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Colorimetry of dehydroalanine residues preserved as ‘lost side chains’ in thyroglobulin
Molecular and Cellular Endocrinology, 57 (1988) 101-106 Elsevier Scientific Pnbhshers Ireland. Ltd.
101
MCE 01848
Colorimetry of dehydroala~ne residues preserved as “lost side chains’ in thyroglobulin Toshihiko Kondo, Yoichi Kondo and Nobuo Ui * Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi 371, Japan (Received
Key wora!r: Thyroid hormone
28 October 1987; accepted 19 January 1988)
synthesis, mechanism;
Thyroglobuhn,
structure, function;
Dehydroalanine,
analysis
We developed a new assay method for dehydroalanine residues in thyroglobulin, which had been proposed to be the ‘lost side chains’ during thyroid hormonogenesis. Thyroglobulin preparations were labeled with 4aminothiophenol at 30 o C for 10 days. Under the conditions, the reagent reacted only with dehydroalanine and cysteine residues. The 4arninothiophenol bound to cysteine was eliminated by reductive cleavage. The 4-~ot~ophenol-labeled dehydroalanine (~a~nophenylcysteine) residues were liberated by acidic hydrolysis, converted to a colored derivative by the ~ratton-M~sh~l reaction and quantified colorimetricaily. The number of dehydroalanine residues was the same as that of hormone residues in each thyroglobulin preparation. The results indicate that when one hormone residue is produced by the coupling of two iodotyrosine residues, the ‘lost side chain’ is preserved as one dehydroalanine residue in the thyroglobuhn molecule.
Intmhtion
Thyroglobulin, a thyroidal glycoprotein of high molecular weight (6~~), is a pro-hormone of the thyroid hormones thyroxine and triiodothyronine. These iodothyronines are synthesized within the peptide matrix of the protein by the iodination of tyrosine residues, followed by the transfer of an iodinated phenoxyl group from a ‘donor’ iodotyrosine residue to an ‘acceptor’ diiodotyrosine residue (Taurog, 1985). The ‘acceptor’ residues which exist as thvroid hormone res-
Address for correspondence: To&.ihiko Kondo, Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan. * Passed away on February 19, 1985.
0303-7207/88/$03.50
0 1988 Elsevier Scientific
idues have been located, one close to the Nterminus and the other near the C-terminus, by chemical and gene techuological analyses (Rawitch et al., 1984; DiLauro et al., 1985; Malthiery and Lissitzky, 1985; Mercken et al., 1985). On the other hand, the fate of ‘donor’ residues after hormone production, or of the ‘lost side chains’ has been a controversial issue (see Cody, 1984). Gavaret et al. (1979, 1980, 1981) proposed that the ‘lost side chain’ was dehydro~a~e, from their finding of radioactive alanine or other derivatives in the thyroglobulin hydrolyzate which had been predicted as being derived from dehydroalanine residues. We also found pyruvic acid, one of the predicted derivatives of dehy~oal~ne by HPLC analyses of the enzymatic hydrolyzate of thyroglobulin (Vi et al., 1982). Although these findings have suggested the formation of dehydro-
Publishers Ireland, Ltd.
102
alanine residues, there is no direct and quantitative proof of the preservation of dehydroalanine residues as the ‘lost side chains’. In the present paper, we describe a new colorimetric assay method for dehydroalanine residues of proteins, in which 4-aminothiophenol-labeled dehydroalanine is converted to a colored derivative and quantified calorimetrically. Analysis of thyroglobulin preparations by the method proved the preservation of all of the ‘lost side chains’ as dehydroalanine residues. Materials and methods Materials 4-Aminothiophenol was purchased from Aldrich Chemical Co., guanidine hydrochloride (grade I) from Sigma Chemical Co., N-ethylmorpholine and dithiothreitol from Nakarai Chemicals, iodoacetic acid and N-(l-napthyl)ethylenediamine dihydrochloride from Wako Pure Chemical Co., crystalline bovine serum albumin from Armour Pharmaceutical Co., pronase from Calbiochem-Behring Corp., and aminopeptidase M from Pierce Chemical Co. Nisin was a generous gift from Dr. Kurahashi of the Institute of Protein Science, Osaka University. Human goitrous thyroglobulin preparations were prepared and kindly provided by Dr. Tarutani of our Department. All other reagents were analytical grade. Purification of thyroglobulin Porcine thyroglobulin was purified by ammonium sulfate precipitation, size-exclusion high performance liquid chromatography (HPLC) and DEAE chromatography as reported previously (Vi, 1980). Determination of protein concentration The concentration of thyroglobulin was determined spectrophotometrically assuming the absorbance of a 1% solution at 280 nm to be 10.0 (Ui and Tarutani, 1961), or by measuring the total amino acids in protein hydrolyzate by the use of an amino acid analyzer. Determination of iodine content The iodine content of thyroglobulin was determined by the Sandell-Kohltoff method using a
Technicon Autoanalyzer, according recommended by the factory.
to the method
Iodoamine acid analysis Thyroglobulin was digested with pronase for 24 h and aminopeptidase M for a further 24 h at 37 o C. Liberated iodoamino acids were separated by ion-exchange chromatography and analyzed with a Technicon Autoanalyzer as previously reported (Sorimachi and Ui, 1974). Reduction of thyroglobulin with sodium borohydride Fifty molar excess of sodium borohydride was reacted with thyroglobulin in a 10% N-ethylmorpholine/acetate buffer (pH 8.5) containing 6 M guanidine-HCl, at 30” C for 2 days with gentle shaking. Assay procedure for dehydroalanine residues in thyroglobulin (1) Labeling of dehydroalanine residues with 4aminothiophenol. Thyroglobulin was treated with 4-aminothiophenol (50 mol/mol of cysteine residue) at 30°C in a 10% N-ethylmorpholine/acetate buffer (pH 8.5) containing 6 M guanidine-HCl (Fig. 1, Step 1). This treatment was carried out in a closed vessel purged with nitrogen gas and stopped by the addition of equimolar iodoacetic acid. Unreacted 4-aminothiophenol was removed by dialysis. The labeled thyroglobulin was then reduced by dithiothreitol to remove 4aminothiophenol bound to cysteine residues which were then alkylated with iodoacetic acid (Fig. 1, Step 2). On the other hand, the reagent bound to dehydroalanine residues remained as S-(4aminophenyl)cysteine residues. (2) Preparation of sample for calorimetry (Fig. I, Step 3). The labeled thyroglobulin was hydrolyzed with 6 N HCl for 24 h at 110 ’ C, resulting in the liberation of S-(4-aminophenyl)cysteine from peptides. The hydrolyzate was diluted with distilled water to 1 N HCl and centrifuged at 10000 t-pm for 10 min. The supematant was applied to a SEP-PAK C,, cartridge (Waters Associate), and washed thoroughly with 0.1% trifluoroacetic acid (TFA) to remove the brown-colored humin derived from carbohydrate components in glycoproteins. The S-(4-aminophenyl)cysteine was eluted from the column with 80% acetonitrile in 0.1% TFA and dried in a vacuum desiccator.
103
H-N
i I F=““2
“=::
pH 8.5
UH
. HS
2
-----mm-
dehydroalanine residue
I
pH
H-N
I
H-c4t~SH
l
i
“-7
HS
“2
8.5
--
Cystein residue
I H-N I H-C-CH~S-S I
o=c
_P
o-
NH2
reduction carboxymethylat
ion
i H-N
H+CtiTS O=
0
NH2
HCL hydrolysis
t
H
H
I
pti +
NaN02
-
1 z=
l
H-N-H H-CXH~S I O=C I 0 k
Fig. 1. Strategy of dehydroalanine analysis.
NtN
104
We used the Bratton-Mashall (3) Calorimetry. reaction (Bratton and Marshall, 1939) to convert the S-(4-aminophenyl)cysteine to a colored derivative. The dried sample was dissolved in 1 ml of 1 N HCl and mixed with 0.5 ml of 0.25% sodium nitrite (Fig. 1, Step 4). Fifteen minutes later, 0.5 ml of 2.5% ammonium sulfamate was added to the reaction mixture and shaken vigorously for a few minutes. One ml of 3 M sodium acetate and 0.5 ml of 0.1% N-(1-naphthyl)ethylenediamine were then added. After a 5 min reaction, the mixture was acidified by the addition of 0.25 ml of concentrated HCl and the absorbance at 550 nm was measured (Fig. 1, Step 5). The dehydroalanine content of the sample was calculated from a standard curve prepared using 4-aminothiophenol as a standard substrate for the color reaction. Results Strategy for labeling and calorimetry of dehydroalanine residues The sulfhydryl group of 4aminothiophenol reacts with the beta-carbon of a dehydroalanine residue and produces an S-(4+uninophenyl)cysteine residue (Fig. 1, Step 1). Although the 4aminothiophenol can react also with cysteine residues (Step l), the double S bonds thus formed can be removed by reductive cleavage and alkylation (Step 2). The Bratton-Marshall reaction is used for coloring S-(4aminophenyl)cysteine since this reaction is specific for aromatic amines (Step 4, 5) which do not exist in native proteins. Therefore, we can specifically label and measure the dehydroalanine residues in thyroglobulin by the present method. Linearity of color development with the increased doses of I-aminothiophenol The absorption peak of the colored derivative of 4-aminothiophenol was found at 550 nm. Although a slight red shift of the peak was observed when the 4-aminothiophenol-labeled thyroglobulin was subjected to this reaction, the thyroglobulin hydrolyzate containing free S-(4-aminophenyl)cysteine gave the same spectrum and intensity in the visible region as those of 4aminothiophenol. These facts justify the use of 4aminothiophenol as a standard for this colorim-
0.8
as
c 3 m
0.4
% Q, 0.3 E 2 b
0.2
3 0.1
0
Fig. 2. Standard curve for dehydroalanine assay. 4Aminothiophenol was used as a standard substance instead of dehydroalanine which is unstable in its free form. The substrate was treated with nitrite and N-(l-napthyl)ethylenediamine, and converted to colored derivatives. The color intensity at 550 nm was measured and plotted against the molar concentration of Caminothiophenol.
etry of dehydroalanine residues. The color intensity at 550 nm linearly increased with the concentration of 4aminothiophenol at least up to 20 pg/rnl or 160 PM (Fig. 2). Reaction conditions for the labeling of dehydroalanine residues of thyroglobulin Thyroglobulin was treated with 4-aminothiophenol in the presence of 6 M guanidine-HCl at 30” C and pH 8.5. As shown in Fig. 3, the reaction proceeded very slowly for 10 days and reached a plateau at about 5 mol/mol of thyroglobulin. On the other hand, no reactive residue was detected in bovine serum albumin which is known to have no dehydroalanine residue, and in borohydride-treated thyroglobulin in which dehydroalanine residues should be reduced and converted to aline residues. These results indicate that the present conditions were sufficient for labeling dehydroalanine residues specifically and completely, if the reaction time was long enough.
105
TABLE
1
DEHYDROALANINE (TG) PREPARATIONS Sample
Iodine content
CONTENT
OF THYROGLOBULIN
Ts+T4 (mol/TG)
AAla * (mol/TG)
A Ala/ Ts + T4
(W
BSA
so
5
10
IS
Incubation time (day)
Fig. 3. Time course of the labeling of thyroglobulin with 4-aminothiophenol. Thyroglobulin was treated with 4aminothiophenol at 30 o C in the presence of 6 M guanidineHCl. The HCl-hydrolyzate was subjected to the color reaction described in Materials and Methods. Molar ratio of 4aminothiophenol to thyroglobulin was plotted against the time for labeling with Qaminothiophenol.
Reliability of quantitative assay values Reliability of assay values was examined by analyzing nisin, which contains 2-dehydroalanine and l/6methyldehydroalanine with the same reactivity as that of dehydroalanine (Kurahashi and Nishio, 1984). The mean dehydroalanine content (n = 4) of kin, 3.15 k 0.13 residues/m01 of peptide, closely coincided with the formula value. Analysis of dehydroalanine residues in various thyroglobulin preparations with different iodothyronine content Table 1 shows the dehydroalanine content of various thyroglobulin preparations. Each value corresponds to the total iodothyronine (T3 + T4) content of the sample, except in the samples with extremely low hormone content. In such exceptional cases, dehydroalanine content is also very low, but not as low as hormone content, and this results in the considerable increase in dehydroalanine/hormone ratio. Although we do not neglect a possibility that the excess dehydroalanine exists as a result of the preferential release of hormones from early iodinated thyroglobulin molecules, further confirmation is required since the observed values were so low and close to the
TGhBl TGhB2 TGhB3 TG327 TG328 TG308 TG312 TG316 TGhB4
0.07 0.18 0.19 0.65 0.67 0.75 0.93 1.07 1.21
0.1 0.3 1.0 4.3 4.9 4.4 5.3 5.8 6.7
0.3 0.6 0.8 4.4 4.5 3.5 5.4 5.1 5.7
3.0 2.0 0.8 1.02 0.92 0.80 1.02 0.88 0.85
TG201 reduced
ND
5.0
0.1
0.02
* Dehydroalanine.
minimum measurable amount in the present assay procedures. Practically no dehydroalanine was found in borohydride-treated thyroglobulin in which dehydroalanine residues were reduced and converted to alanine residues. In conclusion, the present results are quite consistent with the idea that all the ‘lost side chains’ are preserved as dehydroalanine residues in the protein.
Discussion We determined the dehydroalanine content of various thyroglobulin preparations. The observed values showed good correspondence with the thyroid hormone content of the preparation, indicating that the formation of one iodothyronine residue was accompanied by the formation of a dehydroalanine residue. This result provided direct evidence for not only a previous assumption of the production of equimolar dehydroalanine residues with hormone residues based on the incorporation of radioactive tyrosine into alanine (Gavaret et al., 1979, 1980, 1981) but also the preservation of dehydroalanine residues in peptides after the hormonogenic reaction. Such stability of dehydroalanine in peptidyl linkages is understandable when we consider the presence of some peptides and proteins which contain dehydroalanine and other unsaturated
106
amino acid residues as native constituents. In addition to kin, a few antibiotic peptides have dehydroalanine and/or P-methyldehydroalanine residues (Kurahashi and Nishio, 1984), and histidine- (Consevage and Phillips, 1985) and phenylalanine- (Hodgins, 1971) ammonia lyases contain dehydroalanine residues probably localized in the active center of the enzymes. Before our study, an attempt had been made to determine the number of dehydroalanine residues in peptides (Gross and Kiltz, 1973). In that study, the dehydroalanine was converted to benzyl cysteine residues and measured by the use of an amino acid analyzer since benzyl cysteine cannot be colored. Our assay method has advantages over the earlier one. First, we can directly measure the colored derivative of dehydroalanine in the hydrolyzate, using a conventional calorimeter. Second, the use of 6 M guanidine-HCl in the labeling mixture causes the denaturation of rigid globular protein such as thyroglobulin and allows to label all dehydroalanine residues. In addition, a more important aspect of our method is that the principle of the method can be applied for the isolation of protein fragments containing dehydroalanine residues when the reactor in the Bratton-Marshall reaction is replaced by an immobilized reagent (see Step 5 in Fig. 1). This may give a way to locate the ‘lost side chains’, hence, the counter-part of hormonogenic tyrosine residues within the primary structure of thyroglobulin. The only disadvantage of this method is that rather long incubation (10 days) is required for the labeling of dehydroalanine residues. However, such long incubation at lower temperature is necessary to avoid non-specific reactions of 4-aminothiophenol with other amino acid residues. Finally, the analytical method we developed is not only for assaying dehydroalanine residues in thyroglobulin, but also for opening a new way to approach the structure and function problems of dehydroamino acid containing proteins and peptides in general.
Acknowledgements The authors wish to thank Dr. Kurahashi for his invaluable discussion and giving us a pure preparation of nisin, Dr. Tarutani of our Department for providing human goitrous thyroid glands and Dr. Hayashi of our Institute for amino acid analysis. We also thank Ms. Kimura for her skillful assistance in the preparation of the manuscript. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. References Bratton, A.C. and Marshall, E.K. (1939) J. Biol. Chem. 128, 537-550. Cody, V. (1984) Endocr. Res. 10, 73-88. Consevage, M.W. and Phillips, A.T. (1985) Biochemistry 24, 301-308. DiLauro, R., Obici, S., Condliffe, D., Ursini, V.M., Musti, A., MoscatelIi, C. and Awedimento, V.E. (1985) Eur. J. Biothem. 148, 7-11. Gavaret, J.-M., Cahnmann, H.J. and Nunez, J. (1979) J. Biol. Chem. 254, 11218-11222. Gavaret, J.-M., Nunez, J. and Cahnmamr, H.J. (1980) J. Biol. Chem. 255, 5281-5285. Gavaret, J.-M., Cahnmarm, H.J. and Nunez, J. (1981) J. Biol. Chem. 256, 9167-9173. Gross, E. and Kiltz, H.H. (1973) Biochem. Biophys. Res. Commun. 50, 559-565. Hodgins, D.S. (1971) J. Biol. Chem. 246, 2977-2985. Kurahashi, K. and Nishio, C. (1984) in The Cell Membrane (Haber, E., ed.), pp. 55-66, Plenum Publishing Co., New York. Malthiery, Y. and Lissitzky, S. (1985) Eur. J. Biochem. 147, 53-58. Mercken, L., Simons, M.-J., Swillens, S., Massaer, M. and Vassart, G. (1985) Nature 316, 647-651. Rawitch, A.B., Mercken, L., Hamilton, J.W. and Vassart, G. (1984) Biochem. Biophys. Res. Commun. 119, 335-342. Sorimachi, K. and Ui, N. (1974) J. B&hem. 76, 39-45. Taurog, A. (1986) in The Thyroid (Ingbar, S. and Braverman, L.E., eds.), pp. 57-97, (5th edn.), J.B. Lippincott Co., London-New York. Ui, N. (1980) in Thyroid Research (Stockigt, J.R. and Nagataki, S., eds.), Vol. 8, pp. 182-185, Australian Academy of Science, Canberra. Ui, N. and Tarutani, 0. (1961) J. Biochem. 50, 508-518. Ui, N., Hayashi, H. and Fujimoto, D. (1982) in 12th Annual Meeting of the European Thyroid Association, Brussels, Abstract 69A.
101
MCE 01848
Colorimetry of dehydroala~ne residues preserved as “lost side chains’ in thyroglobulin Toshihiko Kondo, Yoichi Kondo and Nobuo Ui * Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, Maebashi 371, Japan (Received
Key wora!r: Thyroid hormone
28 October 1987; accepted 19 January 1988)
synthesis, mechanism;
Thyroglobuhn,
structure, function;
Dehydroalanine,
analysis
We developed a new assay method for dehydroalanine residues in thyroglobulin, which had been proposed to be the ‘lost side chains’ during thyroid hormonogenesis. Thyroglobulin preparations were labeled with 4aminothiophenol at 30 o C for 10 days. Under the conditions, the reagent reacted only with dehydroalanine and cysteine residues. The 4arninothiophenol bound to cysteine was eliminated by reductive cleavage. The 4-~ot~ophenol-labeled dehydroalanine (~a~nophenylcysteine) residues were liberated by acidic hydrolysis, converted to a colored derivative by the ~ratton-M~sh~l reaction and quantified colorimetricaily. The number of dehydroalanine residues was the same as that of hormone residues in each thyroglobulin preparation. The results indicate that when one hormone residue is produced by the coupling of two iodotyrosine residues, the ‘lost side chain’ is preserved as one dehydroalanine residue in the thyroglobuhn molecule.
Intmhtion
Thyroglobulin, a thyroidal glycoprotein of high molecular weight (6~~), is a pro-hormone of the thyroid hormones thyroxine and triiodothyronine. These iodothyronines are synthesized within the peptide matrix of the protein by the iodination of tyrosine residues, followed by the transfer of an iodinated phenoxyl group from a ‘donor’ iodotyrosine residue to an ‘acceptor’ diiodotyrosine residue (Taurog, 1985). The ‘acceptor’ residues which exist as thvroid hormone res-
Address for correspondence: To&.ihiko Kondo, Department of Physical Biochemistry, Institute of Endocrinology, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan. * Passed away on February 19, 1985.
0303-7207/88/$03.50
0 1988 Elsevier Scientific
idues have been located, one close to the Nterminus and the other near the C-terminus, by chemical and gene techuological analyses (Rawitch et al., 1984; DiLauro et al., 1985; Malthiery and Lissitzky, 1985; Mercken et al., 1985). On the other hand, the fate of ‘donor’ residues after hormone production, or of the ‘lost side chains’ has been a controversial issue (see Cody, 1984). Gavaret et al. (1979, 1980, 1981) proposed that the ‘lost side chain’ was dehydro~a~e, from their finding of radioactive alanine or other derivatives in the thyroglobulin hydrolyzate which had been predicted as being derived from dehydroalanine residues. We also found pyruvic acid, one of the predicted derivatives of dehy~oal~ne by HPLC analyses of the enzymatic hydrolyzate of thyroglobulin (Vi et al., 1982). Although these findings have suggested the formation of dehydro-
Publishers Ireland, Ltd.
102
alanine residues, there is no direct and quantitative proof of the preservation of dehydroalanine residues as the ‘lost side chains’. In the present paper, we describe a new colorimetric assay method for dehydroalanine residues of proteins, in which 4-aminothiophenol-labeled dehydroalanine is converted to a colored derivative and quantified calorimetrically. Analysis of thyroglobulin preparations by the method proved the preservation of all of the ‘lost side chains’ as dehydroalanine residues. Materials and methods Materials 4-Aminothiophenol was purchased from Aldrich Chemical Co., guanidine hydrochloride (grade I) from Sigma Chemical Co., N-ethylmorpholine and dithiothreitol from Nakarai Chemicals, iodoacetic acid and N-(l-napthyl)ethylenediamine dihydrochloride from Wako Pure Chemical Co., crystalline bovine serum albumin from Armour Pharmaceutical Co., pronase from Calbiochem-Behring Corp., and aminopeptidase M from Pierce Chemical Co. Nisin was a generous gift from Dr. Kurahashi of the Institute of Protein Science, Osaka University. Human goitrous thyroglobulin preparations were prepared and kindly provided by Dr. Tarutani of our Department. All other reagents were analytical grade. Purification of thyroglobulin Porcine thyroglobulin was purified by ammonium sulfate precipitation, size-exclusion high performance liquid chromatography (HPLC) and DEAE chromatography as reported previously (Vi, 1980). Determination of protein concentration The concentration of thyroglobulin was determined spectrophotometrically assuming the absorbance of a 1% solution at 280 nm to be 10.0 (Ui and Tarutani, 1961), or by measuring the total amino acids in protein hydrolyzate by the use of an amino acid analyzer. Determination of iodine content The iodine content of thyroglobulin was determined by the Sandell-Kohltoff method using a
Technicon Autoanalyzer, according recommended by the factory.
to the method
Iodoamine acid analysis Thyroglobulin was digested with pronase for 24 h and aminopeptidase M for a further 24 h at 37 o C. Liberated iodoamino acids were separated by ion-exchange chromatography and analyzed with a Technicon Autoanalyzer as previously reported (Sorimachi and Ui, 1974). Reduction of thyroglobulin with sodium borohydride Fifty molar excess of sodium borohydride was reacted with thyroglobulin in a 10% N-ethylmorpholine/acetate buffer (pH 8.5) containing 6 M guanidine-HCl, at 30” C for 2 days with gentle shaking. Assay procedure for dehydroalanine residues in thyroglobulin (1) Labeling of dehydroalanine residues with 4aminothiophenol. Thyroglobulin was treated with 4-aminothiophenol (50 mol/mol of cysteine residue) at 30°C in a 10% N-ethylmorpholine/acetate buffer (pH 8.5) containing 6 M guanidine-HCl (Fig. 1, Step 1). This treatment was carried out in a closed vessel purged with nitrogen gas and stopped by the addition of equimolar iodoacetic acid. Unreacted 4-aminothiophenol was removed by dialysis. The labeled thyroglobulin was then reduced by dithiothreitol to remove 4aminothiophenol bound to cysteine residues which were then alkylated with iodoacetic acid (Fig. 1, Step 2). On the other hand, the reagent bound to dehydroalanine residues remained as S-(4aminophenyl)cysteine residues. (2) Preparation of sample for calorimetry (Fig. I, Step 3). The labeled thyroglobulin was hydrolyzed with 6 N HCl for 24 h at 110 ’ C, resulting in the liberation of S-(4-aminophenyl)cysteine from peptides. The hydrolyzate was diluted with distilled water to 1 N HCl and centrifuged at 10000 t-pm for 10 min. The supematant was applied to a SEP-PAK C,, cartridge (Waters Associate), and washed thoroughly with 0.1% trifluoroacetic acid (TFA) to remove the brown-colored humin derived from carbohydrate components in glycoproteins. The S-(4-aminophenyl)cysteine was eluted from the column with 80% acetonitrile in 0.1% TFA and dried in a vacuum desiccator.
103
H-N
i I F=““2
“=::
pH 8.5
UH
. HS
2
-----mm-
dehydroalanine residue
I
pH
H-N
I
H-c4t~SH
l
i
“-7
HS
“2
8.5
--
Cystein residue
I H-N I H-C-CH~S-S I
o=c
_P
o-
NH2
reduction carboxymethylat
ion
i H-N
H+CtiTS O=
0
NH2
HCL hydrolysis
t
H
H
I
pti +
NaN02
-
1 z=
l
H-N-H H-CXH~S I O=C I 0 k
Fig. 1. Strategy of dehydroalanine analysis.
NtN
104
We used the Bratton-Mashall (3) Calorimetry. reaction (Bratton and Marshall, 1939) to convert the S-(4-aminophenyl)cysteine to a colored derivative. The dried sample was dissolved in 1 ml of 1 N HCl and mixed with 0.5 ml of 0.25% sodium nitrite (Fig. 1, Step 4). Fifteen minutes later, 0.5 ml of 2.5% ammonium sulfamate was added to the reaction mixture and shaken vigorously for a few minutes. One ml of 3 M sodium acetate and 0.5 ml of 0.1% N-(1-naphthyl)ethylenediamine were then added. After a 5 min reaction, the mixture was acidified by the addition of 0.25 ml of concentrated HCl and the absorbance at 550 nm was measured (Fig. 1, Step 5). The dehydroalanine content of the sample was calculated from a standard curve prepared using 4-aminothiophenol as a standard substrate for the color reaction. Results Strategy for labeling and calorimetry of dehydroalanine residues The sulfhydryl group of 4aminothiophenol reacts with the beta-carbon of a dehydroalanine residue and produces an S-(4+uninophenyl)cysteine residue (Fig. 1, Step 1). Although the 4aminothiophenol can react also with cysteine residues (Step l), the double S bonds thus formed can be removed by reductive cleavage and alkylation (Step 2). The Bratton-Marshall reaction is used for coloring S-(4aminophenyl)cysteine since this reaction is specific for aromatic amines (Step 4, 5) which do not exist in native proteins. Therefore, we can specifically label and measure the dehydroalanine residues in thyroglobulin by the present method. Linearity of color development with the increased doses of I-aminothiophenol The absorption peak of the colored derivative of 4-aminothiophenol was found at 550 nm. Although a slight red shift of the peak was observed when the 4-aminothiophenol-labeled thyroglobulin was subjected to this reaction, the thyroglobulin hydrolyzate containing free S-(4-aminophenyl)cysteine gave the same spectrum and intensity in the visible region as those of 4aminothiophenol. These facts justify the use of 4aminothiophenol as a standard for this colorim-
0.8
as
c 3 m
0.4
% Q, 0.3 E 2 b
0.2
3 0.1
0
Fig. 2. Standard curve for dehydroalanine assay. 4Aminothiophenol was used as a standard substance instead of dehydroalanine which is unstable in its free form. The substrate was treated with nitrite and N-(l-napthyl)ethylenediamine, and converted to colored derivatives. The color intensity at 550 nm was measured and plotted against the molar concentration of Caminothiophenol.
etry of dehydroalanine residues. The color intensity at 550 nm linearly increased with the concentration of 4aminothiophenol at least up to 20 pg/rnl or 160 PM (Fig. 2). Reaction conditions for the labeling of dehydroalanine residues of thyroglobulin Thyroglobulin was treated with 4-aminothiophenol in the presence of 6 M guanidine-HCl at 30” C and pH 8.5. As shown in Fig. 3, the reaction proceeded very slowly for 10 days and reached a plateau at about 5 mol/mol of thyroglobulin. On the other hand, no reactive residue was detected in bovine serum albumin which is known to have no dehydroalanine residue, and in borohydride-treated thyroglobulin in which dehydroalanine residues should be reduced and converted to aline residues. These results indicate that the present conditions were sufficient for labeling dehydroalanine residues specifically and completely, if the reaction time was long enough.
105
TABLE
1
DEHYDROALANINE (TG) PREPARATIONS Sample
Iodine content
CONTENT
OF THYROGLOBULIN
Ts+T4 (mol/TG)
AAla * (mol/TG)
A Ala/ Ts + T4
(W
BSA
so
5
10
IS
Incubation time (day)
Fig. 3. Time course of the labeling of thyroglobulin with 4-aminothiophenol. Thyroglobulin was treated with 4aminothiophenol at 30 o C in the presence of 6 M guanidineHCl. The HCl-hydrolyzate was subjected to the color reaction described in Materials and Methods. Molar ratio of 4aminothiophenol to thyroglobulin was plotted against the time for labeling with Qaminothiophenol.
Reliability of quantitative assay values Reliability of assay values was examined by analyzing nisin, which contains 2-dehydroalanine and l/6methyldehydroalanine with the same reactivity as that of dehydroalanine (Kurahashi and Nishio, 1984). The mean dehydroalanine content (n = 4) of kin, 3.15 k 0.13 residues/m01 of peptide, closely coincided with the formula value. Analysis of dehydroalanine residues in various thyroglobulin preparations with different iodothyronine content Table 1 shows the dehydroalanine content of various thyroglobulin preparations. Each value corresponds to the total iodothyronine (T3 + T4) content of the sample, except in the samples with extremely low hormone content. In such exceptional cases, dehydroalanine content is also very low, but not as low as hormone content, and this results in the considerable increase in dehydroalanine/hormone ratio. Although we do not neglect a possibility that the excess dehydroalanine exists as a result of the preferential release of hormones from early iodinated thyroglobulin molecules, further confirmation is required since the observed values were so low and close to the
TGhBl TGhB2 TGhB3 TG327 TG328 TG308 TG312 TG316 TGhB4
0.07 0.18 0.19 0.65 0.67 0.75 0.93 1.07 1.21
0.1 0.3 1.0 4.3 4.9 4.4 5.3 5.8 6.7
0.3 0.6 0.8 4.4 4.5 3.5 5.4 5.1 5.7
3.0 2.0 0.8 1.02 0.92 0.80 1.02 0.88 0.85
TG201 reduced
ND
5.0
0.1
0.02
* Dehydroalanine.
minimum measurable amount in the present assay procedures. Practically no dehydroalanine was found in borohydride-treated thyroglobulin in which dehydroalanine residues were reduced and converted to alanine residues. In conclusion, the present results are quite consistent with the idea that all the ‘lost side chains’ are preserved as dehydroalanine residues in the protein.
Discussion We determined the dehydroalanine content of various thyroglobulin preparations. The observed values showed good correspondence with the thyroid hormone content of the preparation, indicating that the formation of one iodothyronine residue was accompanied by the formation of a dehydroalanine residue. This result provided direct evidence for not only a previous assumption of the production of equimolar dehydroalanine residues with hormone residues based on the incorporation of radioactive tyrosine into alanine (Gavaret et al., 1979, 1980, 1981) but also the preservation of dehydroalanine residues in peptides after the hormonogenic reaction. Such stability of dehydroalanine in peptidyl linkages is understandable when we consider the presence of some peptides and proteins which contain dehydroalanine and other unsaturated
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amino acid residues as native constituents. In addition to kin, a few antibiotic peptides have dehydroalanine and/or P-methyldehydroalanine residues (Kurahashi and Nishio, 1984), and histidine- (Consevage and Phillips, 1985) and phenylalanine- (Hodgins, 1971) ammonia lyases contain dehydroalanine residues probably localized in the active center of the enzymes. Before our study, an attempt had been made to determine the number of dehydroalanine residues in peptides (Gross and Kiltz, 1973). In that study, the dehydroalanine was converted to benzyl cysteine residues and measured by the use of an amino acid analyzer since benzyl cysteine cannot be colored. Our assay method has advantages over the earlier one. First, we can directly measure the colored derivative of dehydroalanine in the hydrolyzate, using a conventional calorimeter. Second, the use of 6 M guanidine-HCl in the labeling mixture causes the denaturation of rigid globular protein such as thyroglobulin and allows to label all dehydroalanine residues. In addition, a more important aspect of our method is that the principle of the method can be applied for the isolation of protein fragments containing dehydroalanine residues when the reactor in the Bratton-Marshall reaction is replaced by an immobilized reagent (see Step 5 in Fig. 1). This may give a way to locate the ‘lost side chains’, hence, the counter-part of hormonogenic tyrosine residues within the primary structure of thyroglobulin. The only disadvantage of this method is that rather long incubation (10 days) is required for the labeling of dehydroalanine residues. However, such long incubation at lower temperature is necessary to avoid non-specific reactions of 4-aminothiophenol with other amino acid residues. Finally, the analytical method we developed is not only for assaying dehydroalanine residues in thyroglobulin, but also for opening a new way to approach the structure and function problems of dehydroamino acid containing proteins and peptides in general.
Acknowledgements The authors wish to thank Dr. Kurahashi for his invaluable discussion and giving us a pure preparation of nisin, Dr. Tarutani of our Department for providing human goitrous thyroid glands and Dr. Hayashi of our Institute for amino acid analysis. We also thank Ms. Kimura for her skillful assistance in the preparation of the manuscript. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. References Bratton, A.C. and Marshall, E.K. (1939) J. Biol. Chem. 128, 537-550. Cody, V. (1984) Endocr. Res. 10, 73-88. Consevage, M.W. and Phillips, A.T. (1985) Biochemistry 24, 301-308. DiLauro, R., Obici, S., Condliffe, D., Ursini, V.M., Musti, A., MoscatelIi, C. and Awedimento, V.E. (1985) Eur. J. Biothem. 148, 7-11. Gavaret, J.-M., Cahnmann, H.J. and Nunez, J. (1979) J. Biol. Chem. 254, 11218-11222. Gavaret, J.-M., Nunez, J. and Cahnmamr, H.J. (1980) J. Biol. Chem. 255, 5281-5285. Gavaret, J.-M., Cahnmarm, H.J. and Nunez, J. (1981) J. Biol. Chem. 256, 9167-9173. Gross, E. and Kiltz, H.H. (1973) Biochem. Biophys. Res. Commun. 50, 559-565. Hodgins, D.S. (1971) J. Biol. Chem. 246, 2977-2985. Kurahashi, K. and Nishio, C. (1984) in The Cell Membrane (Haber, E., ed.), pp. 55-66, Plenum Publishing Co., New York. Malthiery, Y. and Lissitzky, S. (1985) Eur. J. Biochem. 147, 53-58. Mercken, L., Simons, M.-J., Swillens, S., Massaer, M. and Vassart, G. (1985) Nature 316, 647-651. Rawitch, A.B., Mercken, L., Hamilton, J.W. and Vassart, G. (1984) Biochem. Biophys. Res. Commun. 119, 335-342. Sorimachi, K. and Ui, N. (1974) J. B&hem. 76, 39-45. Taurog, A. (1986) in The Thyroid (Ingbar, S. and Braverman, L.E., eds.), pp. 57-97, (5th edn.), J.B. Lippincott Co., London-New York. Ui, N. (1980) in Thyroid Research (Stockigt, J.R. and Nagataki, S., eds.), Vol. 8, pp. 182-185, Australian Academy of Science, Canberra. Ui, N. and Tarutani, 0. (1961) J. Biochem. 50, 508-518. Ui, N., Hayashi, H. and Fujimoto, D. (1982) in 12th Annual Meeting of the European Thyroid Association, Brussels, Abstract 69A.