TREJOUBNAL OPBIOLOOICALCHEMISTRY Vol. 238, No.10. Odober1963

Printed in U.S.A.

Thyroxine Degradation

III. COMPETITIVE INHIBITION OF THYROXINE DEGRADATION; RELATIONSHIP OF STRUCTURE TO INHIBITION *

JAMES WYNN AND ROBERT GIBBS

From the Department of Medicine, Duke University Medical Center, Durham, North Carolina

(Received for publication, March 21, 1963)

Few attempts have been made to relate the structure of substances deiodinated by the systems in vitro which degrade thyroxine to specific structural requirements necessary for the reaction. In general, it has been found that several thyronine derivatives may be degraded by the rabbit muscle or rat liver microsomal thyroxine-degrading systems, but that none is more efficiently degraded than thyroxine itself (1, 2). The present study was undertaken in attempt to delineate the critical structure necessary for competition with thyroxine for initial reactions in the rat liver microsomal thyroxine-degrading system. These’ investigations indicate that 1,4-hydroquinone is the simplest reactive structure and that the initial reaction involves microsomal binding of substrate distinct from the later reaction of deiodination.

EXPERIMENTAL PROCEDURE

Materials

n-Thyroxine and DL-3,5-dibromothyronine were purchased from California Corporation for Biochemical Research. L-

Thyroxine, L-3,5,3’-triiodothyronine, L-3,5-diiodothyronine, and L-thyronine were purchased from Mann Research Labora- tories. 3,5,3’, 5’-Tetraiodothyroacetic and propionic acids were supplied by Smith, Kline, and French Laboratories. 3,5-Diiodo4-hydroxyphenylpyruvic acid, 4-hydroxyphenyl- formic acid, and 4-hydroxyphenylacetic acid were purchased from the Aldrich Chemical Company. 3,5-Diiodo-4-hydroxy- phenylsulfonic acid was purchased from K and K Laboratories. 4-Methoxyphenol, 4-nitrophenol, and 2-tcrt-butyl-1,4-hy- droquinone were purchased from Distillation Products Industries. 2-t&-Butyl-4-methoxyphenol was supplied by Eastman Organic Chemicals. 4-Aminophenol was purchased from Matheson, Coleman, and Bell.

I-Methoxy-l , 4-hydroquinone-This compound was purchased from K and K Laboratories, supplied as a dark brown powder. It was sublimed at 83”, yielding approximately 50% of the starting material as a white, crystalline material with a melting point of 85” (recorded melting point, 84” (3)). Elemental analysis’ gave

* This work was supported in part by Grants AM-04130 and Training Grant 285074 from the National Institutes of Health to Duke University.

1 These analyses were performed by Gailbrath Laboratories in Knoxville, Tennessee.

G&O, Calculated: C SO.OO%, H 5.72%, 0 34.30% Found : C 60.08%, H 5.79%, 0 34.20%

.Z , 6-Diiodo-l , ~-hydroquinone-This compound was purchased from K and K Laboratories and had a melting point of 144” (recorded melting point, 144” (4)). Elemental analysis gave

C&LLO~ Calculated: C 19.87%, H l.ll%, I 70.02y0, 0 8.85% Found : C 19.75%, H 1.26%, I70.14%, 08.65y0

N-AcetyWlyroxine-n-Thyroxine (2.0 g) was treated according to the procedure of Ashley and Harington (5). The product was precipitated three times from ethanol by addition of water. After drying this weighed 1.4 g, representing a 66% yield, and had a .melting point of 209-214” (recorded melting point, 210- 215” (5)). This material was chromatographically pure in the three solvents used.

0-Methyl-N-acetylthyrozine-N-Acetylthyroxine (1 g) in 100 ml of ethyl ether containing 10% (v/v) methanol was treated with 0.01 mole of diazomethane in ether at room temperature. The ether-methanol was then evaporated to dryness. The residue was boiled in 100 ml of 0.5 M Na.&Oa for 2 hours. The boiling solution was filtered removing a brown gum and then reduced in volume to about 25 ml at 50” in a vacuum. This was acidified to pH 6.0 with 1 N HCl and cooled to 4”. A white precipitate was collected by centrifugation, precipitated five times from ethanol by addition of water, and treated with charcoal during the final solution in ethanol. A white, amor- phous powder (250 mg) was obtained which, after drying, repre- sented a 24% yield. This material was soluble in hot Na2C03, gave a negative Kendall test for di-o-phenolic iodine substitution, and had a melting point of 215216” (recorded melting point, 214-217” (6)). It was chromatographically pure and distinct from either thyroxine or N-acetylthyroxine in butanol-NBOH- Hz0 and collidine-NHr-HzO. Elemental analysis gave

CdLLNOa Calculated: C 26.030/o, H 1.91%, I Sl.ll%, N 1.41’j&

0 9.39% Found: C 25.92%, H l.SO%, I Sl.OO’& N l.SS%,

0 9.60%

3,6,b’, 6’-Tetraiodothyrocaprooic Ac&-Alleged 3,5,3’, 5’-tetra-

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October 1963 J. Wynn and R. Gibbs 3491

iodothyrobutyric acid was purchased from Cycle Chemical Company. As supplied, it was a gray-white powder which was not chromatographically pure. After precipitation from water made alkaline for solution and acid for precipitation, a white amorphous material was obtained with a melting point of 176-180”, softening at 160”. After such precipitation it was chromatographically homogenous. This material was said to have been synthesized by the procedure of Kharasch and Sarkis (7). However, if so, the melting point should have been 195 196’. It gave a positive Kendall test for di-o-phenolic iodine substitution. Elemental analysis gave

Calculated: C 24.89%, H 1. 56y0, I 65.25%, 0 8.29%

Calculated: C 27.00%, H 2.OC@o, 163.50%, 0 8.90% Found : C 27.100/o, H l.S9%, 162.89%

C 26.9270, H l.SS%, I63.01%, 0 8.64%

Because of this discrepancy on elemental analysis, the acidic groups in the compound were titrated to pH 6.8 (the approximate pK of the phenolic hydroxyl in a di-o-iodine-substituted phenol; see “Results”). This titration was compatible with a com- pound of molecular weight 800 containing one carboxyl and one phenolic group. This data supported the elemental analysis since the molecular weight of the butyric acid derivative is 772 and of the caproic acid derivative, 800. Further data supporting the impression that this is the caproic acid derivative and not the butyric are given below under the derivatization of this compound to the methyl ester.

S,6, .!I’, 6’-Tetraiodothyrocapoic Acid Methyl Ester-The al- leged 3,5,3’, 5’tetraiodothyrobutyric acid supplied by Cycle Chemical Company (800 mg) in 100 ml of ethyl ether made 10% (v/v) with methanol was treated with 0.001 mole of diazo- methane in ether solution at room temperature for 3 hour. Ether-methanol was evaporated. The residue was dissolved in a few milliliters of ethanol; 20 ml of 0.5 M Na2C03 were added, and the mixture was brought to boiling. A gum collected on the bottom of the vessel and on the stirrer. The Na2C03 solution was discarded and the gum was saved. This was dissolved in a few milliliters of ethanol and precipitated with water, and the sediment was collected by centrifugation. This precipitation was repeated five times. After the fourth and fifth solutions in alcohol the material was treated with charcoal before precipitation. White, amorphous powder (174 mg), representing a 21 y0 yield, was obtained. This material was not soluble in hot Na2C03, gave a positive Kendall test, and had a melting point of 144-148’. Elemental analysis gave

Calculated: C 25.950/ H 1.78%, 164.10%, 0 8.14%

Calculated: C 28.00%, H 2.21%, I61.95%, 0 7.86% Found: C 28.27%, H 2.3670, I 61.51y0

C 28.02%, H 2.4770, I61.39%, 0 7.96%

Again it is obvious that this starting material is not compatible with the butyric acid derivative of thyroxine, but is compatible with a caproic acid derivative. Four pieces of evidence support

this: the melting point of the alleged 3,5,3’, 5’-tetraiodothyro- butyric acid is not the reported melting point for this compound; the elemental analysis is compatible with the caproic acid rather than the butyric acid derivative of thyroxine; the elemental analysis of the methyl ester derivative is compatible with the caproic acid derivative rather than the butyric; and the titra- tion of the acidic groups, if one assumes that there is one phenolic group with di-o-iodine substitution (positive Kendall test), is compatible with a compound of molecular weight 800, the caproic, rather than 772, the butyric acid derivative.

L-Y-Momiodothyrmine and L-3’,5’-Diiodothyronine-These were prepared during a single iodination procedure. n-Thyronine (1.0 g) was dissolved in 100 ml of 20% (v/v) aqueous ethylene- diamine, and 1.9 g of iodine in Lugol’s solution were added slowly over a 5-hour period. The entire mixture was then added to each of three resin columns as previously described and eluted with formic acid (8). Three ninhydrin-positive products were obtained. These were unchanged thyronine in Fractions 17 to 18, 3’-monoiodothyronine in Fractions 21 to 23, and 3’,5’- diiodothyronine in Fractions 28 to 30. Each pooled fraction was dried in a vacuum at 50”, dissolved in a few milliliters of 20% ethylenediamine, and subjected to a second such resin chromatography. The products again located by the ninhydrin reaction were dried as before; 350 mg of thyronine, 309 mg of 3’-monoiodothyronine, and 275 mg of 3’) 5’-diiodothyronine were recovered. This represented a loss of 285 mg of starting material altered in such a way that it was not recoverable from the resin chromatography. The compounds eluded from the resin column were chromatographically pure with Rp numbers in the three solvents used consistent with their respect.ive structures.

L-Q ,5’,5’-Triiodothyronine-This was prepared by a modifi- cation of the procedure of Meltzer and Stanaback (9). The synthesis is the same as that described for thyroxine except that 1 X lo-’ mole of L-3-monoiodotyrosine and 1.4 X 10m4 mole of 3,4-diiodo4-hydroxyphenylpyruvic acid were used as the starting materials. The yield was 21% of the theoretical recovery on the basis of L-3’-monoiodotyrosine used. The Kendall test was positive. Chromatographic characteristics in the three solvents used were those of the expected product. Elemental analysis gave

GsHJaNO, Calculated: C 27.70%, H 1.69%, N 2.15%, I 58.600/,,

0 9.84oJ, Found : C 27.510/,, H 2.05%, N 1.9970, 158.54x,

0 9.91%

~-S,6-Diiodo-3’,6’-dib70mothyronine-~-3,5-Diiodothyronine (1 g) in 100 ml of a 20% (v/v) solution of ethylenediamine waz treated with 17.5 ml of a saturated aqueous solution of bromine at 25’. After this mixture had stood for 30 minutes, 6 N HCl was added to pH 6.0. The amorphous yellow precipitate was collected by centrifugation, dissolved in hot ethanol, and precipi- tated by addition of an equal volume of water. After three such precipitations, the fourth alcohol solution was treated with charcoal before precipitation with water. After drying, 990 mg of amorphous white powder representing a 76% yield were obtained. This was chromatographically pure and had charac- teristics in the solvents used here which did not permit its separation from thyroxine. Its upper ultraviolet absorption

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3492 Thyroxine Degradation. III Vol. 238, No. 10

maximum was 325 ml*. Elemental analysis gave

C15HllBr&NO,

Calculated: C 26.330/,, H l.Sl%, N 2.05%, Br 23.4170, 137.18c/,, 0 9.37%

Found: C 26.50%, H l.Sl’%, N 1.98%, Br 23.15?& I 36.98y0, 0 9.5570

LJL-9,5-Dibromo-S’ ,5’-diiodothyronine-This was synthesized in a manner exactly analogous to that used for the preceding compound. DL-3,5-Dihromothyronine (1.0 g) was used as starting material, and 1.1 g of iodine in Lugol’s solution were used to iodinate. The separation and purification of the com- pound were carried out as above. White, amorphous powder (535 mg) was obtained, representing a 417, yield. This was chromatographically pure and could not be distinguished from thyroxine in the solvent systems used. The upper ultraviolet absorption maximum occurred at 325 mp. Elemental analysis gave

Calculated: C 26.33y0, H 1.61%, N 2.05%, Br 23.41’%, 137.1870, 0 9.37%

Found : C 26.49%, H 1.80%, N 2.11%, Br 23.21%, 137.05%, 0 9.19%

2,6-Diiodo-4-nitropheno2-4-Nitrophenol (1.4 g) in 50 ml of 2O70 (v/v) ethylenediamine was treated with 5.1 g of iodine in Lugol’s solution. Then 6 N HCl was added to a pH of less than 3.0, and the mixture was refrigerated at 4’ for 24 hours. A yellow precipitate was collected by filtration, washed with hot 0.1 N HCl, and dried. This was then precipitated five times from alkaline aqueous solution by addition of acid. After drying, 3.1 g of a yellow powder were obtained, representing a 79% yield. The product was chromatographically pure and had a melting point of 153-155” (recorded melting point, 153- 155” (10)).

SJ-Diiodo-.J-hydroxypheny~ormti Acid, 3,5-Diiodo+hy- droxybenzaldehyde, and 3,6-Diiodo+hydroxyphenylacetic Acid- All of these were synthesized according to methods described by Matsuura and Cahnman, except that ethylenediamine rather than methylamine was used as base (11).

1125 Exchange Labeling of 2,6-Diiodo-1 ,Q-hydroquinone-In 2 ml of p-dioxane and 1 ml of 0.1 N sodium acetate buffer at pH 4.8 were dissolved 4 mg of this compound; 1 mg of P5 in 1.1 ml of chloroform (specific activity, 1 mc per mg, purchased from Volk Radiochemical Company) was added to the solution, which then was allowed to stand for 1 hour. The entire volume was then applied to the origin of a large, preparative paper

IjlkrUIIrd~MT. i%i- wax L&YAW kir sUtinn&&ti &ii& water. After development, location of radioactivity on the paper was approximated with a Geiger-Miiller tube probe. The iodinated compound itself was located by observation of an absorption band on the paper under ultraviolet light. The band visualized and marked under ultraviolet light was cut from the main paper, radioactive material was eluted with ethanol, and this eluate was applied to papers for rechroma- tography first in formic acid-water (1: 100) and finally in formic acid-water-methanol (2 : 100 : 100). The separated radioactive material was chromatographically pure, and was inseparable from marker reference standard on paper. Specific activity was estimated on the basis of ultraviolet extinction at the absorption

maximum and the counting rate of an aliquot of the compound compared to an P reference standard. The specific activity was approximately 1 mc per 5 x 1OF mole. This material was stored in absolute ethanol at -20” before use.

Z13* Exchange Labeling of S’-Monoiodothyronine and 3,6,3’, 5’- Tetraiodothyrocaproic Acid Methyl Ester-The 3’-monoiodo- thyronine (3 mg) and 3,5,3’, 5’-tetraiodothyrocaproic acid methyl ester (7 mg) were each dissolved in 2 ml of p-dioxane and 1.0 ml of 0.1 N sodium acetate buffer at pH 4.8. Then 3 mc of NaP (carrier-free, purchased from Abbott Laboratories) which had been acidified to pH less than 1 with 6 N HCl were added to each of these solutions. Iodine (0.5 mg) in Lugol’s solution was added and the solutions were allowed to stand for 16 hours. As in the above labeling procedure, the methyl ester was separated by paper chromatography. The 3’-monoiodo- thyronine was isolated by resin chromatography. After these purifications, the formic acid eluate containing 3’-monoiodo- thyronine was dried and the residue was dissolved in 95% ethanol. The methyl ester had been eluted from its last chromatography in absolute ethanol. Both were assayed spectrophotometrically to determine the amount of material recovered, and I’s1 was assayed by comparing the counting rate of an aliquot to the counting rate of a reference standard. Specific activity of the 3’-monoiodothyronine was approsimately 1 mc per 1 X lop5 mole and of the 3,5,3’, 5’-tetraiodothyrocaproic acid methyl ester, approsimately 1 mc per 7 X 1OP mole.

Methods

The preparation of microsomes, reaction vessels, method of assaying for iodide produced from thyroxine, paper chroma- tography systems, and resin chromatography have been previ- ously described (12, 13). -411 compounds investigated as possible inhibit&s were prepared in stock solutions in 95% ethanol or water which were 1 X lop3 M. Appropriate aliquots of these solutions were used as indicated below.

Inhibition Studies

Duplicate flasks were prepared containing 1,2, and 5 mpmoles of thyroxine to which a standard tracer amount of thyroxine (3’,5’-1131) had been added. Simultaneously similar flasks were prepared with the same constituents plus the possible inhibitor. The amount of each inhibitor added varied according to its capacity to inhibit, but in no instance were more than 200 mMmoles added to the 10 ml of incubation medium. After 4 minutes of incubation with 0.2 ml of microsomes added, the reaction was stopped by the addition of serum and trichloroacetic acid as previously described (12). Iodide-131 produced was determined, and results were plotted according to the methods 69 &n?vL%$L¶* *ti &I+ \/1f$\ similar .st&& .vc?v SW&~ ,futi. with 2,6-diiodo-1,4-hydroquinone and 1,4-hydroquinone as the labeled substrates but with added thyroxine as the inhibitor. In these instances the measured observation was the amount of radioactivity bound to microsomal protein as indicated under the description of protein binding studies below. The amount of thyroxine added to flasks as inhibitor was 10 rnpM per flask. The amounts of labeled 2,6-diiodo-1,4-hydroquinone and 1,4- hydroquinone added to flasks were 5, 10, and 25 rnpM per flask. Lesser amounts of these labeled substances could not be used because their specific activities were too low.

LX-Monoiodothyronine and 3,5,3’, 5’-Tetraiodothyrocapoic Acid Methyl Ester as Substrate-These compounds were labeled

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October 1963 J. Wynn and R. Gibbs

by exchange as previously indicated. Both were added to reaction flasks in amounts of 50 mpmoles and 10 mpmoles, respectively. The amount of iodide produced from these compounds was determined as previously described with thyroxine (3’, 5’-P3’) as substrate (12).

Protein Binding of d, 6-Diiodo-l , .&hydroquinone (1’25) and 1 ,4-Hydroquinone (C14)-These labeled compounds (5 to 25 mpmoles each) were added to the usual reaction flask. After 2 to 5 minutes of incubation, 3 ml of 50% (v/v) trichloroacetic acid were added, the mixture was centrifuged, and the sediment was saved. This sediment was extracted three times with 3 ml of 1-butanol, which was discarded. Protein containing C14- labeled 1,4-hydroquinone was resuspended in 3.0 ml of 25% (v/v) ethanol.2 Duplicate l-ml aliquots of this were added to diosane-dimethoxyethane-anisole solution containing phosphor, and protein was suspended by addition of thixotropic gel powder (Cab-0-Sil, purchased from Packard Instrument Company). These samples were then assayed for radioactive content as indicated below. The P26-labeled protein was dissolved in 2 ml of 1 N NaOH and diluted to 3.0 ml, and all of this solution was then counted as indicated below.

Attempted Demonstration of Deiodination of F-Labeled d,6- Diiodo-1 , .J-hydroquinone-Reaction flasks containing 10 mpmoles of this labeled material were prepared as usual. Observations exactly similar to those previously carried out with thyroxine (3’,5’-P31) were made. This substance, like thyroxine, is entirely protein-precipitated in the presence of serum and trichloroacetic acid. Attempts to measure iodide-125 released were made in the supernatant solution after such protein precipitation.

Measurement of Solubility of Thyronine Derivatives in Reaction Medium-Each of the examined compounds (200 mpmoles) was added to 10 ml of the usual reaction medium without microsomes. The solutions were then centrifuged at 105,000 x

g for 1 hour. The supernatant solutions were discarded and the sediment was dissolved in 2 ml of 0.1 N NaOH. The optical density of each of these NaOH solutions at the absorption maxi- mum of each of the compounds in question was determined. These values were compared with extinction values of known dilutions of the compound, and the amount of each compound sedimented by this centrifugation was determined.

Determination of pK of Phenolic Hydroxyl-This was ac- complished by a modification of the method of Flexser, Hammett, and Dingwall (15). Gemmill has published work indicating that pK values were determined for thyroxine and 3,5-diiodo- thyronine in aqueous solution by these methods (16). Similar results could not be obtained in this laboratory. The problem encountered was that thyroxine is not soluble in aqueous solu- tions below pH 9.0 in the concentrations at which Gemmill worked. Therefore, below pH 9.0, attempts to measure dissocia- tion of the phenolic hydroxyl by extinction of the ultraviolet absorption maximum were markedly influenced by turbidity of aqueous solutions. Furthermore, for similar reasons, extinction in acid medium was not obtainable even in 50% ethanol. Thus no attempt to develop an entire extinction curve as a function of pH with the tetraiodinated thyronine derivatives or thyroxine was successful. On the other hand, in ethanol solution above 30% (v/v) concentration at a test material concentration of

* Ethanol was added to disperse small amounts of butanol lay- ered above the aqueous mixture.

1 X 10-d M, accurate estinction data could be obtained above pH 7.5 at 25”. For this reason, extinction as a function of apparent pH in 30,40,50, and occasionally 60% ethanol solutions was determined. pH was adjusted by addition of 0.01 to 0.10 ml of 2 N NaOH or 2 N HCl to 10 ml of the test solution. The volume of acid or base added was insufficient to change these measurements significantly. Extinction data were collected at pH 12 and above and below the value which represented half- maximal optical density after adjustment of pH with acid and base. The pK value was assumed to be the pH value at half- maximal optical density. Assuming that maximal extinction represented ultraviolet light absorption developed by nearly 100% dissociation of the phenolic hydroxyl, the fractional dissociation of this group was calculated from the observed extinction at each pH. The logarithmic function of this fraction was then added to the observed pH according to the formula

pK = pH - log observed extinction

maximal extinction - observed extinction

The validity of the assumption that half-maximal extinction occurs at the pH of the pK value without correction for sig- nificant extinction at the absorption maximum in acid pH was supported by the observation that points symmetrically above and below the calculated pK gave nearly identical values for log fractional dissociation except that signs were appropriately reversed. Thus apparent pK values were measured in various concentrations of alcohol. These points were plotted and extrapolated back to zero alcohol concentration. The validity of this procedure is supported by the fact that the calculated phenolic pK value in aqueous solution for 3,5-diiodotyrosine was very near to that obtained by Crammer and Neuberger with similar methods and by Dalton, Kirk, and Schmidt with entirely different methods (17, 18).

illeaswment of Oxidation Potentials-A procedure similar to that described for analysis of 1,4-hydroquinone by Koltoff was used (19). The apparatus was a Sargent model XV continuous recording polarograph and the cell was a dropping mercury electrode above a pool of mercury. Test substances were prepared in a 1 X 10-S M concentration in 0.1 N sodium phosphate buffer. All procedures were carried out from -0.5 volt to +0.3 volt. Half-wave potentials were calculated directly from the curves developed. Correction for IR drop was calcu- lated. The reference electrode was a standard Weston cell.

Radioactive Assays--Samples containing Pa1 were assayed at the 0.360 m.e.v. y-peak, and those containing 1126, at the 0.035 m.e.v. y-peak in an Autogamma scintillation well. CY4- Containing samples were assayed in a liquid scintillation spectrometer.

RESULTS

Inhibition of Deiodination of Thyroxine-In Table I are presented the results of attempted inhibition studies with a number of compounds. The reaction measured was the pro- duction of iodide-131 from thyroxine (3’,5’-Im). The apparent K, for this deiodination was 1.7 X 10s6 M (&to.52 = two standard deviations). All of those compounds that inhibited were competitive inhibitors. I f a compound was found not to inhibit at lesser concentrations, the concentration was increased until significant inhibition could be measured or until a concen- tration of 20 X 10-e M was achieved. If no inhibition could be

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3494 Thyroxine Degradation. III Vol. 238, No. 10

TABLE I

Determination of K: of compounds related to thyroxine in inhibiting deiodination of thyroxine

The concentrations of thyroxine used were 0.1, 0.2, and 0.5 X 10-S M. The concentration of each inhibitor is indicated below.

Compound Concentration

at which Ki was measured

-- x 10-s Y

D-Thyroxine........................... N-Acetylthyroxine . Tetraiodothyroacetic acid. . Tetraiodothyropropionic acid. Tetraiodothyrocaproic acid. . Tetraiodothyrocaproic acid methyl ester 3,3’,5’-Triiodothyronine . . 3’,5’-Diiodothyronine. 3,5-Dibromo-3’,5’-diiodothyronine. 3,5,3’-Triiodothyronine, . 3’-Monoiodothyronine 3,5-Diiodothyronine . Thyronine........ .._........._._._. 3,5-Diiodo-3’,5’-dibromothyronine . 0-Methoxy-N-acetylthyroxine. 1,4-Hydroquinone . 2,6-Diiodo-1,4-hydroquinone.. 4-Methoxyphenol . . . 2-Methoxy-1,4-hydroquinone. 2-tert-Butylhydroquinone . 2-tert-Butyl+methoxyphenol . 3,5-Diiodotyrosine. 3,5-Diiodo-4-hydroxyphenylformic acid. 3,5-Diiodo-4-hydroxyphenylacetic acid. 3,5-Diiodo-4-hydroxybenzaldehyde . . 2,6-Diiodo-4-nitrophenol. 3,5-Diiodo-4-hydroxyphenylsulfonic

2.5 2.5 2.5 1.0 2.5 2.5 5.0 5.0 1.0

10.0 20.0 20.0 20.0

5.0 20.0 20.0 2.0

20.0 20.0

1.0 1.0

20.0 20.0 20.0 20.0 20.0

acid................................. 20.0 4-Aminophenol 20.0 Cysteine............................... 20.0 Sodium bisulfite....................... 20.0

Ki

2.4 2.6 1.8 1.5 2.0 1.6 5.9

10.6 1.8

15.3 52.0 69.0

:9 m 9.6 1.4

;2 3.2 4.5 m m co co m

lt.0 00 00

TABLE II Amounts of 3’-monoiodothyronine and 3,5,3’,6’-tetraiodothyro-

caproic acid methyl ester deiodinated to yield iodide-13i from prime positions

3’-Monoiodothyronine was added to reactions in 5 X UP M concentration, and 3,5,3’,5’-tetraiodothyrocaproic acid methyl ester, in 1 X We M concentration. Labeled thyroxine in the same concentrations was added to simultaneous incubations for comparison. Reactions were carried out for 2 minutes.

Thyroxine................................. 1.0 Thyroxine................................. 5.0 3’-Monoiodothyronine . 5.0 Tetraiodothyrocaproic acid methyl ester. . 1.0

Amount degraded to yield iodide

VlZ~?lWl~S

2.5 6.7 0.75 2.3

-

observed at this concentration, it was felt that a significant Ki

could not be measured. Deiodi~tion of L-S’-Monoiodothyronine and S,6,$’ ,6’-Tetra-

iodothyrocaproic Acid Methyl Ester-In Table II are shown

results of studies of deiodination of these labeled compounds by microsomes. The 3’-monoiodothyronine was least active as substrate. The amount of each compound degraded is com- pared with the amount of thyroxine degraded at the same concentration.

Attempt to Dewmnstrate Deiodination of 2,6-Ditido-1,4- hydroquinone-This compound was added to the usual micro- somal preparation in 1 X 10B6 M concentration; it was not deiodinated. When 0.4 ml rather than the usual 0.2 ml of microsomes was used, again no deiodination was shown.

Protein Binding of 1,6-Diiodo-l ,&hydroquinone and 1,4- Hydroquinone-Thyroxine labeled with 04 or Pa1 in the cr-phenyl ring forms a firm microsomal protein complex (13). Since 2,6-diiodo-1,4-hydroquinone does not lose its iodine and 1,4- hydroquinone has none to begin with, it seemed possible that the initial reaction might be a protein-combining reaction which these compounds blocked. Because they too might form such microsomal complexes, they were added to the reaction as labeled substances in amounts which varied from 5 to 25 mpmoles. The quantity of each which remained bound to the microsomes after butanol extraction was measured. These results are shown in Table III. Only 2,6-diiodo-1,4-hydroquinone was shown to form such a firm protein complex. This complex was similar to that demonstrated after incubation of thyroxine with micro- somes (13). It could not be dissociated by alcohol extraction, brief treatment in alkaline or acid aqueous solution, electro- phoresis, or dialysis against 2,6-diiodo-1,4-hydroquinone or thyroxine. It was not, therefore, a product of simple equi- librium binding at a surface. It appeared to have joined the microsomal particle in an irreversible complex.

InhiMion of Microsomal Binding of 2,6-Diiodo-1 ,$hydro- quinone by Thyroxine-In order to determine whether reversible binding of 2,6-diiodo-1,4-hydroquinone occurred before the irreversible step, the amount of this substance bound in the presence of thyroxine was measured. As noted previously, the average K,,, for the deiodination of thyroxine was 1.7 X 1OP M

TABLE III Amounts of labeled i,6-diiodo-1 ,.&hydroquinone and 1 ,.$-hydro-

quinone bound to microsomes in acid-butanol-insoluble complex

Results are indicated at various concentrations of each of the labeled compounds.

Compound

2,6-Diiodo-1,4-hydroquinone

1,4-Hydroquinone*

Concentration Amount b0”nd to microsomes

x 10-s Af ?lJpf?k7lCS

0.5 0.45 1.0 1.0 2.5 2.4

0.5 0.1 1.0 0.2 2.5 0.3

* The counting rate of these samples was very low. Reproduc- ibility was poor. The numbers given are averages of five experi- ments at each concentration, but no significance is attached to the absolute values. In general, approximately 1% of the radio- activity was protein-bound regardless of concentration within the limits of investigations at these concentrations. It is felt that by the technique described, this amount of material is accounted for as 1,4-hydroquinone bound nonspecifically to the microsomal protein.

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October 1963 J. Wynn and R. Gibbs 3495

(ho.52 = two standard deviations). The apparent K, for the microsomal binding of 2,6-diiodo-l , 4-hydroquinone was 1.0 X 10m6 M. Thyroxine served as a competitive inhibitor of this reaction, and the apparent Kc for thyroxine in inhibiting this reaction was 2.4 x lo+ M.

Aerobic versus Anaerobic Microsomal Binding of d,B-Diiodo- 1, &hydroquinune (1’2”) and !Z’hyrozine (S ,6-Z126)-Microsomal binding of thyroxine (3’,5’-Pi) cannot be shown aerobically since as soon as thyroxine is bound, both the 3’- and Y-iodine atoms are lost (12, 13). Because it now seemed that a micro- somal combining reaction preceded deiodination and because it is known that the reactions leading to deiodination of thyroxine are oxygen-dependent, it seemed pertinent to determine whether the microsomal combining reactions are oxygen-dependent.

TABLE IV

Solubility of various thyronine derivatives in incubation medium to which no microsomes were added

Results are expressed as the percentage of each compound sedimented at lo5 X g when present in a 20 X 10-6 M concentration in the incubation medium, and as the amount soluble (in molar concentration) under these same conditions.

Compound

L-Thyroxine............................ n-3,5,3’-Triiodothyronine. . n-3,3’,5’-Triiodothyronine . n-3’,5’-Diiodothyronine.. n-3,5-Diiodothyronine . . . Tetraiodothyroacetic acid.. Tetraiodothyropropionic acid. . Tetraiodothyrocaproic acid .

6C )-.

)-.

)-.

Q+= 6.0

Sedimented Solubility

% x 10-e M

72 5.6 75 5.0 6 18.8 5 19.0

25 15.0 33 13.3 74 5.2 73 5.4

-

7.0 8.0 9.0 10.0 apparent pH of phenolic pK

II.0

FIG. 1. Graph of apparent pK values of several halogenated thyronine derivatives and 3,5-diiodotyrosine in different con- centrations of ethanol and water. The numbers at the top of each line refer to the numbers given each compound in Table V. Com- pounds 5 and 6 had a common point at 300Jo ethanol concentration, and this point is therefore represented by only one X mark.

TABLE V

Apparent pK of phenolic hydroxyl of various thyronine derivatives in aqueous solution

Values were determined by extrapolation of lines developed in Fig. 1 for the apparent pK of the phenolic hydroxyl in various concentrations of alcohol and water. The numbers preceding each compound refer to numbers given each compound in Fig. 1.

Compound Apparent pK of phenolic hydroxyl

1. Thyroxine. ......................... 6.95 2. 3,5,3’-Triiodothyronine . ............ . . . . 9.22 3. 3’-Monoiodothyronine . .............. 8.40 4. 3,5-Diiodothyronine. ................ . 10.60 5. 3’,5’-Diiodothyronine . .............. 6.92 6. 3,3’,5’-Triiodothyronine. ............ . 6.90 7. 3,5-Dibromo-3’,5’-diiodothyronine ... 6.90 8. 3,5-Diiodo-3’,5’-dibromothyronine. . . . 6;82 9. 3,5-Diiodotyrosine . ................. . . 6.55

Cm

200

z . l5cl zi $ 2 0

=i IO.0 2

50

0

__71 t 4 A YUB t

\ C 4 I 0 t.30

Volts FIG. 2. Graphs of current voltage waves measured for 1,4-hy-

droquinone (A), 2-methoxyhydroquinone (B), and 2,6-diiodo-1,4- hydroquinone (C). Estimated points of half-wave are indicated by +. The sharp increase in current flow at about +0.2 volt in the 1,4-hydroquinone curve has been observed and commented upon by others (19, 29). The estimated half-wave potentials are: 1,4-hydroquinone, +0.031 volt; 2-methoxyhydroquinone, +0.031 volt; and 2,6-diiodo-1,4-hydroquinone, +0.049 volt. Although all measurements were done at a 1 X 1P M concentration of test substance in 0.1 hl phosphate buffer, it is apparent that the cur- rent voltage wave of 2,6-diiodo-1,4-hydroquinone is smaller than that of the other two hydroquinones. This is probably due to a difference in solubility. 2,6-Diiodo-1,4-hydroquinone was diffi- cult to solubilize at this pH. It seems probable that when meas- urements were made, this compound was not entirely in true solution.

When 2,6-diiodo-l , 4-hydroquinone and thyroxine (3,5-P) were added to the microsomal preparation, neither was bound under anaerobic conditions.

Solubility of Thyroxine Derivatives in Incubation Medium--In Table IV are shown the percentages of various thyronine deriva- tives soluble in the incubation medium, when present in a 20.0 X lee M concentration. It can be seen by comparison

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Thyroxine Degradation. III Vol. 238, No. 101

with Table I that there is no relationship between Ki and solubility.

Apparent Phenolic pK of Various Thymnine Derivatives--In Fig. 1 is shown a composite of the data collected in alcohol solutions for approximation of phenolic pK values. Table V gives the values of pK when the lines projected from these data were extrapolated to zero alcohol concentration. Again it can be seen that the correlation between Ki of these compounds and apparent pK of phenolic hydroxyl is not absolute.

Illeasuremcnt of Oxidation PotentialsThe oxidation potentials of 1,4-hydroquinone, 2-methoxy-1,4-hydroquinone, and 2,6- diiodo-1,4-hydroquinone were measured. An attempt was made to demonstrate whether 4-methoxyphenol might have a current voltage wave below +0.2 volt. None was found. The current voltage waves measured for the first three compounds are illustrated in Fig. 2. The apparent half-wave potentials were estimated from geometric approximation of the midpoints of the almost symmetrical curves. These values are: 1,4- hydroquinone, +0.031 volt; 2-methoxy-1,4-hydroquinone, +0.031 volt; and 2,6-diiodo-1,4-hydroquinone, $0.049 volt. The value for 1,4-hydroquinone compares with Koltoff’s observed value of to.037 volt (19).

DISCUSSION

A number of similar compounds will competitively inhibit the degradation of thyroxine by rat liver microsomes. For reasons detailed below, it is thought that a common structural charac- teristic renders these compounds susceptible to the same initial reactions with the microsome. The primary aim of this work has been to delineate this reactive structure.

All of those compounds which competitively inhibit, except 4-aminophenol, may be considered derivatives of 1 ,4-hydro- quinone. Alkylation of 1,4-hydroquinone, yielding 4-methoxy- phenol, or arylation, yielding thyronine, destroys capacity to inhibit. The methyl and phenylalanine moieties are analogous in their deactivating function. Both form ethers. Changes in the aliphatic side chain of thyronine have no effect on inhibition activity. Methylation of the free phenolic hydroxyl of a thyroxine derivative completely destroys capacity to inhibit the reaction measured. The common structure of all inhibiting compounds is that of a phenol in which there is an electron- donating p-substituent. I f p-substituent hydroxyl is conjugated to form an ether, capacity to inhibit is lost; this may be restored by substitution of the positions ortho to the phenolic hydroxyl with either a halogen or tertiary butyl group. Since o-phenolic tertiary butyl substitution is as effective as halogen substitution in facilitating inhibition, there is no feature specific to the halogens themselves which increases capacity to inhibit. o-Sub- stituted methosy also facilitates the reaction. Similarly, the differences in facilitation noted when either bromine or iodine is the substituent are quantitative rather than qualitative. Bromine and iodine substitution in the cu-phenyl ring of thyronine produce equivalent facilitation. However, iodine substitution in the P-phenyl ring facilitates inhibition to a greater extent than does similar bromine substitution.

As noted, competitive inhibition of deiodination of thyroxine is exhibited by compounds, such as 1,4-hydroquinone, which contain no halogens at all. Pertinent to an inquiry regarding reactions which might precede deiodination, 1,4-hydroquinone and 2,6-diiodo-l,4-hydroquinone have been examined not only as inhibitors but also as substrates in the reaction. The latter

has been found to form an irreversible complex with microsomes similar to that formed when thyroxine is used as substrate (13). Furthermore, the apparent K, for this reaction approximately equals Ki in displacing thyrosine from the deiodination reaction, and the apparent K, for the deiodination of thyroxine approxi- mately equals K i in displacing the 2,6-diiodo-l , 4-hydroquinone from its complexing reaction. These observations imply that 2,6-diiodo-l , 4-hydroquinone and thyroxine are competing for common, initial, reversible binding sites on the microsomes. It should be noted, however, that the irreversible microsome complex separated when either thyroxine or 2,6-diiodo-l , 4- hydroquinone is used as substrate is not the initial, transient combining form. The dependence of the irreversible complex on the prior reversible complex is inherent in the competitive inhibition studies. 1,4-Hydroquinone could not be demon- strated to form an irreversible complex with the microsomes. Inhibition by this compound may simply be related to the formation of the proposed reversible or dissociable initial complex. Inhibition does not seem simply to be related to a nonspecific reduction since neither cysteine nor bisulfite, in the concentrations used, influenced the reaction,

Although the initial binding sites may be common, there is a significant difference between the subsequent reactions of thyroxine and 2,6-diiodo-1,4-hydroquinone. As soon as thyroxine is shown to have formed the irreversible microsomal complex, both /3-phenyl ring iodine atoms are lost (13). 2,6- Diiodo-1,4-hydroquinone does not lose its iodine at all. This implies that the sequence of reactions involves first microsomal complexing followed by iodine removal from the P-phenyl ring of thyroxine. The requisite structure for the second reaction must involve a conjugated p-substituted oxygen atom, and it is probable that all of the inhibiting phenoxy derivatives containing iodine in the P-phenyl ring are deiodinated. This is inferred from the fact that 3’-monoiodothyronine and 3’) 5’) 3,5-tetra- iodothyrocaproic acid methyl ester both served as substrate for deiodination. These two compounds represent extremes of modification of thyroxine by partial halogenation and aliphatic derivation. All other similar compounds are intermediate between these and thyroxine itself.

ilt a time when it was not appreciated that irreversible protein complexing and deiodination were sequential reactions, it was shown that deiodination was oxygen-dependent (12). The studies in regard to irreversible binding of 2,6-diiodo-1,4- hydroquinone indicate that the reactions which lead to the formation of this complex are oxygen-dependent. Thus the initial, reversible binding complex or the later irreversible combining reaction, or both, may be oxygen-dependent. The deiodination reaction itself may not be oxygen-dependent at all.

The combination of p-substituted oxygen conjugation and various o-substitutions will have certain predictable influences on physical characteristics of these hydroquinone derivatives. Solubility, pK of the phenolic hydroxyl, oxidation potentials, resonance probabilities, and steric hindrance are all variables which might alter susceptibility of the basic hydroquinone structure for the reversible microsomal complexing reaction. Of these, solubility and pK of the phenolic hydroxyl seem clearly not to be related to inhibition.

2-Methoxy-1,4-hydroquinone and 1,4-hydroquinone are among the least effective inhibitors, and they have the lowest oxidation potentials among those compounds examined. 2,6- Diiodo-1,4-hydroquinone has an oxidation potentia.1 only

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October 1963 J. Wynn and R. Gibbs

slightly higher than these compounds but lower than those of 4-methoxyphenol, thyronine, and other phenols. These latter values are inferred from the observations made on phenol with a graphite electrode (20). Similarly, the iodinated p-phenoxy phenolic compounds would be expected to have even higher oxidation potentials than the uniodinated compounds. Thus, there is no absolute correlation between observed and projected oxidation potentials and inhibition.

Inhibition is not related to the electron-accessive charac- teristics of the o-substituent. The differences noted among 1,4-hydroquinone, 2,6-diiodo-l , 4-hydroquinone, and a-ted- butyl-1,4-hydroquinone emphasize this point. Whereas the net electron density of the phenyl ring of 2,6-diiodo-1 ,Chydro- quinone is decreased relative to 1,4-hydroquinone, the net electron density of the phenyl ring of 2-tert-butyl-1,4-hydro- quinone is virtually unaffected (21). Yet both substituted compounds are far better inhibitors than 1 ,4-hydroquinone. These observations indicate that the effect of the substituent is to add a feature which facilitates and that such an addition is independent of the electron-accessive characteristics of the substituent. This suggests that a steric influence of the various &ho substituents on the adjacent phenolic hydroxyl may facilitate the initial reaction. Thus tertiary butyl and o-iodine substitutions have the greatest influence in facilitating inhibition; bromine has intermediate, and methoxyl has least influence. This order of effect conforms in general to the size of the o- substituted group and thus perhaps to the magnitude of the steric effect, but does not correlate with influence of the sub- stituents on the electron density of the substituted ring.

There is obviously a requisite relating to the p-substituent of the phenol as well. This substituent cannot be electron- accessive (viz. sulfonate, nitro, carboxyl, or aldehyde). It may not be simply an electron-dense group with an inductive ortho- para directing influence since the derivatives formed by (CH2) COOH do not inhibit. Only p-hydroxyl, amino, or p-ether oxygen with associated o-substitution promote the participation of a phenol in these reactions.

In seeking some characteristic which will relate these con- ditions, the problem is directed by summing these observations. First, the reactions require a compound with a free phenolic hydroxyl. This seems to be the only reactive group of thyroxine which is inviolable. Second, although deiodination of thyroxine is the reaction initially chosen to follow, it has been shown that inhibition of this reaction by these compounds is related to another reaction preceding deiodination which is oxygen- dependent. Third, the fact the differing o-substituents will promote these prior reactions indicates that the o-substituent itself does not participate directly. Fourth, the characteristic of the effect of various o-substituents suggests that facilitation of inhibition is correlated with magnitude of steric effect on the phenolic hydroxyl. And finally, there is a requirement for an electron-donating p-substituent. All of these features point to a primary reaction involving oxidation of a phenol containing certain o- and p-substituents. The similarity of these observa- tions to those of Cook, Kuhn, and Fianu (22) relating to the production of stable free radicals after oxidation of o-, p- substituted phenols seems more than a coincidence.

A brief review of the influence of various substituents on the stability of free radicals of substituted benzene derivatives reveals the following. Swain, Stockmayer, and Clarke (23) and Blom- quist and Berstein (24) correlated the molecular stability of

various p-substituted benzoyl peroxides with the known Ham- mett s@ma function of the substituents. They found a direct relationship between the rate of dissociation of peroxides to form free radicals and the influence of the p-substituent as predicted by the Hammett sigma function. The most electron- accessive or electrophilic substituents were related to slow rates of dissociation of the peroxides to the free radical; the electron- donating substituents were related to rapid rates of dissociation to the free radical. It follows that the activation energies for these dissociation reactions were least when groups with negative Hammett sigma numbers were the substituents and were largest when groups with positive tigma numbers were the substituents. Thus p-nitro substitution increased stability of the peroxide and did not favor dissociation to the free radical; p-methoxy substitution decreased stability of the peroxide and favored dissociation to the free radical. From these observations it can be inferred that electron-donating p-substituents enhance resonance stability of the free radical whereas electrophilic p-substituents decrease resonance stability of the free radical and promote the formation of more stable peroxides. The concept essential to this discussion is that these demonstrations have related free radical stability to the predictable influence of the p-substituent on the electron cloud of the phenyl ring.

Cook, Kuhn, and Fianu (22), investigating the nature of substituents necessary to stabilize free radicals generated by oxidation of phenols, have drawn the following conclusions. Relatively stable free radicals may be generated if the ortho and para positions of the phenol are hindered by substitution and if none of these substitutions contain labile hydrogen. Furthermore, free radical stability is related in part to the size of the o-substituent and therefore in all likelihood is related at least in part to a steric effect. The p-substituent may be tertiary butyl, but p-alkoxy substituents formed the most stable p-substituted free radicals studied by these investigators. These of course are better electron donors than the tertiary butyl group and therefore this finding is consistent with the generali- zations made above regarding Hammett sigma numbers. Al- though the effects of several alkoxy groups were studied, no relationship was found between the size of the alkoxy p-substi- tution and the stability of the free radical.

Returning to the summary of features found to be common to all of the inhibiting compounds, it is apparent that these are simply a statement of the requisites necessary for the pro- duction of a stable phenoxyl free radical. Furthermore, the negative observations regarding p-substituent (CH&COOH compounds containing labile hydrogen and those concerning electrophilic para substitutions are also consistent with the hypothesis that a stable phenoxyl free radical is involved in the inhibition reactions. Despite the apparent dissimilarity of the p-alkoxy and p-aryloxy compounds from the 1,4-hydroquinones and 4-aminophenol, both of these types of compound would provide a source of stable phenoxyl free radical through the rapidly reversible hydroquinone-quinone and aminophenol- quinonimine oxidation-reduction systems. Furthermore, conju- gation of the 4-hydroxyl to form methoxyphenol or thyronine without substitution of the o-phenolic positions would im- mediately disrupt this free radical stability for several reasons. The electron-donating influence of the p-substituent would be markedly reduced. The ortho positions would be subject to attack after the first electron withdrawal. The phenoxyl radical itself would not be sterically hindered and therefore might

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Thyroxine Degradation. III Vol. 238, No. 10

form peroxides or other slowly dissociated products. Thus the ability of all of these derivatives of 1,4-hydroquinone to inhibit the reversible binding reactions preceding the deiodination of thyroxine can be closely correlated with the predicted tendency of these compounds to form stable phenoxyl free radicals.

The facilitation of inhibition effected by halogen substitution in the cu-phenyl ring of thyronine may relate to a steric effect about the ether oxygen. After electron withdrawal from the hydroxyl, ether oxygen may contribute to the resonating system by donating 1 electron. This would leave an unshared electron on ether oxygen and cause a relative positive charge to reside there. Under these circumstances, dimer or polymer reactions might involve the et,her oxygen itself. However, models of thyroxine and other phenoxyphenols iodinated in the a-phenyl ring clearly show that a 3 ,&iodine-substituted cr-phenyl ring shields more than three-fourths of the space about the ether oxygen. Thus, any tendency of the ether oxygen to participate in polymer reactions is hindered to a small extent by the cr-phenyl ring and to a large extent by iodine substitutions in this ring. For these reasons, it seems reasonable to suggest that the facilitation of inhibition effected by 3,5-halogen substitution in the cr-phenyl ring is also related to free radical stabilization.

The demonstration of a reversible binding phenomenon, the dependence of binding of any sort on oxygen, and the evidence that a stable phenoxyl free radical may be involved early in the reactions has led to the formulation that these three observations may be related. It is presumed that the initial reaction may be the oxidation of the phenolic hydroxyl to form the phenoxyl free radical. It is this free radical which may then form a reversible complex with the microsome, competing with other phenoxyl free radicals for the initial binding sites.

SUMMARY

The competitive inhibition of deiodination of the &phenyl ring of thyroxine by liver microsomes has been studied. A group of 1,4-hydroquinones and their derivatives have been shown to displace thyroxine from oxygen-dependent initial reactions which precede deiodination. These inhibiting com- pounds are structurally related to thyroxine in that all are phenols containing electron-donating pars substitutions. Ortho substitution of these phenols greatly increases their ability to inhibit deiodination of thyroxine. From the specific nature of the various ortho and para substituents which facilitate the inhibition, and because oxygen is requisite for the reactions which precede deiodination, it is surmised that the reactive structure which competes with thyroxine for the initial binding site may be a stable phenoxyl free radical. Ability to inhibit deiodination is not related to solubility, pK of the phenolic hydroxyl, or oxidation potential of the phenolic group.

The sequence of reactions involved in thyroxine degradation by microsomes has been further delineated. 2,6-Diiodo-1,4- hydroquinone forms an oxygen-dependent, irreversible, micro- somal complex similar to that formed by thyroxine (13). Whereas thyroxine and other thyronine derivatives iodinated in t,he P-phenyl ring are deiodinated so rapidly that it has not been possible to separate the complexing and deiodination reactions, 2,6-diiodo-1,4-hydroquinone is not deiodinated at all. Thus a reversible microsomal combining reaction involving the phenoxyl free radical appears to occur first, followed by irreversible combination and deiodination. A requisite for the deiodination reaction itself is the presence of p-substituent ether or, even more specifically, p-phenoxy itself.

1. 2. 3. 4. 5.

6. 7.

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13. 14.

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James Wynn and Robert GibbsDEGRADATION; RELATIONSHIP OF STRUCTURE TO INHIBITION

Thyroxine Degradation: III. COMPETITIVE INHIBITION OF THYROXINE

1963, 238:3490-3498.J. Biol. Chem. 

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