concentration ratio (T/S[I-1) was determined (11). In the Separation of “‘Br-labeled products by paper chromatography. first part of this study, paper chromatography was used to separate mono- and dibromotyrosine from unreacted bromide. The solvent was collidine-NH,OH, and the procedure was exactly as described previously for TPO-catalyzed iodination (12). After chromatography the dried paper strips were placed in contact with X-ray film for 15~-18 h, and the areas on the paper corresponding to the bands on the film were excised and counted in a gamma counter, together with origin, front, and intermediate areas between the visible bands. The energy window was set at 50&1000 keV. This yielded a background count of approximately 50 cpm and about 1.1 X lo6 cpm per &i “Br. With tyrosine as the acceptor there were two major bands on the radioautogram. These were assumed to correspond to mono- and dibromotyrosine. The position of the latter (R, = 0.16) was identified by comigration with an authentic sample of dibromotyrosine, visualized hy spraying the filter paper strip with diazotized sulfanilic acid. The monobromotyrosine (R, = 0.34) was identified by exclusion. When thyroglobulin or BSA was the acceptor, the incubation sample was digested with pronase plus aminopeptidase (12) before it was applied to the filter paper strip. The radioautogram again showed hands corresponding to mono- and dibromotyrosine. However, the monobromotyrosine band was split and appeared to contain an additional component. We suspected that this might be monohromohistidine, which Manthey et al. had previously reported (13) was formed when human serum albumin was brominated with bromoperoxidase or myeloperoxidase. For better separation of the products of LPO-catalyzed bromination of t,hyroglobulin and BSA, therefore, we used the HPLC procedure described in the following section. The pronase digest (generally Analysis ofprotein digests by HPLC. 200 ~1) was mixed with 4 vol of ethanol in a small plastic, conical centrifuge tube and the mixture was kept in an ice bath for 20 min. After centrifugation at 12,OOOg for 10 min the supernate was withdrawn and concentrated in a Speed-Vat to a volume of 150 al or less. One hundred microliters of the concentrated solution was applied to the HPLC column. Analysis by HPLC was performed using a reverse phase Clx ultrasphere column (250 X 4.6 mm, particle size, 5 am; Beckman Instruments). The pumps (Model 114M). uv detector (Model 164), and uv integrator were also products of Beckman Instruments. Gradient elution was employed, with 0.02 M KH,PO,, pH 4.8, as solution A and methanol as solution B. The elution program was as follows: 0 to 10 min, 5%B:95%A; 10 to 15 min, linear increase to 30%B:70%A; 15 to 25 min, continue at 3O’%B: 7O%A; 25-37 min, linear increase to lOO%B; 37-42 min, continue at lOO%B. The flow rate was 1 ml/min. The column eluant, after emerging from the uv detector, was led through a flow counter (Model HS, Radio Analytic Inc.), which was connected to a recorder. The ““Br activity peaks were recorded, and the elution times of the peaks and the fraction

290

TAUROG

AND TABLE

Bromination

of Tyrosine

Catalyzed

DORRIS I by TPO,

% s*Br distribution Enzyme TPO TPO TPO LPO LPO LPO CPO

PH

5.4 6.0 7.0 5.4 6.0 7.0 4.8

Origin 0.02 0.02 0.02 3.1 4.7 5.6 0.9

Dibromotyrosine

LPO,

and CPO

on chromatogram

Monobromotyrosine

Bromide

Front

99.9 99.9 99.9 84.6 81.5 82.8 82.1

1.7 1.8 0.8 3.1

0.04 0.03 0.04 6.4 6.4 5.3 10.2

0.02 0.02 0.02 4.1 5.4 5.5 3.7

Note. The incubation system contained 50 nM peroxidase, 118 PM “Br-, 150 FM tyrosine, 1 mg/ml glucose, and 0.5 or 1.0 kg/ml glucose oxidase in 67 mM phosphate buffer (pH 5.4, 6.0, or 7.0) or 0.1 M acetate buffer (pH 4.8). The incubation temperature was 37°C and the reaction time was 30 min.

of the total “Br in each peak were numerically indicated by the builtin-printer of the flow counter. Retention times for expected products were established with known compounds. Dibromotyrosine was purchased from Sigma Chemical Co. Tetrabromothyronine, 3’,3,5,-tribromothyronine, and 3’,5’,3-tribromothyronine were kindly made available through the courtesy of Dr. Vivian Cody, Medical Foundation of Buffalo. Monobromotyrosine and monobromohistidine were prepared by LPO-catalyzed bromination of tyrosine and histidine, respectively, using “Br. These compounds were not isolated, but retention times could readily be established by s*Br peaks. Retention times for the compounds of interest in this study were: bromide, 2.8 min; monobromohistidine, 4.3 min; monobromotyrosine, 16.7 min; dibromotyrosine, 21.0 min; 3’3,5-tribromothyronine, 36.1 min; 3’5’$tribromothyronine, 36.7 min; and tetrabromothyronine, 37.3 min.

Bromination of bovine serum albumin catalyzed by thyroid peroxidase, lactoperoxidase, and chloroperoxidase. These results are shown in Table II. As in the case of tyrosine, TPO displayed negligible ability to catalyze bromination of BSA in the pH range 5.4-7.0. This occurred whether H202 was added directly or generated by glucose-glucose oxidase. Lactoperoxidase, on the other hand, was very active in catalyzing bromination of BSA, especially at pH 5.4. The brominating activity of LPO toward BSA was much greater at pH 5.4 than at pH 7.0, TABLE

RESULTS

In Vitro Experiments Bromination of tyrosine catalyzed by thyroid peroxidase, These results are lactoperoxidase, and chloroperoridase. shown in Table I. Thyroid peroxidase displayed negligible ability to catalyze bromination of tyrosine in the pH range 5.4-7.0. Lactoperoxidase, on the other hand, showed significant brominating activity under the same incubation conditions. There was little effect of pH on LPO-catalyzed bromination of tyrosine. When the LPO concentration was reduced from 50 to 10 nM, bromination of tyrosine was greatly reduced (results not shown). In addition to mono- and dibromotyrosine, a product was formed that remained at the origin of the paper chromatogram. By analogy with peroxidase-catalyzed tyrosine (14), this product may represent the condensation product, dimonobromotyrosine. A small amount of product was also observed at or near the solvent front. The nature of this material is unknown. The brominating activity of CPO was quantitatively similar to that of LPO under the incubation conditions used. However, the distribution of 82Br was somewhat different. There was less formation of origin product and more formation of solvent front product with CPO.

II

Bromination of BSA Catalyzed by Thyroid Peroxidase, Lactoperoxidase, and Chloroperoxidase % s2Br organically PH

TPO

LPO

bound CPO

Glucose + glucose oxidase (30 min) 5.4 6.0 7.0 4.8 4.8

0.03 0.1 0.06

61.7 30.5 10.6 75.6 28.5"

H,O1 (10 min) 5.4 6.0 7.0 4.8 4.8

0.02 0.03

0.02

14.1 4.3 2.7 11.6 58.2"

Note. The incubation system contained 50 nM enzyme, 500 pg/ml BSA, 118 FM Br-, and either glucose-glucose oxidase or H202. The LPO and TPO samples contained 1 mg/ml glucose and 0.5 pg/ml glucose oxidase. The CPO samples contained 1 mg/ml glucose and 1 pg/ml glucose oxidase. H,O:, in all samples was 100 PM. a Samples contained 100 mM NaCl.

PEROXIDASE-CATALYZED TABLE

III

Effect of pH on LPO-Catalyzed Bromination BSA, and Goiter Thyroglobulin % of *‘Br organically PH 5.4 6.0 7.0

Tyrosine 13.2 16.6 15.2

BROMINATION

of Tyrosine,

bound

BSA

Tg

51.2 34.5 10.4

15.1 1.4 1.1

Note. The incubation mixture contained 50 nM LPO, 134 KM *‘Br , 1 mg/ml glucose, 0.5 fig/ml glucose oxidase, and 150 pM tyrosine, 1 pM thyroglobulin, or 7.5 @M BSA. The reaction was started at 37°C with glucose oxidase and stopped after 30 min by addition of MM1 (final concentration 5 mM).

to the results obtained with tyrosine (Table I). The reaction was more sustained when H,O, was generated by glucose-glucose oxidase, compared to results with directly added Hz02. Similar results were previously observed with TPO-catalyzed iodination (15) and were attributed to more rapid inactivation of the peroxidase by the initially high concentration of directly added HzOz. Chloroperoxidase was also more active in catalyzing bromination of BSA than of tyrosine. The effect of 0.1 N chloride was tested, because in a previous study (16) we observed that Cll markedly enhanced CPO-catalyzed iodination. Interestingly, CPO-catalyzed bromination of BSA was inhibited by 0.1 N Cll when H202 was generated by glucose-glucose oxidase. However, when Hz02 was in contrast

TABLE

Comparison

of TPO- and LPO-Catalyzed

OF TYROSINE

AND

291

PROTEIN

added directly, CPO-catalyzed bromination of BSA was greatly enhanced in the presence of 0.1 N Cl-. Effect of pH on LPO-catalyzed bromination. As indicated above, bromination of tyrosine was much less affected by pH than bromination of BSA. This experiment was repeated with the addition of another protein, goiter thyroglobulin. As shown in Table III, LPO-catalyzed bromination of tyrosine was again little affected by pH, whereas LPO-catalyzed bromination of both BSA and thyroglobulin occurred much more readily at pH 5.4 than at pH 6.0 or 7.0. Presumably, therefore, the shift to a more acid pH has a much greater effect on tyrosyl residues in protein than on free tyrosine as substrates for LPOcatalyzed bromination. Effect of varying conditions on TPO-catalyzed bromination of thyroglobulin. Table IV shows results of experiments in which the bromide concentration was varied over the range 10-500 PM, and in which thyroglobulin was present at 0.3 or 1 PM. The TPO concentration was 100 nM, twice as great as in the preceding experiments, and H,O, was either generated by glucose-glucose oxidase or added directly in a bolus. No significant TPO-catalyzed bromination was observed under any of these conditions. Shown for comparison in Table IV are results obtained with LPO, with H,O, generated by glucose-glucose oxidase. In contrast to TPO, LPO displayed brominating activity under all conditions. The activity increased markedly between 10 and 100 PM bromide, but relatively little between 100 and 500 PM bromide. Very high degrees of bromination were attained when Tg was present at only 0.3 PM.

IV

Bromination

of Thyroglobulin

under Various Conditions

% of added s2Br bound to Tg TPO Br- concn hM)

Tg concn (FM)

10 25 100 500 10 25 100 500

0.3 0.3 0.3 0.3 1.0 1.0 1.0 1.0

H,O, generated by glucose-g.0.”

LPO H,O, directly addedb

H,O, generated by glucose-g.0.”

Atoms of Br bound per molecule of Tg in LPO samples

2.6 7.5 34.2 8.2 2.8 3.3 24.7 5.4

0.9 6.3 114 137 0.3 0.8 25 27

0

0.02 0.04 0 0 0.01 0 0.03

Note. Incubations were performed at 37”C, either with 100 nM TPO at pH 7.0 or with 100 were added at 1 mg/ml and 0.5 pg/ml, respectively. H202 was added at 100 pM. u 30 min incubation. ’ 10 min incubation.

nM

LPO at pH 5.4. Glucose and glucoseeoxidase

292

TAUROG

AND TABLE

Distribution

Protein

Protein concn

Bromide concn

(FM)

(PM)

Tg

0.3 0.3

100 100

Tg Tg

1.0 1.0

BSA BSA

7.5 7.5

200 200 200 200

Tg

67

Atoms Br bound per molecule protein 127 127 a7 84

19 18

DORRIS V

of s2Br in LPO-Brominated

Thyroglobulin

(Tg) and BSA

Percentage distribution Bromide 14 13 7 6 5 5

Monobromotyrosine

Dibromotyrosine

17 17 30 31 21 22

of s2Br in pronase digest Monobromohistidine

0.6” 0.8”

22 21 25 24 24 24

21 20 26 24 38 38

Tetrabromothyronine

0.1 O2

Note. Values are means ? SD. Four animals per group. Low iodine diet (LID) for 44 or 49 days. Animals killed 1 h after ‘“‘I~. p value calculated using Student’s t test,

294

TAUROG

demonstrate unequivocally, therefore, that TPO is not a bromoperoxidase. Studies previously performed in this laboratory (3) indicated that TPO readily catalyzes oxidation of the pseudohalogen, CNS. As indicated in the introduction, the standard oxidation-reduction potential of the couple 2CNS t--t (CNS)2 + 2e is intermediate between those of 21I ++ I2 + 2e and 2Br- c--)(Br)z + 2e. Based on the values listed in the introduction, these results suggest that the standard oxidation-reduction potential of the couple TPO-native enzyme * TPO-Compound I + 2e lies between -0.77 and -1.07 V. On this basis, TPO-Compound I does not have sufficient thermodynamic potential to oxidize Br , an essential requirement for covalent binding to carbon. Results obtained with LPO in the present study confirm previous reports (1) that LPO acts as a bromoperoxidase. With tyrosine as acceptor, LPO catalyzed the formation of both mono- and dibromotyrosine. The reaction was rather limited in extent; only about 11% of the added “Br- was bound to tyrosine under the conditions shown in Table I. There was little or no effect of pH in the range of 5.4-7.0. With BSA as the acceptor, LPO was a more efficient catalyst of bromination. There was also a very marked effect of pH, in contrast to the results with tyrosine bromination. As shown in Table II, 61.7% of the added ‘*Br- was covalently bound to BSA at pH 5.4, compared to 30.5% at pH 6.0 and 10.6% at pH 7.0. Thyroglobulin was brominated less readily than BSA by LPO, but it too was a better acceptor at pH 5.4 than at pH 7.0 (Table III). In the case of LPO-catalyzed iodination of BSA, it was shown previously (18), and confirmed in the present study (Table VII), that this reaction is also faster at pH 5.4 than at pH 7.0. It is not apparent why the pH profile for LPO-catalyzed bromination of tyrosine is so different from that for tyrosy1 residues of protein. One possibility we considered is that at the more acid pH’s, bromination of histidyl residues may become relatively more important. We studied LPO-catalyzed iodination of histidine (data not shown) and observed that this reaction is faster at pH 7.0 than at pH 5.4. Assuming that LPO-catalyzed bromination of histidyl residues in protein is similarly affected by pH, these results do not support the explanation suggested above. It is usually stated that in both chemical (20) and peroxidase-catalyzed (21) iodination of tyrosine (and presumably therefore of tyrosyl residues in protein), that phenolate anion is more rapidly iodinated than the undissociated molecule. On this basis it might be expected that the rate of LPO-catalyzed bromination of tyrosine would progressively decrease with decreasing pH. This was not observed in the present study (Table III), suggesting that the phenolate form of tyrosine is not the

AND

DORRIS

preferred acceptor for the active brominating species. With BSA as acceptor, the much higher rate of LPOcatalyzed iodination (Table VII) as well as bromination (Tables II and III) at pH 5.4, compared to pH 7.0, also suggests that dissociated tyrosyl residues are not the preferred substrate for LPO-catalyzed halogenation. The nature of the covalently bound ‘*Br in LPO-brominated thyroglobulin and BSA was also examined in the present study (Table V). The largest part of the bound “Br was distributed between mono- and dibromotyrosine. In the case of thyroglobulin (1 FM), the fraction of “Br present as monobromotyrosine exceeded that in the form of dibromotyrosine. This contrasts with results obtained with enzymatically iodinated thyroglobulin, in which, at comparable levels of iodination, the fraction of radioiodine present as diiodotyrosine greatly exceeds that as monoiodotyrosine. In radiobrominated BSA, the fraction of “Br present as dibromotyrosine exceeded that in the form of monobromotyrosine, in contrast to the results obtained with thyroglobulin. An unexpectedly large fraction (2125%) of the “Br was also bound as monoiodohistidine both in thyroglobulin and in BSA. This is much higher than the value of 10% previously reported by Manthey et al. (13) in digests of human serum albumin after bromination with bromoperoxidase from Penicillus capitatus. The higher value observed in the present study may be related to the high concentration of bromide carrier that we used. The bromide concentration used by Manthey et al. was not indicated, but their observation that 81% of the radiobromine was present as monobromotyrosine suggests a low degree of bromination under their reaction conditions. McElvany and co-workers used bromoperoxidase (22) or myeloperoxidase (23) to label proteins with 77Br and claimed that proteins labeled in this manner displayed some advantages over proteins labeled with radioiodine. It is well known (18,19) that LPO catalyzes iodination of thyroglobulin with substantial formation of thyroxine (tetraiodothyronine). With the HPLC procedure used in the present study, we detected a small percentage of the “Br in the pronase digest in a peak that coeluted with carrier tetrabromothyronine. This was observed with BSA as well as with thyroglobulin. These results were obtained at extremely high levels of bromination, far beyond the levels of iodination observed in thyroglobulin under physiological conditions. In thyroglobulin containing 127 atoms Br per molecule, a mean average of 0.7% of the “Br was observed in the form of tetrabromothyronine, equivalent to 0.2 residue per molecule. This represents significant tetrabromothyronine formation, although it is much less than the thyroxine (3 residues) formed in comparably iodinated thyroglobulin (17). The percentage of “Br in the tetrabromothyronine peak in BSA digests was comparable to that observed for thyroglobulin (Table V), but the calculated number of residues per molecule is only

PEROXIDASE-CATALYZED

BROMINATION

about 0.02. This relates to the much lower molecule weight of BSA (67,000), compared to that of thyroglobulin (660,000). Some evidence was also obtained for the formation of 3’,3,5-tribromothyronine, but the data are too few and variable to be convincing. It has long been known that the thyroid gland possesses a mechanism for concentrating iodide many fold above the level in the circulation (11). Studies reported many years ago (24-26) suggested that the thyroid may also concentrate bromide to a slight extent. In the present study we measured the uptake of “2Br~ by the thyroids of rats fed either a normal stock diet or a special low iodine diet (LID). The iodide transport system was greatly stimulated in the rats maintained on LID. However, the thyroid:serum concentration ratio for %‘Br~ was very low (

Peroxidase-catalyzed bromination of tyrosine, thyroglobulin, and bovine serum albumin: comparison of thyroid peroxidase and lactoperoxidase.

A recent paper (Buchberger, W., 1988, J. Chromatogr. 432, 57) on lactoperoxidase-catalyzed bromination of tyrosine and thyroglobulin stated, without e...
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