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Ion-exchange chromatographic analysis of iodothyronines
ANALYTICAL
67, 157- 165 ( 19755)
BIOCHEMISTRY
Ion-Exchange
Chromatographic
Analysis
of Iodothyronines KENJI SORIMACHI AND NOBUO UI Department
of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi, 371 Japan
Received September 23, 1974: accepted March 3, 1975 An ion-exchange chromatographic procedure for the analysis of iodothyronine mixtures containing iodotyrosines is described. Eight different iodothyronines are separated into five peaks and one shoulder, the order of their elution being related to the number and position of iodine substitution in the compounds. The resolution of monoiodotyrosine and diiodotyrosine from each other and from iodothyronines is excellent. Quantitation of iodoamino acids is carried out by the automatic analysis of the effluent based on the ceric-arsenite reaction. This procedure has been applied to the determination of the iodoamino acid distribution in hog thyroglobulin and to the analysis of photodegradation products of thyroxine and triiodothyronine.
Ion-exchange chromatography, as combined with the automatic iodine analysis of the effluent by the ceric-arseneous reaction, has proven useful for the quantitative determination of iodoamino acids (l-3). However, hitherto-reported studies employing this procedure are concerned almost exclusively with the four major constituent iodoamino acids in thyroglobulin, i.e., monoiodotyrosine (MIT),’ diiodotyrosine (DIT), 3,3’,Striiodothyronine (T3) and thyroxine (TJ. It has not been known whether this method can also be adopted for the analysis of several other iodoamino acids, some of which might be contained in thyroglobulin as minor iodoamino acid residues. In their study on the iodoamino acid distribution in rat or human thyroglobulin, labeled either in viva (equilibrium labeled) or in vitro with radioactive iodine, Ogawara et al. (4) employed an ion-exchange chromatographic procedure to fractionate thyroglobulin digests into the total iodothyronine fraction and iodotyrosines and subsequently analyzed the former fraction by means of thin layer chromatography. They reported the presence of a small but not negligible amount of minor iodoamino acids in addition to the four major iodoamino acids in thyroglobulin. In this paper we report a simple chromatographic procedure that permits the analysis of most unlabeled iodothyronines in a single step. ’ See Table 1 for abbreviation of iodoamino acids used. 1.57 Copyright All rights
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
158
SORIMACHI
AND
UI
The method has been applied to assess the possible content of minor iodoamino acids in hog thyroglobulin and also to analyze the degradation products formed by exposure of T4 or T, to ultraviolet irradiation. MATERIALS
AND
METHODS
Zodoamino acids. MIT, DIT, T3 and Td were the same commercial products as used in the previous study (3); 3,5-diiodothyronine (3,5-TZ) was purchased from Sigma. Most other iodothyronines, i.e., 3monoiodothyronine (3-T,), 3’-monoiodothyronine (3’-T,), 3,3’-diiodothyronine (3,3’-T,) and 3,3’,5’-triiodothyronine (T& and iodohistidines, i.e., monoiodohistidine (MIH) and diiodohistidine (DIH), were kindly supplied by Dr. H. J. Cahnmann of the National Institutes of Health, Bethesda, MD. The iodohistidines had been prepared by Dr. J. C. Savoie of the Hopital de la PitiC, Paris. The preparation of 3’,5’diiodothyronine (3’,5’-T2) was a kind gift of Dr. M. Nakano of Gunma University School of Medicine. The purity of all these preparations of iodoamino acids was verified by ion-exchange chromatography carried out under the standard conditions employed in the present study. A few preparations were found to be very slightly contaminated with iodide (6 1% of the total iodine). The concentration of each iodoamino acid solution was determined from the iodine analysis, which was performed with a Technicon AutoAnalyzer according to the procedure recommended by the manufacturer for the analysis of protein-bound iodine. Thyroglobulin. Hog thyroglobulin was prepared by the method previously reported (5). In the present investigation, a chromatographically purified but not subfractionated preparation with an average iodine content of 0.71% was used. Before the chromatographic analysis of iodoamino acids, thyroglobulin was hydrolyzed enzymatically (2) by the consecutive use of Pronase and aminopeptidase M as reported previously (3). Chromatographic analysis of iodoamino acids. A slight modification of a previously reported method (3) was adopted. Samples were applied to a column (1.0 X 15 cm) of a cation-exchange resin, AG 5OW-X4 (30-35 pm), which had been equilibrated with 0.04 M ammonium acetate buffer, pH 4.7, containing 30% (v/v) ethanol at 5O”C, and a gradient of increasing pH was prepared from 100 ml of the starting buffer and 100 ml of 0.65 N NH,OH. The effluent was analyzed continuously by the iodine-catalyzed ceric ammonium sulfate-arseneous acid reaction with a Technicon AutoAnalyzer. Photodegradation. A solution of T, or ‘I; dissolved in 0.04 M ammonium acetate buffer, pH 4.7, containing 30% ethanol, was placed in a quartz spectrophotometer cuvette and irradiated for 3 min with two low-
ANALYSIS
159
OF IODOTHYRONINES
pressure mercury lamps (19 W) having an emission peak at 253.7 nm. The distance between the cuvette and the lamps was approximately 15 cm. The irradiated solution was applied directly to the column for analysis. RESULTS Chromatographic Separation of Various Iodothyronines and Iodotyrosines
In our previous study (3) aiming at the ion-exchange chromatographic separation of the four major iodoamino acids in thyroglobulin, a pH
-
-
-
-
FIG. 1. Elution profiles in the cation-exchange chromatography of iodoamino acids. (A) A mixture of authentic iodoamino acids (2 nmoles each except for DIH of which 20 nmoles were used); (B) an enzymatic digest of hog thyroglobulin. For the latter experiment, 1 ml of a 0.106% thyroglobulin solution in 0.1 M Tris-HCl buffer, pH 8.0, was mixed with 100 ~1 of a 1% Pronase solution, 10 ~1 of 0.5 M I-methyl-2-mercaptoimidazole and 10 ~1 of toluene and incubated at 37°C for 24 hr; subsequently, 10 ~1 of 0.1% aminopeptidase M solution was added and the incubation was continued for an additional 24 hr. The digest was mixed with 1 ml of 0.2 M ammonium acetate buffer, pH 4.7, containing 30% ethanol, and 1 ml was charged to the column for chromatographic analysis. The flow rate for elution was 0.56 ml/min.
160
SORIMACHI
AND UI
gradient was prepared using equal volumes (90 ml) of 0.04 M ammonium acetate buffer, pH 4.7, and 1 N NH,OH. When iodothyronines other than TS and Ta were included in the sample and subjected to analysis under the same conditions, they were not separated well from either the TZj or the TJ peak. The increase in the total volume of eluant, which gives a shallower pH gradient, did not improve the resolution but brought about broadening of the peaks. However, the use of a lower concentration of NH,OH, i.e., 0.65 N, and a slightly increased total eluant volume (200 ml) was found to give excellent separation of most iodothyronines. The concentration of NH,OH was not very critical and could be increased up to 0.85 N without great change in the resolution.g Further increase in eluant volume did not improve the resolution. Figure IA shows a typical chromatographic pattern obtained when a mixture containing 2 nmoles each of eight iodothyronines and two iodotyrosines was analyzed under the modified conditions mentioned above. The time required for complete elution of the iodoamino acids was approximately 3 hr. Each peak was identified by a number of separate runs made with a mixture of one unknown plus one or two known iodoamino acids. As shown in this figure, the separation of MIT and DIT from each other and from other iodoamino acids is excellent. Iodothyronines give five peaks and one shoulder. No separation was attained, however, between 3-T, and 3,5-T, and between 3’-T1 and Ti. Addition of a large amount of DIH results in the appearance of a small peak that is eluted slightly before MIT. The behavior of another iodohistidine, MIH, could not be investigated, because the sensitivity of the analysis for this particular iodoamino acid was too low. Table 1 summarizes the elution volume of each iodoamino acid and iodide as well as the relative peak area, which was measured separately using solutions each of which contained one kind of iodoamino acid of known concentration. A remarkable variation in the catalytic effect of different iodoamino acids on the reduction of ceric ions by arseneous acid, even after correction was made for the difference in the number of iodine atoms in each molecule, is to be noticed. The iodine atom substituted in the phenolic ring of the diphenyl ether group in thyronines is generally less effective than that in the nonphenolic ring. Thus, 3’-T, shows a peak area much smaller than that of 3-T, when the same amount of each compound was applied to the column. In the case of diiodothyronines, the relative area decreases in the order of 3,5-T,, 3,3’-T, and 3’,5’-T2. However, T3, having two iodine atoms in the p In some experiments employing a column used for a long time without repacking, 0.85 NH,OH rather than 0.65 N NH,OH gave a slightly better separation of iodothyronines. Hence, exploration of the most suitable concentration of NH,OH for each column is recommended for critical investigations. N
ANALYSIS
OF
TABLE ELUTION
VOLUMES
AND
CHROMATOGRAPHIC
Compound Monoiodotyrosine Diiodotyrosine 3-Monoiodothyronine 3 ‘-Monoiodothyronine 3,5-Diiodothyronine 3,3 ‘-Diiodothyronine 3 ’ .5 ‘-Diiodothyronine 3.3’,5-Triiodothyronine 3,3 ’ ,5 ‘-Triiodothyronine Thyroxine Monoiodohistidine Diiodohistidine Iodide
PEAK
ANALYSIS
OF IODOAMINO
MIT DIT 3-T, 3’-T, 3,5-T, 3,3’-T, 3 ‘,5 I-T, T3 T; T4 MIH DIH I-
1
RELATIVE
Abbreviation
161
IODOTHYRONINES
AREAS
Approx elution volume (ml) 31 50 80 96 80 113 88 106 96 91 21 6”
THE CATION-EXCHANGE
IN
ACIDS
AND
IODIDE
Peak area measured (cm*/nmole)
Relative peak area per mole
1.52 3.82 1.53 0.50 2.56 2.32 2.04 2.94 3.27 5.72 0.005 0.07 2.96
1 .oo 2.50
1.00 0.33
1.68 1.53 1.34
1.93 2.15 3.75 ‘co.01 0.04 1.94
Relative peak area per iodine atom 1.00 1.25 1.00 0.33 0.84 0.76 0.67 0.64 0.72 0.94
CO.01 0.02
1.94
“ Iodide was not retarded in this chromatogram
nonphenolic ring, gives a slightly lower relative peak area than that of TA with two iodine atoms in the phenolic ring. DIH and especially MIH show a very weak catalytic effect on the reduction (Table 1). Therefore, a chromatographic analysis of unlabeled iodohistidines by the present procedure does not seem to be useful. Analysis of lodothyronines
in Thyroglobulin
In order to see whether thyroglobulin contains iodothyronines other than T, and T, in appreciable amounts, a large amount of hog thyroglobulin digest was applied to the column and analyzed under the present conditions. As shown in Fig. lB, the elution profile observed is quite similar to that obtained by the chromatographic procedure described previously for the analysis of the four major iodoamino acids in thyroglobulin (3). In the iodothyronine region, no peak or shoulder other than the T, and T3 peaks can be detected, and the shape of the curve is exactly the same as that for a mixture of T4 and T, prepared in accordance with their contents in thyroglobulin. From this and other similar experiments, it has been concluded that most of iodothyronines other than T, and T, are not contained in amounts exceeding 0.05 residue per molecule; however, 3’,5’-T, that is eluted, if present, as a shoulder of the large T4 peak and 3’-T, that gives an exceptionally small peak will es-
162
SORIMACHI
AND
UI
cape detection at this low level. This conclusion was confirmed by several other chromatographic runs made with samples in which small increments of the minor iodothyronines were added to a thyroglobulin digest or to an equivalent mixture of T, and T,. Even in the case of Tj, which intervenes between the T, and T, peaks, the valley between the two peaks was elevated significantly when TA was added in an amount corresponding to 0.05 residue per molecule of thyroglobulin; on further addition, the extent of elevation increased in proportion to the amount of TA added. On the other hand, the content of the four major iodoamino acids in this preparation of hog thyroglobulin (Fig. 1B) coincided with the value expected from the results of our previous investigation on the relationship between the iodoamino acid distribution and the total iodine content in hog thyroglobulin (6). The mean values obtained in two independent experiments were: MIT, 7.17; DIT, 7.47; T,. 0.93: and Tq, 3.33 residues per molecule of thyroglobulin.
c--cc&& I. _ c-L -L& : -,i L i~~~L7 FIG. 2. Elution profiles in the cation-exchange chromatographic analysis of photodegradation products of (A) T4 and (B) T3. For details, see Table 2 and text.
ANALYSIS
OF
163
IODOTHYRONINES
A fairly large peak that follows the unretarded I- peak, which shows the deiodination of iodoamino acids occurring during the storage and enzymatic hydrolysis of thyroglobulin, is due to 1-methyl-2-mercaptoimidazole added to the sample to prevent the degradation of iodoamino acids (Fig. 1B). In addition to the four major iodoamino acid peaks eluted later, several tiny peaks are seen in the regions between l-methyl2-mercaptoimidazole and MIT and also between DIT and T4. They are attributed to undigested iodine-containing peptides. The sum of these areas is approximately 2% of the total area excluding that of I-methyl-2mercaptoimidazole. This is somewhat higher than that observed usually. Analysis
of Photodegradation
Products
of T, and T,
Figure 2 shows the elution profiles obtained when a solution of T, or T, exposed to ultraviolet irradiation was subjected to the analysis. In addition to the huge peak of II, several peaks can be seen. They represent, besides the starting compounds, several iodothyronines formed by deiodination and iodotyrosines formed by rupture of the diphenyl ether bond in iodothyronines. Table 2 summarizes the results of these analyses. Since the amount of iodide contained in the samples was too large to be measured in the TABLE 2 ION-EXCHANGE CHROMATOGRAPHIC ANALYSIS OF THE PHOTODEGRADATION PRODUCTS OF T, AND TV Amount found after exposure” (nmolesl I-containing compound
From T,
T, T3 Ti 3,5-T,* 3.3’-T, 3’,5’-T, 3’-T, DIT MIT I-
2.22 1.17 1.05 0.20 0.49 0.63 0.29 12.2
6.02 1.21 1.49 1.11 0.33 0.21 16.0
Recovery of iodine
104%
93%
-
From T,
-
(LFor this analysis, 7.4 nmoles of T, or 14.9 nmoles of T, dissolved in 1 ml of 0.04 M ammonium acetate buffer, pH 4.7, containing 30% ethanol, were exposed to ultraviolet irradiation, and 1 ml of each solution was charged to the column. b This component may also contain 3-T,. but the value was calculated as though no 3-T, was contained in the samples.
164
SORIMACHI
AND
UI
same run as used for the analysis of iodoamino acids (Fig. 2), iodide was determined separately using much smaller sample sizes. DISCUSSION
The results presented in this paper show that the ion-exchange chromatographic procedure can be used successfully not only for the analysis of the four major iodoamino acids in thyroglobulin, i.e., MIT, DIT, T, and T,, but also for the analysis of various iodothyronines other than T3 and T,. The sensitivity of analysis, even though considerably different with different iodothyronines, is high (Table 1). Usually less than 1 nmole of each iodothyronine is enough for its quantitation, although the presence of a huge nearby peak of other iodothyronines will make the determination inaccurate or impossible. Iodohistidines cannot be analyzed at such low levels due to their extremely low sensitivity. However, the present chromatographic procedure could possibly be useful for all iodoamino acids including iodohistidines, if they are isotopically labeled. Resolution of iodothyronines in this chromatography was generally good, although some of them could not be separated from others. There is some relationship between the order of elution and the difference in iodine substitution in iodothyronine molecules. Iodothyronines without iodine substitution in the phenolic ring of the diphenyl ether group, i.e., 3-T, and 3,5-T,, are eluted earliest, whereas those with two iodine substitutions in the phenolic ring, 3’S’-T,, T4 and TA, come off the column later, and those with one substitution in the phenolic ring 3’-T,, T3 and 3,3’-T,, belong to the group retained most strongly. Furthermore, among the group with two iodine substitutions in the phenolic ring, iodothyronine with no iodine substitution in the nonphenolic ring, 3’S’-T,, is eluted first, followed then by that with two substitutions, T,, and finally by that with one substitution, T& This order is also the same within the group of iodothyronines with only one iodine substitution in the phenolic ring, i.e., 3-T,, T3 and 3,3’-T2. The presence in thyroglobulin of minor iodoamino acid residues other than those of the four major iodoamino acids has been shown qualitatively by several investigators employing tracer experiments. In the quantitative investigation by Ogawara et al. (4), various differences in the distribution of iodoamino acids were noticed between thyroglobulin iodinated in viva and in vitro. They reported that almost 15% of the total iodothyronines belonged to those other than T, and TY, even in equilibrium-labeled, native rat thyroglobulin. However, none of these minor iodothyronines, at least those excluding 3’,5’-T2, was present in hog thyroglobulin in amounts exceeding 0.05 residue per molecule (Fig. 1B). Although further investigation with rat and other thyroglobulins is
ANALYSIS
OF IODOTHYRONINES
165
needed, it is to be emphasized that the enzymatic digestion of thyroglobulin was almost complete and the extent of deiodination was very low in the present study. We believe, therefore, that this result at least provides a rationale for our previous investigation (6) on the relationship between the iodoamino acid distribution and the total iodine content in hog thyroglobulin in which an analytical method capable of measuring only four major iodoamino acids was used. The usefulness of this chromatographic procedure has been shown also in the analysis of photodegradation products of T, and T3. The results indicate that the ultraviolet radiation not only deiodinates iodothyronines successively (7) but also results in the cleavage of the diphenyl ether bond leading to the formation of DIT or MIT. The latter finding confirms the observation by Koya (8) who analyzed similar products by paper and thin-layer chromatography. No attempt was made in this study to determine the dependence of the composition of the irradiation products on the experimental conditions of the irradiation. However, the chromatographic method described in this paper will be useful in such investigations which may provide a better understanding of the mechanism of the photodegradation of the thyroid hormones. ACKNOWLEDGMENT We are greatly indebted to Dr. H. J. Cahnmann of the National Institutes of Health for his helpful discussions and advice.
REFERENCES 1. 2. 3. 4.
Block, R. J., and Mandl, R. H. (1962) Ann. N. Y. Acad. Sci. 102, 87-95. Rolland, M., Aquaron, R., and Lissitzky, S. (1970) Anal. Biochem. 33, 307-3 17. Sorimachi, K., and Ui, N. (1974) J. Bid&em. (Tokyo) 76, 39-45. Ogawara, H., Bilstad, J. M., and Cahnmann, H. J. (1972) Biochim. Biophys. Acra 339-349.
5. Ui, N., and Tarutani, 0. (1961) J. Biochem. (Tokyo) 50, 508-5 18. 6. Sorimachi, K., and Ui, N. (1974) Biochim. Biophys. Acta 342, 30-40. 7. Lein, A., and Michel, R. (1959) C. R. Sot. Biol. 153, 538-540. 8. Koya, S. (1973) J. Pharm. Sot. Japan 93,117-728.
257,
67, 157- 165 ( 19755)
BIOCHEMISTRY
Ion-Exchange
Chromatographic
Analysis
of Iodothyronines KENJI SORIMACHI AND NOBUO UI Department
of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi, 371 Japan
Received September 23, 1974: accepted March 3, 1975 An ion-exchange chromatographic procedure for the analysis of iodothyronine mixtures containing iodotyrosines is described. Eight different iodothyronines are separated into five peaks and one shoulder, the order of their elution being related to the number and position of iodine substitution in the compounds. The resolution of monoiodotyrosine and diiodotyrosine from each other and from iodothyronines is excellent. Quantitation of iodoamino acids is carried out by the automatic analysis of the effluent based on the ceric-arsenite reaction. This procedure has been applied to the determination of the iodoamino acid distribution in hog thyroglobulin and to the analysis of photodegradation products of thyroxine and triiodothyronine.
Ion-exchange chromatography, as combined with the automatic iodine analysis of the effluent by the ceric-arseneous reaction, has proven useful for the quantitative determination of iodoamino acids (l-3). However, hitherto-reported studies employing this procedure are concerned almost exclusively with the four major constituent iodoamino acids in thyroglobulin, i.e., monoiodotyrosine (MIT),’ diiodotyrosine (DIT), 3,3’,Striiodothyronine (T3) and thyroxine (TJ. It has not been known whether this method can also be adopted for the analysis of several other iodoamino acids, some of which might be contained in thyroglobulin as minor iodoamino acid residues. In their study on the iodoamino acid distribution in rat or human thyroglobulin, labeled either in viva (equilibrium labeled) or in vitro with radioactive iodine, Ogawara et al. (4) employed an ion-exchange chromatographic procedure to fractionate thyroglobulin digests into the total iodothyronine fraction and iodotyrosines and subsequently analyzed the former fraction by means of thin layer chromatography. They reported the presence of a small but not negligible amount of minor iodoamino acids in addition to the four major iodoamino acids in thyroglobulin. In this paper we report a simple chromatographic procedure that permits the analysis of most unlabeled iodothyronines in a single step. ’ See Table 1 for abbreviation of iodoamino acids used. 1.57 Copyright All rights
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
158
SORIMACHI
AND
UI
The method has been applied to assess the possible content of minor iodoamino acids in hog thyroglobulin and also to analyze the degradation products formed by exposure of T4 or T, to ultraviolet irradiation. MATERIALS
AND
METHODS
Zodoamino acids. MIT, DIT, T3 and Td were the same commercial products as used in the previous study (3); 3,5-diiodothyronine (3,5-TZ) was purchased from Sigma. Most other iodothyronines, i.e., 3monoiodothyronine (3-T,), 3’-monoiodothyronine (3’-T,), 3,3’-diiodothyronine (3,3’-T,) and 3,3’,5’-triiodothyronine (T& and iodohistidines, i.e., monoiodohistidine (MIH) and diiodohistidine (DIH), were kindly supplied by Dr. H. J. Cahnmann of the National Institutes of Health, Bethesda, MD. The iodohistidines had been prepared by Dr. J. C. Savoie of the Hopital de la PitiC, Paris. The preparation of 3’,5’diiodothyronine (3’,5’-T2) was a kind gift of Dr. M. Nakano of Gunma University School of Medicine. The purity of all these preparations of iodoamino acids was verified by ion-exchange chromatography carried out under the standard conditions employed in the present study. A few preparations were found to be very slightly contaminated with iodide (6 1% of the total iodine). The concentration of each iodoamino acid solution was determined from the iodine analysis, which was performed with a Technicon AutoAnalyzer according to the procedure recommended by the manufacturer for the analysis of protein-bound iodine. Thyroglobulin. Hog thyroglobulin was prepared by the method previously reported (5). In the present investigation, a chromatographically purified but not subfractionated preparation with an average iodine content of 0.71% was used. Before the chromatographic analysis of iodoamino acids, thyroglobulin was hydrolyzed enzymatically (2) by the consecutive use of Pronase and aminopeptidase M as reported previously (3). Chromatographic analysis of iodoamino acids. A slight modification of a previously reported method (3) was adopted. Samples were applied to a column (1.0 X 15 cm) of a cation-exchange resin, AG 5OW-X4 (30-35 pm), which had been equilibrated with 0.04 M ammonium acetate buffer, pH 4.7, containing 30% (v/v) ethanol at 5O”C, and a gradient of increasing pH was prepared from 100 ml of the starting buffer and 100 ml of 0.65 N NH,OH. The effluent was analyzed continuously by the iodine-catalyzed ceric ammonium sulfate-arseneous acid reaction with a Technicon AutoAnalyzer. Photodegradation. A solution of T, or ‘I; dissolved in 0.04 M ammonium acetate buffer, pH 4.7, containing 30% ethanol, was placed in a quartz spectrophotometer cuvette and irradiated for 3 min with two low-
ANALYSIS
159
OF IODOTHYRONINES
pressure mercury lamps (19 W) having an emission peak at 253.7 nm. The distance between the cuvette and the lamps was approximately 15 cm. The irradiated solution was applied directly to the column for analysis. RESULTS Chromatographic Separation of Various Iodothyronines and Iodotyrosines
In our previous study (3) aiming at the ion-exchange chromatographic separation of the four major iodoamino acids in thyroglobulin, a pH
-
-
-
-
FIG. 1. Elution profiles in the cation-exchange chromatography of iodoamino acids. (A) A mixture of authentic iodoamino acids (2 nmoles each except for DIH of which 20 nmoles were used); (B) an enzymatic digest of hog thyroglobulin. For the latter experiment, 1 ml of a 0.106% thyroglobulin solution in 0.1 M Tris-HCl buffer, pH 8.0, was mixed with 100 ~1 of a 1% Pronase solution, 10 ~1 of 0.5 M I-methyl-2-mercaptoimidazole and 10 ~1 of toluene and incubated at 37°C for 24 hr; subsequently, 10 ~1 of 0.1% aminopeptidase M solution was added and the incubation was continued for an additional 24 hr. The digest was mixed with 1 ml of 0.2 M ammonium acetate buffer, pH 4.7, containing 30% ethanol, and 1 ml was charged to the column for chromatographic analysis. The flow rate for elution was 0.56 ml/min.
160
SORIMACHI
AND UI
gradient was prepared using equal volumes (90 ml) of 0.04 M ammonium acetate buffer, pH 4.7, and 1 N NH,OH. When iodothyronines other than TS and Ta were included in the sample and subjected to analysis under the same conditions, they were not separated well from either the TZj or the TJ peak. The increase in the total volume of eluant, which gives a shallower pH gradient, did not improve the resolution but brought about broadening of the peaks. However, the use of a lower concentration of NH,OH, i.e., 0.65 N, and a slightly increased total eluant volume (200 ml) was found to give excellent separation of most iodothyronines. The concentration of NH,OH was not very critical and could be increased up to 0.85 N without great change in the resolution.g Further increase in eluant volume did not improve the resolution. Figure IA shows a typical chromatographic pattern obtained when a mixture containing 2 nmoles each of eight iodothyronines and two iodotyrosines was analyzed under the modified conditions mentioned above. The time required for complete elution of the iodoamino acids was approximately 3 hr. Each peak was identified by a number of separate runs made with a mixture of one unknown plus one or two known iodoamino acids. As shown in this figure, the separation of MIT and DIT from each other and from other iodoamino acids is excellent. Iodothyronines give five peaks and one shoulder. No separation was attained, however, between 3-T, and 3,5-T, and between 3’-T1 and Ti. Addition of a large amount of DIH results in the appearance of a small peak that is eluted slightly before MIT. The behavior of another iodohistidine, MIH, could not be investigated, because the sensitivity of the analysis for this particular iodoamino acid was too low. Table 1 summarizes the elution volume of each iodoamino acid and iodide as well as the relative peak area, which was measured separately using solutions each of which contained one kind of iodoamino acid of known concentration. A remarkable variation in the catalytic effect of different iodoamino acids on the reduction of ceric ions by arseneous acid, even after correction was made for the difference in the number of iodine atoms in each molecule, is to be noticed. The iodine atom substituted in the phenolic ring of the diphenyl ether group in thyronines is generally less effective than that in the nonphenolic ring. Thus, 3’-T, shows a peak area much smaller than that of 3-T, when the same amount of each compound was applied to the column. In the case of diiodothyronines, the relative area decreases in the order of 3,5-T,, 3,3’-T, and 3’,5’-T2. However, T3, having two iodine atoms in the p In some experiments employing a column used for a long time without repacking, 0.85 NH,OH rather than 0.65 N NH,OH gave a slightly better separation of iodothyronines. Hence, exploration of the most suitable concentration of NH,OH for each column is recommended for critical investigations. N
ANALYSIS
OF
TABLE ELUTION
VOLUMES
AND
CHROMATOGRAPHIC
Compound Monoiodotyrosine Diiodotyrosine 3-Monoiodothyronine 3 ‘-Monoiodothyronine 3,5-Diiodothyronine 3,3 ‘-Diiodothyronine 3 ’ .5 ‘-Diiodothyronine 3.3’,5-Triiodothyronine 3,3 ’ ,5 ‘-Triiodothyronine Thyroxine Monoiodohistidine Diiodohistidine Iodide
PEAK
ANALYSIS
OF IODOAMINO
MIT DIT 3-T, 3’-T, 3,5-T, 3,3’-T, 3 ‘,5 I-T, T3 T; T4 MIH DIH I-
1
RELATIVE
Abbreviation
161
IODOTHYRONINES
AREAS
Approx elution volume (ml) 31 50 80 96 80 113 88 106 96 91 21 6”
THE CATION-EXCHANGE
IN
ACIDS
AND
IODIDE
Peak area measured (cm*/nmole)
Relative peak area per mole
1.52 3.82 1.53 0.50 2.56 2.32 2.04 2.94 3.27 5.72 0.005 0.07 2.96
1 .oo 2.50
1.00 0.33
1.68 1.53 1.34
1.93 2.15 3.75 ‘co.01 0.04 1.94
Relative peak area per iodine atom 1.00 1.25 1.00 0.33 0.84 0.76 0.67 0.64 0.72 0.94
CO.01 0.02
1.94
“ Iodide was not retarded in this chromatogram
nonphenolic ring, gives a slightly lower relative peak area than that of TA with two iodine atoms in the phenolic ring. DIH and especially MIH show a very weak catalytic effect on the reduction (Table 1). Therefore, a chromatographic analysis of unlabeled iodohistidines by the present procedure does not seem to be useful. Analysis of lodothyronines
in Thyroglobulin
In order to see whether thyroglobulin contains iodothyronines other than T, and T, in appreciable amounts, a large amount of hog thyroglobulin digest was applied to the column and analyzed under the present conditions. As shown in Fig. lB, the elution profile observed is quite similar to that obtained by the chromatographic procedure described previously for the analysis of the four major iodoamino acids in thyroglobulin (3). In the iodothyronine region, no peak or shoulder other than the T, and T3 peaks can be detected, and the shape of the curve is exactly the same as that for a mixture of T4 and T, prepared in accordance with their contents in thyroglobulin. From this and other similar experiments, it has been concluded that most of iodothyronines other than T, and T, are not contained in amounts exceeding 0.05 residue per molecule; however, 3’,5’-T, that is eluted, if present, as a shoulder of the large T4 peak and 3’-T, that gives an exceptionally small peak will es-
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cape detection at this low level. This conclusion was confirmed by several other chromatographic runs made with samples in which small increments of the minor iodothyronines were added to a thyroglobulin digest or to an equivalent mixture of T, and T,. Even in the case of Tj, which intervenes between the T, and T, peaks, the valley between the two peaks was elevated significantly when TA was added in an amount corresponding to 0.05 residue per molecule of thyroglobulin; on further addition, the extent of elevation increased in proportion to the amount of TA added. On the other hand, the content of the four major iodoamino acids in this preparation of hog thyroglobulin (Fig. 1B) coincided with the value expected from the results of our previous investigation on the relationship between the iodoamino acid distribution and the total iodine content in hog thyroglobulin (6). The mean values obtained in two independent experiments were: MIT, 7.17; DIT, 7.47; T,. 0.93: and Tq, 3.33 residues per molecule of thyroglobulin.
c--cc&& I. _ c-L -L& : -,i L i~~~L7 FIG. 2. Elution profiles in the cation-exchange chromatographic analysis of photodegradation products of (A) T4 and (B) T3. For details, see Table 2 and text.
ANALYSIS
OF
163
IODOTHYRONINES
A fairly large peak that follows the unretarded I- peak, which shows the deiodination of iodoamino acids occurring during the storage and enzymatic hydrolysis of thyroglobulin, is due to 1-methyl-2-mercaptoimidazole added to the sample to prevent the degradation of iodoamino acids (Fig. 1B). In addition to the four major iodoamino acid peaks eluted later, several tiny peaks are seen in the regions between l-methyl2-mercaptoimidazole and MIT and also between DIT and T4. They are attributed to undigested iodine-containing peptides. The sum of these areas is approximately 2% of the total area excluding that of I-methyl-2mercaptoimidazole. This is somewhat higher than that observed usually. Analysis
of Photodegradation
Products
of T, and T,
Figure 2 shows the elution profiles obtained when a solution of T, or T, exposed to ultraviolet irradiation was subjected to the analysis. In addition to the huge peak of II, several peaks can be seen. They represent, besides the starting compounds, several iodothyronines formed by deiodination and iodotyrosines formed by rupture of the diphenyl ether bond in iodothyronines. Table 2 summarizes the results of these analyses. Since the amount of iodide contained in the samples was too large to be measured in the TABLE 2 ION-EXCHANGE CHROMATOGRAPHIC ANALYSIS OF THE PHOTODEGRADATION PRODUCTS OF T, AND TV Amount found after exposure” (nmolesl I-containing compound
From T,
T, T3 Ti 3,5-T,* 3.3’-T, 3’,5’-T, 3’-T, DIT MIT I-
2.22 1.17 1.05 0.20 0.49 0.63 0.29 12.2
6.02 1.21 1.49 1.11 0.33 0.21 16.0
Recovery of iodine
104%
93%
-
From T,
-
(LFor this analysis, 7.4 nmoles of T, or 14.9 nmoles of T, dissolved in 1 ml of 0.04 M ammonium acetate buffer, pH 4.7, containing 30% ethanol, were exposed to ultraviolet irradiation, and 1 ml of each solution was charged to the column. b This component may also contain 3-T,. but the value was calculated as though no 3-T, was contained in the samples.
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same run as used for the analysis of iodoamino acids (Fig. 2), iodide was determined separately using much smaller sample sizes. DISCUSSION
The results presented in this paper show that the ion-exchange chromatographic procedure can be used successfully not only for the analysis of the four major iodoamino acids in thyroglobulin, i.e., MIT, DIT, T, and T,, but also for the analysis of various iodothyronines other than T3 and T,. The sensitivity of analysis, even though considerably different with different iodothyronines, is high (Table 1). Usually less than 1 nmole of each iodothyronine is enough for its quantitation, although the presence of a huge nearby peak of other iodothyronines will make the determination inaccurate or impossible. Iodohistidines cannot be analyzed at such low levels due to their extremely low sensitivity. However, the present chromatographic procedure could possibly be useful for all iodoamino acids including iodohistidines, if they are isotopically labeled. Resolution of iodothyronines in this chromatography was generally good, although some of them could not be separated from others. There is some relationship between the order of elution and the difference in iodine substitution in iodothyronine molecules. Iodothyronines without iodine substitution in the phenolic ring of the diphenyl ether group, i.e., 3-T, and 3,5-T,, are eluted earliest, whereas those with two iodine substitutions in the phenolic ring, 3’S’-T,, T4 and TA, come off the column later, and those with one substitution in the phenolic ring 3’-T,, T3 and 3,3’-T,, belong to the group retained most strongly. Furthermore, among the group with two iodine substitutions in the phenolic ring, iodothyronine with no iodine substitution in the nonphenolic ring, 3’S’-T,, is eluted first, followed then by that with two substitutions, T,, and finally by that with one substitution, T& This order is also the same within the group of iodothyronines with only one iodine substitution in the phenolic ring, i.e., 3-T,, T3 and 3,3’-T2. The presence in thyroglobulin of minor iodoamino acid residues other than those of the four major iodoamino acids has been shown qualitatively by several investigators employing tracer experiments. In the quantitative investigation by Ogawara et al. (4), various differences in the distribution of iodoamino acids were noticed between thyroglobulin iodinated in viva and in vitro. They reported that almost 15% of the total iodothyronines belonged to those other than T, and TY, even in equilibrium-labeled, native rat thyroglobulin. However, none of these minor iodothyronines, at least those excluding 3’,5’-T2, was present in hog thyroglobulin in amounts exceeding 0.05 residue per molecule (Fig. 1B). Although further investigation with rat and other thyroglobulins is
ANALYSIS
OF IODOTHYRONINES
165
needed, it is to be emphasized that the enzymatic digestion of thyroglobulin was almost complete and the extent of deiodination was very low in the present study. We believe, therefore, that this result at least provides a rationale for our previous investigation (6) on the relationship between the iodoamino acid distribution and the total iodine content in hog thyroglobulin in which an analytical method capable of measuring only four major iodoamino acids was used. The usefulness of this chromatographic procedure has been shown also in the analysis of photodegradation products of T, and T3. The results indicate that the ultraviolet radiation not only deiodinates iodothyronines successively (7) but also results in the cleavage of the diphenyl ether bond leading to the formation of DIT or MIT. The latter finding confirms the observation by Koya (8) who analyzed similar products by paper and thin-layer chromatography. No attempt was made in this study to determine the dependence of the composition of the irradiation products on the experimental conditions of the irradiation. However, the chromatographic method described in this paper will be useful in such investigations which may provide a better understanding of the mechanism of the photodegradation of the thyroid hormones. ACKNOWLEDGMENT We are greatly indebted to Dr. H. J. Cahnmann of the National Institutes of Health for his helpful discussions and advice.
REFERENCES 1. 2. 3. 4.
Block, R. J., and Mandl, R. H. (1962) Ann. N. Y. Acad. Sci. 102, 87-95. Rolland, M., Aquaron, R., and Lissitzky, S. (1970) Anal. Biochem. 33, 307-3 17. Sorimachi, K., and Ui, N. (1974) J. Bid&em. (Tokyo) 76, 39-45. Ogawara, H., Bilstad, J. M., and Cahnmann, H. J. (1972) Biochim. Biophys. Acra 339-349.
5. Ui, N., and Tarutani, 0. (1961) J. Biochem. (Tokyo) 50, 508-5 18. 6. Sorimachi, K., and Ui, N. (1974) Biochim. Biophys. Acta 342, 30-40. 7. Lein, A., and Michel, R. (1959) C. R. Sot. Biol. 153, 538-540. 8. Koya, S. (1973) J. Pharm. Sot. Japan 93,117-728.
257,