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Human thyroglobulin: Studies on the native and S-carboxymethylated protein
BIOCHIMICA ET BIOPHYSICA ACTA
247
BBA 25415 HUMAN T H Y R O G L O B U L I N : S T U D I E S ON T H E NATIVE AND S-CARBOXYMETHYLATED PROTEIN j. G. PIERCE,
A. B. RA~rlTCH,
D. M. BROWN
AND
P. G. STANLEY*
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles ( U.S.A .) and Wellcome Laboratory, Royal North Shore Hospital, Crows Nest, N.S.W. (Australia)
(Received April I2th, 1965)
SUMMARY I. The properties of human thyroglobulin prepared from both normal thyroid glands and from subjects with non-toxic colloid goiters have been studied. No significant differences in composition were found with respect to the non-iodinated amino acids, the total amount of carbohydrate, and the kinds of sugar residues found nor were differences observed in electrophoretic and centrifugal properties. The amino acid compositions are also compared to those of sheep and of hog thyroglobulin. 2. The amino acid analyses showed the presence of approximately 18o halfcystine residues per molecular weight of 66oooo. Within experimental error all the half-cystine residues were found to be in disulfide linkage as shown b y amino acid analyses of preparations which had been treated at p H 8.6 in 8 M urea with sodium iodoacetate with and without prior reduction b y mercaptoethanol. This conclusion was supported by results of amperometric titrations. 3. Evidence concerning subunit structure has been obtained b y physical studies of the S-carboxymethyl derivative of reduced thyroglobulin. In the presence of sodium dodecyl sulfate at p H 9.6 this derivative undergoes dissociation and sediments as a single component having an s °20, w of 3.2-3.4 S. The molecular weight of the dissociated species was estimated to be in the range of 110000-125000, thus indicating at least 5 or 6 physically identical subunits or peptide chains in the native thyroglobulin molecule.
INTRODUCTION Although thyroglobulin has been the subject of extensive stucty with considerable emphasis placed on determination of physical parameters and investigation of the iodinated amino acids, much remains to be learned concerning the number and structure of the peptide chains and oligosaccharide moieties that constitute this glycoprotein. The molecular weight of the native protein from several species has been found to be between 66ooo0 and 69oooo 1-5. It has been found, however, that under certain conditions, including high p H or in the presence of anionic detergents or urea, the native protein with a sedimentation coefficient of 19 S undergoes dissociation and * Present address: C.S.R. Research Laboratories. Box 39, Roseville, N.S.W. (Australia). Biochim. Biophys. Acta, I I i (1965) 247-257
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intramolecular unfolding 2, v-7. Several molecular species have been observed including one with a sedimentation coefficient of 12 S and a molecular weight half that of the native protein 5. Evidence of further dissociation has come from several sources including physical studies in 9 M urea 5 but no data have been available concerning the molecular weight of components other than the material with a sedimentation coefficient of 12 S. Proteins of relatively low molecular weight (sedimentation coefficients between 3 S and 8 S) have been found to be precursors of thyroglobulin during its biosynthesis and the component of 12 S is apparently the immediate precursor s. The present paper provides further data concerning the subunit structure of human thyroglobulin. The freshly prepared S-carboxymethyl derivative of reduced thyroglobulin has been found to dissociate in the presence of sodium dodecyl sulfate at p H 9.6 to a subunit which is physically homogeneous. Its s%0, ~ is 3.2-3.4 S and a molecular weight in the range of I IO ooo-125 ooo has been calculated from sedimentation and diffusion data. Whether or not the subunits or peptide chains of thyroglobulin are identical in amino acid sequence is not known. Two markedly different types of oligosaccharide units have been recognized 9 but their location on the peptide chains has not been studied. Prerequisite to a study of these questions is a knowledge of the total amino acid and carbohydrate composition of thyroglobulin and these data are presented both for human thyroglobulin from normal and goitrous glands as well as for hog and sheep thyroglobulin. The two human thyroglobulins do not differ significantly either in carbohydrate or amino acid composition (other than iodinated amino acids) and it thus appears t h a t goitrous glands, with their far greater content of thyroglobulin, are good sources of material for structural studies. At the beginning of the investigation little definitive data were available concerning the amino acid composition of thyroglobulins of any species particularly in respect to total recovery of amino acids and no data were found for the human material. Recently the complete amino acid compositions of calf s and sheep thyroglobulin ~° have appeared from other laboratories. MATERIALS AND METHODS
Thyroid glands for the preparation of "normal" human thyroglobulin were obtained on autopsy and showed no macroscopic or microscopic abnormality. Sheep and hog thyroids were obtained from freshly killed animals. Glands were quickly frozen and stored at --7 o°. For preparation of thyroglobulin the glands were allowed to come to o °, cut into slices of 3-4 m m thickness, and the slices were extracted overnight with 0. 9 % NaC1 (3 ml/g of tissue) containing a few drops of toluene. All procedures were done at 4 °. After removal of the tissue and centrifugation, the extract was fractionated with ammonium sulfate b y the method of DERRIEN et al 11. Modified ammonium sulfate concentrations (37-43 % of saturation) were used in the case of the human protein, due to its slightly higher solubility TM. After the final precipitation, the thyroglobulin was dissolved in sufficient o.15 M NaC1 to yield a protein concentration of about 2 % and the solution was immediately frozen. Further purification was achieved b y chromatography on Sephadex G - 2 o o , a s described b y PERELMUTTER et al. 13. The major peak from the Sephadex (detected by its absorption at 280 m/z) was pooled, dialysed against distilled water for 40 h in the presence of a trace of toluene, Biochim. Biophys. Acta, i i i (1965) 247-257
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and freeze-dried. The dry protein was stored in vacuo at 4 ° over P20~. Material for ultra-centrifugal analysis was taken before dialysis to eliminate the partial dissociation and aggregation previously observed to result from this procedure 14,15. The protein from goitrous glands was prepaxed as above except that three preparations were made, each utilizing a single goitrous thyroid gland*. The preparations were freeze dried from o.15 M NaC1 and stored in this form until chromatographed on Sephadex. A yield of about 17 g was obtained from a goitrous gland weighing 15o g. Analytical methods The several thyroglobulins were subjected to starch gel electrophoresis as previously described TM at pH 8.6 in 0.025 M borate buffer. The gels were run for 18 h at 4 ° with a voltage gradient of 7 V/cm. Preparations of human thyroglobulin were examined for contamination with serum proteins by agar gel diffusion against antibodies to normal human serum** in a micromodification of the technique of OUCHTERLONY17, Antiserum to normal human thyroglobulin was prepared by inoculation of two healthy male rabbits. A total of 18 mg of protein per rabbit was given in three equal injections over a period of three weeks. Each protein sample was administered in I ml of Freund's complete adjuvant. Antiserum was prepared six weeks after the initial injection. Amino acid analyses were carried out on a Beckman model 12o amino acid analyser by the method of SPACKMANet al. TM. The freeze-dried samples were washed with ether and absolute ethanol and air dried before analyses. Hydrolysis was carried out in 6 N HC1 in evacuated glass ampules for 2o, 4 ° or 72 h at i i o °. Tyrosine and tryptophan were determined by the method of GOOI)WIN AND MORTON19 without correction for iodo-amino acids. Cysteine was determined by amino acid analysis for S-carboxymethylcysteine following treatment of the protein with iodoacetate in 8 M urea at pH 8.6 and by amperometric titration as described by BENESCH et al. 2°. The 12 content of "normal" and "goitrous" human samples was determined by Bioscience Laboratories, Los Angeles 25, California. Analyses for carbohydrate content and chromatographic identification of individual sugars were essentially as previously described 21. Sialic acid was determined by the procedure of WARREN22. Hexose values were corrected for fucose content. Reduction and carboxymethylation The procedure of A N F I N S E N AND HABER23 was modified as described below. One hundred milligrams of thyroglobulin were dissolved in 3 ml of 0.05 M NaC1 in a polyethylene vial, solid urea (3.6 g of reagent grade) was added, and the volume of the solution brought to 7.5 ml with water. The vial was arranged on a pH stat (Radiometer, Copenhagen) with magnetic stirring and a stream of nitrogen was passed over the solution. The pH was brought to and maintained at 8.6 by addition of 5 % methylamine./3-Mercaptoethanol (IOO t~l) was then added and the reaction allowed to proceed for 4 h at 25-3 o°. The reaction mixture was transferred to a beaker, diluted with 23 ml of 0. 5 M Tris-acetate buffer (pH 8.6), and 2.62 g of recrystallized sodium iodo* Goitrous protein was obtained from individual multinodular, n o n m a l i g n a n t goiters, containing large colloid-filled follicles. A n t i - h u m a n s e r u m protein a n t i s e r u m was obtained from Colorado Serum Co., Denver, Colo.
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acetate were added slowly with stirring. Small amounts of buffer and IO % methylamine were used to maintain the pH. The alkylation was allowed to proceed for 15 min at room tempeIature and was stopped by the addition of IO ml of fl-mercaptoethanol. The reaction mixture was then dialyzed against distilled water for 40 h at 4 °. The protein was freeze dried and stored at 4 ° in vacuo. Completeness of the reduction and S-carboxymethylation was established by an amino acid analysis. With all samples, the cystine peak was absent and S-carboxymethylcysteine was equal within experimental error to the number of half cystine residues found before reduction and alkylation. Physical methods Measurements of sedimentation coefficients were conducted as previously described 2~-~6 with a Spinco Model E analytical ultracentrifuge at a rotor speed of 59780 rev./min. Diffusion measurements were carried out at 20 ° in the ultracentrifuge by the method of EHRENBERG27. Calculation of the diffusion coefficient was based on the average value obtained from tracings of the schlieren patterns outlining the inside and outside images. Experiments utilizing proteins of known diffusion constants showed this procedure to give more precise coefficients, particularly when the phase plate angle was decreased for greater magnification. The partial specific volume of the protein was calculated from the amino acid composition and carbohydrate composition as previously described ~. The sodium dodecyl sulfate bound to the protein was determined by the method of SCHACHMAN29. Protein concentrations were determined from the index of refraction of the solutions. RESULTS
The patterns obtained after electrophoresis in starch gels of samples of thyroglobulin from normal and goitrous glands are given in Fig. I. No marked differences were seen between the preparations from the three species oi between any of the three preparations from individual goitrous human glands and the patterns are essentially identical to those reported by SPIRO14 for human, calf and sheep thyroglobulins. Double diffusion precipitin tests showed the thyroglobulin from normal and goitrous glands to give a single continuous precipitin line against antiserum to the normal human preparation. A preparation from a goitrous gland did not react with antiserum to human serum proteins although a trace of contamination with serum proteins (less than 1%) was observed with the preparation from normal glands. Immunoelectro-
Fig. i. Starch gel electrophoresis of human thyroglobulins at pH 8.6 in 0.025 1V[ borate buffer. a, normal human thyroglobulin; b, thyroglobulin prepared from goiterous human thyroid glands.
Biochim. Biophys. Acta, I I i (1965) 247-257
25I
PROPERTIES OF HUMAN THYROGLOBULIN TABLE I COMPOSITION OF THYROGLOBULINS
Component
Normal Goitrous Hog human human Lysine Histidine Amide NH 3 Arginine Aspartic acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine** Tryptophan** Phenylalanine Glucosamine*** Hexose Fucose Sialic acid Recovery
residues/mole (mol. wt. 660 ooo)
g residue/zoo g protein* Sheep
3.4 1.4 0.9 5.9 5.9 3 .8 5.3 12.4 4.4 3 .2 3 .8 2.9 4 .6 1. 3 2. 4 7.5 3.2 -5.3 2. 7 7-3 i.o 1.2
3.4 1.6 0.9 6.7 6.1 3 .6 5 .8 12. 4 4 .o 3 .2 3 .8 2.8 4-4 i .2 2.1 7 .6 3.2 2.6 5 .1 3.3 6-7 o.7 1. 4
3 .0 1. 3 -7.9 6. 4 4 .1 6.o 13.5 6.0 3.7 5 .1 2.1 4.9 1.o 2.3 9.9 2.6 1.8 5 .2 1-7 ----
3 .0 1.2 -7.4 6. 4 3.5 5.7 12.6 5.o 3.3 4.5 2.2 5.9 1.2 2.4 9.2 2.9 2.6 5 .2 1-5 ----
89,8
92.9
87.7
85.7
Normal human
Goitrous human
Hog
Sheep
175 68 408 25o 339 248 4 °2 634 299 367 349 187 3o3 65 14o 437 129 -238 Iii 298 45 27
175 75 408 284 345 238 44 ° 634 272 367 349 181 293 60 122 442 129 83 229 i 35 273 31 32
154 63 -334 366 267 454 691 408 429 473 136 326 5° 134 579 lO5 58 231 7° ----
154 58 -313 366 228 432 645 339 383 418 142 393 60 14o 536 117 83 231 62 ----
* V a l u e s for t h e n o r m a l h u m a n m a t e r i a l are b a s e d on t h r e e s e p a r a t e a n a l y s e s ( h y d r o l y s i s t i m e s 20 a n d 4 ° h ), v a l u e s for t h e m a t e r i a l from g o i t r o u s g l a n d s a re b a s e d on four s e p a r a t e a n a l y s e s ( h y d r o l y s i s t i m e s 2o, 4 ° a n d 72 11). The h o g a n d sheep a n a l y s e s a re e a c h t h e a v e r a g e of t w o s e p a r a t e a n a l y s e s ( h y d r o l y s i s t i m e 20 h). I n t h e case of g o i t r o u s m a t e r i a l t h e serine, t h r e o n i n e a n d a m i d e n i t r o g e n are c o r r e c t e d b y e x t r a p o l a t i o n to zero t i m e of h y d r o l y s i s . V a l ue s are c o r r e c t e d for m o i s t u r e c o n t e n t . D e t e r m i n a t i o n of ash on t h e m a t e r i a l from g o i t r o u s g l a n d s s h o w e d none t o be pre s e nt . ** V a l u e s o b t a i n e d from u l t r a v i o l e t a b s o r p t i o n s p e c t r a b y t h e m e t h o d of GOODWlN AND MORTON 19. *** F o r t h e h u m a n s a m p l e s a s e p a r a t e h y d r o l y s i s w a s p e r f o r m e d a t IOO° for 4 h in 6 N HCI. I n t h e case of t h e h u m a n t h y r o g l o b u l i n t h i s w e i g h t r e c o v e r y g a v e q u a n t i t a t i v e r e c o v e r y of t o t a l n i t r o g e n ( d e t e r m i n e d i n d e p e n d e n t l y ) b y E l e c k M i c r o a n a l y t i c a l L a b o r a t o r i e s , Torrance, Calif.
phoresis in starch gels as described b y POULIK33 showed all three electrophoretic components to give a single continuous precipitin line with no spurs. The amino acid and carbohydrate compositions of thyroglobulin from normal human, goitrous human, hog and sheep glands are given in Table I. Paper chromatography of acid hydrolysates of the normal and goitrous human material demonstrated the presence of fucose, galactose, mannose and glucosamine. The amounts of each sugar in the two preparations appeared to be equal as judged b y the intensity of staining with silver nitrate. In the amino acid analyses of samples which had been treated with iodoacetate in 8 M urea but without prior reduction, the amount of Scarboxymethylcysteine detected was such that the number of cysteine residues was less than two per 660 ooo molecular weight. After reduction and S-carboxymethylation approx. 18o residues of S-carboxymethylcysteine were found per mol. wt. of 660000. Biochim. Biophys. Acta, 111 ( 1 9 6 5 ) 2 4 7 - 2 5 7
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P I E R C E e t al.
The iodine contents of "normal" thyroglobulin and of that from one of the goitrous glands were 0.32 % and 0.07 %, respectively. The sedimentation patterns of the native thyroglobulin were as previously reported (e.g. ref. 6). For example, one preparation from goitrous glands consisted of 95 % of a I8.7-S component and 5 % of a 27.I-S component. A preparation from normal glands contained 9 ° % of a I8.8-S component, 5 % of a 27.4-S component and a small amount of poorly resolved material with slow sedimentation rate.
Physical properties of thyroglobulin following reduction and carboxymethylation Investigation of the solubility properties of the reduced and carboxymethylated preparations showed that it could be dissolved in 95 % acetic acid and remained in solution after dialysis against 0.5 M acetate buffer at p H 3.5. Ultracentrifugal examination, however, showed the material to be polydisperse with a number of fastsedimenting components. In 0.05 M sodium glycinate at pH 9.5, the S-carboxymethyl derivative was readily soluble and ultracentrifugal analysis revealed the presence of two peaks of approximately equal area with sedimentation coefficients of 3.4 and 13.5 S as shown in Fig. 2a. After addition of o.oi M sodium dodecyl sulfate to the cell and allowing the solution to stand I h at room temp., a nearly complete conversion to what appears to be a single species with an s20, w of about 3.0 S was found (Fig. 2b). When the total sodium dodecyl sulfate added was increased to O.Ol7 M complete dissociation to the 3-S component occurred and the material sedimented with a single symmetrical boundary as shown in Fig. 2c. The concentration dependence of the
F i g . 2. S e d i m e n t a t i o n p a t t e r n s of r e d u c e d S - c a r b o x y m e t h y l a t e d h u m a n t h y r o g l o b u l i n . All r u n s a r e in 0.05 M g l y c i n e ( p H 9.6). a, n o s o d i u m d o d e c y l s u l f a t e , p r o t e i n c o n c e n t r a t i o n = 0.57 % , s20. w = 3.4, 13-3 S ; b, o . o i M s o d i u m d o d e c y l s u l f a t e , p r o t e i n c o n c e n t r a t i o n = 0.8o % , s20. w ~ 3.0, 6.8 S; c, O.Ol 7 M s o d i u m d o d e c y l s u l f a t e , p r o t e i n c o n c e n t r a t i o n = 0 . 5 8 % , s~o, w ~ 2.8 S. T i m e i n t e r v a l for p h o t o g r a p h y ; a, 8 r a i n (4 r a i n l a s t t w o e x p o s u r e s ) ; b, 8 m i n ; c, 16 m i n .
Biochim. Biophys. Acta, I I i (1965) 2 4 7 - 2 5 7
PROPERTIES OF HUMAN THYROGLOBULIN
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sedimentation coefficients of the S-carboxymethyl derivatives of two preparations of thyroglobulin (from goitrous glands) in the presence of O.Ol7 M sodium dodecyl sulfate are shown in Fig. 3a, t h a t of their diffusion coefficients in Fig. 3b. In these experiments
f i
0
i
1
i
2
i
3
I
4
i
5
[
6
I
7
I
I
I
i
8 9 1 2 Protein conch. (mg/rnD
I
3
i
4
i
5
i
6
i
7
I
8
i
9
f
Fig. 3. T h e d e p e n d e n c e of s e d i m e n t a t i o n velocity a n d diffusion on p r o t e i n c o n c e n t r a t i o n . Circles a n d s q u a r e s r e p r e s e n t two different p r e p a r a t i o n s of t h e r e d u c e d a n d c a r b o x y m e t h y l a t e d d e r i v a t i v e of t h y r o g l o b u l i n . All m e a s u r e m e n t s were carried o u t in o.o36 M glycine (pH 9.6), a n d in t h e presence of O.Ol 7 M s o d i u m dodecyl sulfate. Left, a; right, b.
the solutions with the highest protein concentration were diluted with O.Ol7 M sodium dodecyl sulfate in the p H 9.5 buffer for the determinations at the lower protein concentrations. Ratios of protein-sodium dodecyl sulfate complex to unbound sodium dodecyl sulfate in the solvent varied from 3.5 : I to 0.6 : I. This variation apparently did not affect the dissociation of the protein as a single symmetrical boundary was formed at all concentrations of protein and the values for each preparation fitted closely a straight line. In another experiment, protein to give a concentration at the lower level studied (2 mg/ml) was dissolved directly in the O.Ol7 M sodium dodecyl sulfate buffer solution and the s20, w was determined within 2 h. Again a single symmetrical boundary formed and the value of s20, w was in agreement with the data in Fig. 3a. This experiment appears to eliminate the possibility that hydrolysis of peptide or glycoside bonds occurred during experiments of longer duration. In the method described b y SCHACHMAN29 for determination of bound sodium dodecyl sulfate, the number of interference fringes contributed by the total amount of sodium dodecyl sulfate and the number of fringes attributed to bound sodium dodecyl sulfate sedimenting with the protein are measured. These data are given in Table I I for one of the preparations from a goitrous gland and show that the amount of sodium dodecyl sulfate bound in the hydrodynamic particles is 30 % by weight of the complex. A value of o.719 was calculated for the partial specific volume based on amino acid and carbohydrate composition. With the value of 0.885 for sodium dodecyl sulfate and correction made for the binding of sodium dodecyl sulfate, the partial specific volume of the complex is 0.769 . This together with the values of the sedimentation coefficients and diffusion coefficients of the glycoprotein-sodium dodecyl sulfate complex extrapolated to zero concentration gave molecular weights of Biochim. Biophys. Acta, i i i (1965) 247-257
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TABLE II T H E B I N D I N G OF SODIUM D O D E C Y L S U L F A T E B Y S - C A R B O X Y M E T H Y L A T E D H U M A N T H Y R O G L O B U L I N *
Material
Method
Concn. in fringes
Conch. (mg/ml)
Rev./min
Thyroglobulin Sodium dodecyl sulfate Thyroglobulin plus sodium dodecyl sulfate Thyroglobulin plus sodium dodecyl sulfate Thyroglobulin plus b o u n d sodium dodecyl sulfate
Synthetic b o u n d a r y cell Synthetic b o u n d a r y cell Calculated
23.66 12.oo 35.66
5.8 4.5
6569 6569
Synthetic b o u n d a r y cell
36.oo
Double sector cell
29.35"*
6569 42 o4o
* All m e a s u r e m e n t s were carried o u t ill a Spinco Model E analytical ultracentrifuge, equipped w i t h interference optics. ** Therefore 6.65 of the 12 available sodium dodecyl sulfate fringes sedimented with the protein or 55 % of the sodium dodecyl sulfate was bound. We therefore assume t h a t °.55 × 4.5 X IOO 5.8 + (0.55 x 4-5) or 3° % of the weight of the sedimenting species is due to b o u n d sodium dodecyl sulfate.
113 ooo and 123ooo for the two preparations (corrected for bound sodium dodecyl sulfate) *. There have been few reports concerning the determination of the molecular weights of proteins by measurement of sedimentation and diffusion in the presence of sodium dodecyl sulfate, fl-Galactosidase has been studied by WALLENFELS gt al. 31 and HOFFMAN AND HARRISONa2 have reported values for the molecular weights of the subunits of apoferritin based on physical measurements in the presence of sodium dodecyl sulfate. In the latter case the data agreed well with data obtained by other methods. Following completion of the observations on thyroglobulin given herein, determinations of the molecular weight of a well characterized protein derivative, S-carboxymethyl ribonuclease, were carried out. Sedimentation, diffusion and determination of bound sodium dodecyl sulfate were done as described above. Extrapolation of the data to zero protein concentration together with correction for binding of sodium dodecyl sulfate (30 %) gave a value of 14400 for the molecular weight of the S-carboxymethyl ribonuclease (in good agreement with the actual value of 1416o). In these experiments a ratio of protein-sodium dodecyl sulfate complex to unbound sodium dodecyl sulfate of 0.8 : I was needed to give values of s20, w and D which yielded the correct molecular weight. When native thyroglobulin from goitrous glands was subjected to treatment with O.Ol7 M sodium dodecyl sulfate at p H 9.5, 9 ° % of the material was converted to * A f u r t h e r dissociation was suggested on two occasions w h e n a p r e p a r a t i o n of the S-carboxym e t h y l derivative gave sedimentation and diffusion d a t a (s~o,w = 2.2; D = 2.76 ) indicating a mol. wt. of a b o u t 5 ° ooo-6o ooo b u t these d a t a could n o t be obtained consistently. The d a t a leading to the values of 113 ooo-123 ooo were obtained with two different p r e p a r a t i o n s of the S-carboxym e t h y l derivative of " g o i t r o u s " thyroglobulin and similar values were obtained from two preparations of derivative from the thyroglobulin of n o r m a l glands. I t should also be noted t h a t storage at --2o ° for 3 m o n t h s of a S - c a r b o x y m e t h y l derivative of " n o r m a l " thyroglobulin led to deterioration in t h a t the material no longer yielded a single peak in the ultracentrifuge as it had s h o r t l y after its preparation.
Biochim, Biophys. Acta, I I i (1965) 247-257
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a species with an s20, w of 5.3 (protein concentration, o.5 %). This result is consistent with previous findings ~. Due to the presence of lO% faster sedimenting material, however, molecular weights could not be determined with any c e r t i n t y by measurements of sedimentation and diffusion. DISCUSSION
The present investigation shows that the thyroglobulin isolated from goitrous glands is identical by several criteria to the protein from normal glands even though the amount synthesized is many times greater. The amino acid compositions of the two types of human thyroglobulins are identical within experimental error but differ somewhat from those of the two other species studied, particularly in their contents of arginine, proline, alanine and leucine and half cystine. The latter appears to be all in disulfide form and the molecule therefore must be extensively cross linked. It is noteworthy that the amount of carbohydrate in the thyroglobulin from goitrous glands is equal to that found in normal thyroglobulin. This finding suggests that the biosynthesis or linking of the carbohydrate moieties is governed by the amount of the protein component present. The amounts of neutral sugar in these experiments are somewhat higher than those recently reported for normal human material by SPIRO AND SPIRO33. As expected the iodine content of the normal material is the greater. Similar electrophoretic patterns in starch gel were found for all preparations with observation of the three-banded patterns attributed by SPIRO14 to the presence of 6-S and 27-S components in addition to the I9-S material. The 6-S and 27-S fractions are diffficult to remove from the I9-S component and their amounts are increased by dialysis and freeze drying 14. The results of the immunoelectrophoresis in starch gel show these three components to be immunologically identical. This provides evidence that most, if not all, the 27-S component of the preparations is an aggregated form of thyroglobulin rather than another species of protein, a conclusion further supported by the sedimentation patterns of the S-carboxymethyl derivative in o.o17 M sodium dodecyl sulfate (see Fig. 2c) where only a single peak was observed. The peptide mapping studies of BOUCHILLOUXet al. ~° provide additional evidence for this conclusion. The number of subunits or peptide c h i n s constituting thyroglobulin is of great importance if detailed structural studies are to be undertaken. Thyroglobulin has been defined as the iodine-contining, saline-extractable protein component of the thyroid gland with a sedimentation coeËficient of 19 S. The molecular weight of the native protein from several species has been estimated to be between 660000 and 690000. Some heterogeneity has been found in I9-S material by chromatographic studies but is presumably due to the existence of a family of proteins which vary only in their iodine and sialic acid content 1°, 34,35. While careful studies of its physical parameters, notably by EDELHOCH and colleagues, have shown that the molecule undergoes complex conformational changes as well as dissociation and aggregation phenomena, the number of peptide c h i n s constituting the molecule is not known. Of particular interest have been studies in the presence of sodium dodecyl sulfate 7 where dissociation readily occurs yielding two components whose sedimentation coefficients have values of approx. 3 and 4 S when extrapolated to zero ionic strength. The persistence of two Biochim. Biophys. Acta, I i i (1965)247-257
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components when native thyroglobulin has been placed in sodium dodecyl sulfate and in several other systems which promote dissociation has hitherto prevented estimation of molecular weight of subunits from centrifugal data 5. Polarization of fluorescence of derivatives of thyroglobulin m also suggests subunits of relatively low molecular weight but interpretation of the data in terms of molecular size alone is complicated b y probable losses in internal rigidity of the molecule. Results of end-group determinations have been variable and also have not given clear information about the number of peptide chains. Difficulties have arisen because of contamination of thyroglobulin preparations with proteolytic enzymes of the thyroid 37. The most recent results (with hog thyroglobulin) 37 have indicated the presence of 2 moles of aspartic acid or asparagine and one mole of glycine per molecular weight of 660 ooo*. Thus in the present investigation it was encouraging to find that the S-carboxymethyl derivatives of both the "goitrous" and "normal" human thyroglobulins were completely soluble in sodium glycinate at p H 9.6 and that the addition of O.Ol7 M sodium dodecyl sulfate caused dissociation to a species which appears homogeneous in the ultracentrifuge. This result enabled measurement of physical parameters with most consistent data obtained with two samples of the carboxymethyl derivative prepared from goitrous material. These two preparations showed sedimentation coefficients which gave a straight line plot over a 4-fold range of protein concentration and extrapolated values for s°20,w of 3.42 and 3.12 S. Values for several samples of the S-carboxymethyl derivative from the normal human thyroglobulin were all within the range of those shown in Fig. 3 although more scatter of points was present, perhaps due in this instance to deterioration of the derivative during storage. I t should also be noted that with the Scarboxymethyl derivative the slope of the plot of sedimentation constant against protein concentration is far less than that reported by EDELI-IOCHAND LIPPOLDT2 for the slower moving of two components seen when native calf thyroglobulin is placed in sodium dodecyl sulfate. The change in slope is most likely due to creation of a more flexible molecule by reduction of the disulfide bridges. The calculated molecular weight of the S-carboxymethyl derivative in the presence of O.Ol7 M sodium dodecyl sulfate is in the range 110000-125000. The determination of molecular weight in the presence of sodium dodecyl sulfate requires a correction for the amount of bound sodium dodecyl sulfate both in the buoyancy factor (partial specific volume) and the determination of the percent by weight of the hydrodynamic particle which is the glycoprotein. The molecular weight values thus m a y be subiect to greater experimental error than if it had been possible to obtain the subunits in the absence of sodium dodecyl sulfate. The most reasonable interpretation of our data, thus far, is that human thyroglobulin after reduction of its disulfide bonds can dissociate to 5 or 6 physically identical subunits. That the observed subunits m a y not represent the total number of peptide chains in thyroglobulin is suggested by the fact that on two occasions the observed values of the sedimentation and diffusion coefficients of material giving symmetrical peaks in the ultracentrifuge indicated a further dissociation. This possibility was also suggested b y the data of STEINER AND EDELHOCH~ concerning the polarization of fluorescence of derivatives of * Preliminary end group determinations carried out in this laboratory by the method of STARK AND S M Y T H 38 gave values of approx. 2 moles of aspartic acid or asparagine per 660000 tool. wt. together with lesser amounts of several other amino acids for the human preparations from both normal and goitrous glands. Biochim. Biophys. Acta, iii (1965) 247-257
PROPERTIES OF HUMAN THYROGLOBULIN
257
thyroglobulin and further work is required to finally identify the number and types of peptide chains in thyroglobulin. ACKNOWLEDGEMENTS
This investigation was supported in part by a U.S. Public Health Service grant C-229o by Cancer Research Funds of the University of California and by the National Health and Medical Research Council, Canberra. The authors express their appreciation for stimulating advice from Dr. A. N. GLAZER and for the capable assistance of Miss T. REIMO. The authors are indebted to Dr. A. LORBER,Wadsworth Veterans Administration Hospital, West Los Angeles, Calif., for the amperometric analyses.
REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 i8 19 2o 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38
M. HEIDELBERGER AND K. O. PEDERSEN, J. Gen. Physiol., 19 (1935) 95. H. P. LUNDGREN AND J. w . WILLIAMS, J. Phys. Chem., 43 (1939) 989. Y. DERRIEN, R. MICHEL, K. O. PEDERSEN AND J. ROCHE, Biochim. Biophys. Acta, 3 (1949) 436. I. J. O'DONNELL, R. L. BALDWIN AND J. w . WILLIAMS, Biochim. Biophys. Acta, 28 (1958) 294. H. EDELHOCH, in R. PITT-RIVERS AND W. R. TROTTER, The Thyroid Gland, Vol. I, B u t t e r w o r t h Inc., W a s h i n g t o n , D.C., 1964, p. 113. H. EDELHOCH, J. Biol. Chem., 235 (196o) 1326. H. EDELHOCH AND R. E. LIPPOLDT, J. Biol. Chem., 235 (196~) 1335. R. V~T. SEED AND I. H. GOLDBERG, J. Biol. Chem., 24o (1965) 764 . R. G. SplRO, New Engl. J. Med., 269 (1963) 566. S. BOUCHILLOUX, M. ROLLAND, J. TORRESANI, M. ROQUES AND S. LlSSlTZKY, Biochim. Biophys. Acta, 93 (1964) 15. Y. DERRIEN, R. MICHEL AND J. ROCHE, Biochim. Biophys. Acta, 2 (1948) 454. P. G. STANLEY, Acta Endocrinol. 45 (1964) 161. L. PERELMUTTER, W. DEVLIN AND N. R. STEPHENSON, Can. J. Biochem. Physiol., 41 (1963) 2493. M. J. SPlRO, J. Biol. Chem., 236 (1961) 29Ol. S. SHULMAN AND J. P. ARMENIA, J. Biol. Chem., 238 (1963) 2723. M. E. CARSTEN AND J. G. PIERCE, J. Biol. Chem., 235 (196o) 78. ~. OUCHTERLONY, in P. I~ALLOS AND B. H. WAKSMAN, Progress in Allergy, Vol. 6, S. Karger, Basel, 1962, p. 30. D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 3° (1958) 119o. T. W. GOODWlN AND R. A. MORTON, Bioehem. J., 4 ° (1946) 628. R. E. BENESCH, H. A. LARDY AND R. BENESCH, J. Biol. Chem., 216 (1955) 663. M. E. CARSTEN AND J. G. PIERCE, J. Biol. Chem., 238 (1963) 1724. L. WARREN, J. Biol. Chem., 234 (1959) 1971C. B. ANFINSEN AND E. HABER, J. Biol. Chem., 236 (1961) 1361. E. L. SMITH AND D. M. BROWN, J. Biol. Chem., 195 (1952 ) 525 • E. L. SMITH, D. M. BROWN AND M. LASKOWSKI, J. Biol. Chem., 191 (1951) 639. E. L. SMITH, D. M. BROWN, J. B. FISHMAN AND A. E. WILHELMI, J. Biol. Chem., 177 (1949) 3o5 . A. EHRENBERG, Acta Chem. Scand., I I (1957) 1257. E. L. SMITH, A. STOCKELL AND J. R. KIMMEL, J. Biol. Chem., 2o 7 (1954) 551. H. K. SCHACHMAN, Biochemislry, 2 (1963) 887. M. D. POULIK, J. Immunol., 82 (1959) 502. K. WALLENFELS, H. SUND AND K. WEBER, Biochem. Z., 338 (1963) 714 . T. HOFMANN AND V. M. HARRISON, J. Mol. Biol., 6 (1963) 256. R. G. SPIRO AND M. J. SPIRO, J. Biol. Chem., 240 (1965) 997. J. ROBBINS, J. Biol. Chem., 238 (1963) 182. N. WI, O. TARUTANI, Y. I~ONDO AND H. TAMURA, Nature, 191 (1961) 1199. R. F. STEINER AND H. EDELHOCH, J. Am. Chem. Soc., 83 (1961) 1435. T. A. A. DOPHEIDE AND V. M. TRIKOJUS, Nature, 2Ol (1964) 1128. G. R. STARK AND D. G. SMYTH, J. Biol. Chem., 238 (1963) 214.
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BBA 25415 HUMAN T H Y R O G L O B U L I N : S T U D I E S ON T H E NATIVE AND S-CARBOXYMETHYLATED PROTEIN j. G. PIERCE,
A. B. RA~rlTCH,
D. M. BROWN
AND
P. G. STANLEY*
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles ( U.S.A .) and Wellcome Laboratory, Royal North Shore Hospital, Crows Nest, N.S.W. (Australia)
(Received April I2th, 1965)
SUMMARY I. The properties of human thyroglobulin prepared from both normal thyroid glands and from subjects with non-toxic colloid goiters have been studied. No significant differences in composition were found with respect to the non-iodinated amino acids, the total amount of carbohydrate, and the kinds of sugar residues found nor were differences observed in electrophoretic and centrifugal properties. The amino acid compositions are also compared to those of sheep and of hog thyroglobulin. 2. The amino acid analyses showed the presence of approximately 18o halfcystine residues per molecular weight of 66oooo. Within experimental error all the half-cystine residues were found to be in disulfide linkage as shown b y amino acid analyses of preparations which had been treated at p H 8.6 in 8 M urea with sodium iodoacetate with and without prior reduction b y mercaptoethanol. This conclusion was supported by results of amperometric titrations. 3. Evidence concerning subunit structure has been obtained b y physical studies of the S-carboxymethyl derivative of reduced thyroglobulin. In the presence of sodium dodecyl sulfate at p H 9.6 this derivative undergoes dissociation and sediments as a single component having an s °20, w of 3.2-3.4 S. The molecular weight of the dissociated species was estimated to be in the range of 110000-125000, thus indicating at least 5 or 6 physically identical subunits or peptide chains in the native thyroglobulin molecule.
INTRODUCTION Although thyroglobulin has been the subject of extensive stucty with considerable emphasis placed on determination of physical parameters and investigation of the iodinated amino acids, much remains to be learned concerning the number and structure of the peptide chains and oligosaccharide moieties that constitute this glycoprotein. The molecular weight of the native protein from several species has been found to be between 66ooo0 and 69oooo 1-5. It has been found, however, that under certain conditions, including high p H or in the presence of anionic detergents or urea, the native protein with a sedimentation coefficient of 19 S undergoes dissociation and * Present address: C.S.R. Research Laboratories. Box 39, Roseville, N.S.W. (Australia). Biochim. Biophys. Acta, I I i (1965) 247-257
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intramolecular unfolding 2, v-7. Several molecular species have been observed including one with a sedimentation coefficient of 12 S and a molecular weight half that of the native protein 5. Evidence of further dissociation has come from several sources including physical studies in 9 M urea 5 but no data have been available concerning the molecular weight of components other than the material with a sedimentation coefficient of 12 S. Proteins of relatively low molecular weight (sedimentation coefficients between 3 S and 8 S) have been found to be precursors of thyroglobulin during its biosynthesis and the component of 12 S is apparently the immediate precursor s. The present paper provides further data concerning the subunit structure of human thyroglobulin. The freshly prepared S-carboxymethyl derivative of reduced thyroglobulin has been found to dissociate in the presence of sodium dodecyl sulfate at p H 9.6 to a subunit which is physically homogeneous. Its s%0, ~ is 3.2-3.4 S and a molecular weight in the range of I IO ooo-125 ooo has been calculated from sedimentation and diffusion data. Whether or not the subunits or peptide chains of thyroglobulin are identical in amino acid sequence is not known. Two markedly different types of oligosaccharide units have been recognized 9 but their location on the peptide chains has not been studied. Prerequisite to a study of these questions is a knowledge of the total amino acid and carbohydrate composition of thyroglobulin and these data are presented both for human thyroglobulin from normal and goitrous glands as well as for hog and sheep thyroglobulin. The two human thyroglobulins do not differ significantly either in carbohydrate or amino acid composition (other than iodinated amino acids) and it thus appears t h a t goitrous glands, with their far greater content of thyroglobulin, are good sources of material for structural studies. At the beginning of the investigation little definitive data were available concerning the amino acid composition of thyroglobulins of any species particularly in respect to total recovery of amino acids and no data were found for the human material. Recently the complete amino acid compositions of calf s and sheep thyroglobulin ~° have appeared from other laboratories. MATERIALS AND METHODS
Thyroid glands for the preparation of "normal" human thyroglobulin were obtained on autopsy and showed no macroscopic or microscopic abnormality. Sheep and hog thyroids were obtained from freshly killed animals. Glands were quickly frozen and stored at --7 o°. For preparation of thyroglobulin the glands were allowed to come to o °, cut into slices of 3-4 m m thickness, and the slices were extracted overnight with 0. 9 % NaC1 (3 ml/g of tissue) containing a few drops of toluene. All procedures were done at 4 °. After removal of the tissue and centrifugation, the extract was fractionated with ammonium sulfate b y the method of DERRIEN et al 11. Modified ammonium sulfate concentrations (37-43 % of saturation) were used in the case of the human protein, due to its slightly higher solubility TM. After the final precipitation, the thyroglobulin was dissolved in sufficient o.15 M NaC1 to yield a protein concentration of about 2 % and the solution was immediately frozen. Further purification was achieved b y chromatography on Sephadex G - 2 o o , a s described b y PERELMUTTER et al. 13. The major peak from the Sephadex (detected by its absorption at 280 m/z) was pooled, dialysed against distilled water for 40 h in the presence of a trace of toluene, Biochim. Biophys. Acta, i i i (1965) 247-257
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and freeze-dried. The dry protein was stored in vacuo at 4 ° over P20~. Material for ultra-centrifugal analysis was taken before dialysis to eliminate the partial dissociation and aggregation previously observed to result from this procedure 14,15. The protein from goitrous glands was prepaxed as above except that three preparations were made, each utilizing a single goitrous thyroid gland*. The preparations were freeze dried from o.15 M NaC1 and stored in this form until chromatographed on Sephadex. A yield of about 17 g was obtained from a goitrous gland weighing 15o g. Analytical methods The several thyroglobulins were subjected to starch gel electrophoresis as previously described TM at pH 8.6 in 0.025 M borate buffer. The gels were run for 18 h at 4 ° with a voltage gradient of 7 V/cm. Preparations of human thyroglobulin were examined for contamination with serum proteins by agar gel diffusion against antibodies to normal human serum** in a micromodification of the technique of OUCHTERLONY17, Antiserum to normal human thyroglobulin was prepared by inoculation of two healthy male rabbits. A total of 18 mg of protein per rabbit was given in three equal injections over a period of three weeks. Each protein sample was administered in I ml of Freund's complete adjuvant. Antiserum was prepared six weeks after the initial injection. Amino acid analyses were carried out on a Beckman model 12o amino acid analyser by the method of SPACKMANet al. TM. The freeze-dried samples were washed with ether and absolute ethanol and air dried before analyses. Hydrolysis was carried out in 6 N HC1 in evacuated glass ampules for 2o, 4 ° or 72 h at i i o °. Tyrosine and tryptophan were determined by the method of GOOI)WIN AND MORTON19 without correction for iodo-amino acids. Cysteine was determined by amino acid analysis for S-carboxymethylcysteine following treatment of the protein with iodoacetate in 8 M urea at pH 8.6 and by amperometric titration as described by BENESCH et al. 2°. The 12 content of "normal" and "goitrous" human samples was determined by Bioscience Laboratories, Los Angeles 25, California. Analyses for carbohydrate content and chromatographic identification of individual sugars were essentially as previously described 21. Sialic acid was determined by the procedure of WARREN22. Hexose values were corrected for fucose content. Reduction and carboxymethylation The procedure of A N F I N S E N AND HABER23 was modified as described below. One hundred milligrams of thyroglobulin were dissolved in 3 ml of 0.05 M NaC1 in a polyethylene vial, solid urea (3.6 g of reagent grade) was added, and the volume of the solution brought to 7.5 ml with water. The vial was arranged on a pH stat (Radiometer, Copenhagen) with magnetic stirring and a stream of nitrogen was passed over the solution. The pH was brought to and maintained at 8.6 by addition of 5 % methylamine./3-Mercaptoethanol (IOO t~l) was then added and the reaction allowed to proceed for 4 h at 25-3 o°. The reaction mixture was transferred to a beaker, diluted with 23 ml of 0. 5 M Tris-acetate buffer (pH 8.6), and 2.62 g of recrystallized sodium iodo* Goitrous protein was obtained from individual multinodular, n o n m a l i g n a n t goiters, containing large colloid-filled follicles. A n t i - h u m a n s e r u m protein a n t i s e r u m was obtained from Colorado Serum Co., Denver, Colo.
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acetate were added slowly with stirring. Small amounts of buffer and IO % methylamine were used to maintain the pH. The alkylation was allowed to proceed for 15 min at room tempeIature and was stopped by the addition of IO ml of fl-mercaptoethanol. The reaction mixture was then dialyzed against distilled water for 40 h at 4 °. The protein was freeze dried and stored at 4 ° in vacuo. Completeness of the reduction and S-carboxymethylation was established by an amino acid analysis. With all samples, the cystine peak was absent and S-carboxymethylcysteine was equal within experimental error to the number of half cystine residues found before reduction and alkylation. Physical methods Measurements of sedimentation coefficients were conducted as previously described 2~-~6 with a Spinco Model E analytical ultracentrifuge at a rotor speed of 59780 rev./min. Diffusion measurements were carried out at 20 ° in the ultracentrifuge by the method of EHRENBERG27. Calculation of the diffusion coefficient was based on the average value obtained from tracings of the schlieren patterns outlining the inside and outside images. Experiments utilizing proteins of known diffusion constants showed this procedure to give more precise coefficients, particularly when the phase plate angle was decreased for greater magnification. The partial specific volume of the protein was calculated from the amino acid composition and carbohydrate composition as previously described ~. The sodium dodecyl sulfate bound to the protein was determined by the method of SCHACHMAN29. Protein concentrations were determined from the index of refraction of the solutions. RESULTS
The patterns obtained after electrophoresis in starch gels of samples of thyroglobulin from normal and goitrous glands are given in Fig. I. No marked differences were seen between the preparations from the three species oi between any of the three preparations from individual goitrous human glands and the patterns are essentially identical to those reported by SPIRO14 for human, calf and sheep thyroglobulins. Double diffusion precipitin tests showed the thyroglobulin from normal and goitrous glands to give a single continuous precipitin line against antiserum to the normal human preparation. A preparation from a goitrous gland did not react with antiserum to human serum proteins although a trace of contamination with serum proteins (less than 1%) was observed with the preparation from normal glands. Immunoelectro-
Fig. i. Starch gel electrophoresis of human thyroglobulins at pH 8.6 in 0.025 1V[ borate buffer. a, normal human thyroglobulin; b, thyroglobulin prepared from goiterous human thyroid glands.
Biochim. Biophys. Acta, I I i (1965) 247-257
25I
PROPERTIES OF HUMAN THYROGLOBULIN TABLE I COMPOSITION OF THYROGLOBULINS
Component
Normal Goitrous Hog human human Lysine Histidine Amide NH 3 Arginine Aspartic acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine** Tryptophan** Phenylalanine Glucosamine*** Hexose Fucose Sialic acid Recovery
residues/mole (mol. wt. 660 ooo)
g residue/zoo g protein* Sheep
3.4 1.4 0.9 5.9 5.9 3 .8 5.3 12.4 4.4 3 .2 3 .8 2.9 4 .6 1. 3 2. 4 7.5 3.2 -5.3 2. 7 7-3 i.o 1.2
3.4 1.6 0.9 6.7 6.1 3 .6 5 .8 12. 4 4 .o 3 .2 3 .8 2.8 4-4 i .2 2.1 7 .6 3.2 2.6 5 .1 3.3 6-7 o.7 1. 4
3 .0 1. 3 -7.9 6. 4 4 .1 6.o 13.5 6.0 3.7 5 .1 2.1 4.9 1.o 2.3 9.9 2.6 1.8 5 .2 1-7 ----
3 .0 1.2 -7.4 6. 4 3.5 5.7 12.6 5.o 3.3 4.5 2.2 5.9 1.2 2.4 9.2 2.9 2.6 5 .2 1-5 ----
89,8
92.9
87.7
85.7
Normal human
Goitrous human
Hog
Sheep
175 68 408 25o 339 248 4 °2 634 299 367 349 187 3o3 65 14o 437 129 -238 Iii 298 45 27
175 75 408 284 345 238 44 ° 634 272 367 349 181 293 60 122 442 129 83 229 i 35 273 31 32
154 63 -334 366 267 454 691 408 429 473 136 326 5° 134 579 lO5 58 231 7° ----
154 58 -313 366 228 432 645 339 383 418 142 393 60 14o 536 117 83 231 62 ----
* V a l u e s for t h e n o r m a l h u m a n m a t e r i a l are b a s e d on t h r e e s e p a r a t e a n a l y s e s ( h y d r o l y s i s t i m e s 20 a n d 4 ° h ), v a l u e s for t h e m a t e r i a l from g o i t r o u s g l a n d s a re b a s e d on four s e p a r a t e a n a l y s e s ( h y d r o l y s i s t i m e s 2o, 4 ° a n d 72 11). The h o g a n d sheep a n a l y s e s a re e a c h t h e a v e r a g e of t w o s e p a r a t e a n a l y s e s ( h y d r o l y s i s t i m e 20 h). I n t h e case of g o i t r o u s m a t e r i a l t h e serine, t h r e o n i n e a n d a m i d e n i t r o g e n are c o r r e c t e d b y e x t r a p o l a t i o n to zero t i m e of h y d r o l y s i s . V a l ue s are c o r r e c t e d for m o i s t u r e c o n t e n t . D e t e r m i n a t i o n of ash on t h e m a t e r i a l from g o i t r o u s g l a n d s s h o w e d none t o be pre s e nt . ** V a l u e s o b t a i n e d from u l t r a v i o l e t a b s o r p t i o n s p e c t r a b y t h e m e t h o d of GOODWlN AND MORTON 19. *** F o r t h e h u m a n s a m p l e s a s e p a r a t e h y d r o l y s i s w a s p e r f o r m e d a t IOO° for 4 h in 6 N HCI. I n t h e case of t h e h u m a n t h y r o g l o b u l i n t h i s w e i g h t r e c o v e r y g a v e q u a n t i t a t i v e r e c o v e r y of t o t a l n i t r o g e n ( d e t e r m i n e d i n d e p e n d e n t l y ) b y E l e c k M i c r o a n a l y t i c a l L a b o r a t o r i e s , Torrance, Calif.
phoresis in starch gels as described b y POULIK33 showed all three electrophoretic components to give a single continuous precipitin line with no spurs. The amino acid and carbohydrate compositions of thyroglobulin from normal human, goitrous human, hog and sheep glands are given in Table I. Paper chromatography of acid hydrolysates of the normal and goitrous human material demonstrated the presence of fucose, galactose, mannose and glucosamine. The amounts of each sugar in the two preparations appeared to be equal as judged b y the intensity of staining with silver nitrate. In the amino acid analyses of samples which had been treated with iodoacetate in 8 M urea but without prior reduction, the amount of Scarboxymethylcysteine detected was such that the number of cysteine residues was less than two per 660 ooo molecular weight. After reduction and S-carboxymethylation approx. 18o residues of S-carboxymethylcysteine were found per mol. wt. of 660000. Biochim. Biophys. Acta, 111 ( 1 9 6 5 ) 2 4 7 - 2 5 7
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P I E R C E e t al.
The iodine contents of "normal" thyroglobulin and of that from one of the goitrous glands were 0.32 % and 0.07 %, respectively. The sedimentation patterns of the native thyroglobulin were as previously reported (e.g. ref. 6). For example, one preparation from goitrous glands consisted of 95 % of a I8.7-S component and 5 % of a 27.I-S component. A preparation from normal glands contained 9 ° % of a I8.8-S component, 5 % of a 27.4-S component and a small amount of poorly resolved material with slow sedimentation rate.
Physical properties of thyroglobulin following reduction and carboxymethylation Investigation of the solubility properties of the reduced and carboxymethylated preparations showed that it could be dissolved in 95 % acetic acid and remained in solution after dialysis against 0.5 M acetate buffer at p H 3.5. Ultracentrifugal examination, however, showed the material to be polydisperse with a number of fastsedimenting components. In 0.05 M sodium glycinate at pH 9.5, the S-carboxymethyl derivative was readily soluble and ultracentrifugal analysis revealed the presence of two peaks of approximately equal area with sedimentation coefficients of 3.4 and 13.5 S as shown in Fig. 2a. After addition of o.oi M sodium dodecyl sulfate to the cell and allowing the solution to stand I h at room temp., a nearly complete conversion to what appears to be a single species with an s20, w of about 3.0 S was found (Fig. 2b). When the total sodium dodecyl sulfate added was increased to O.Ol7 M complete dissociation to the 3-S component occurred and the material sedimented with a single symmetrical boundary as shown in Fig. 2c. The concentration dependence of the
F i g . 2. S e d i m e n t a t i o n p a t t e r n s of r e d u c e d S - c a r b o x y m e t h y l a t e d h u m a n t h y r o g l o b u l i n . All r u n s a r e in 0.05 M g l y c i n e ( p H 9.6). a, n o s o d i u m d o d e c y l s u l f a t e , p r o t e i n c o n c e n t r a t i o n = 0.57 % , s20. w = 3.4, 13-3 S ; b, o . o i M s o d i u m d o d e c y l s u l f a t e , p r o t e i n c o n c e n t r a t i o n = 0.8o % , s20. w ~ 3.0, 6.8 S; c, O.Ol 7 M s o d i u m d o d e c y l s u l f a t e , p r o t e i n c o n c e n t r a t i o n = 0 . 5 8 % , s~o, w ~ 2.8 S. T i m e i n t e r v a l for p h o t o g r a p h y ; a, 8 r a i n (4 r a i n l a s t t w o e x p o s u r e s ) ; b, 8 m i n ; c, 16 m i n .
Biochim. Biophys. Acta, I I i (1965) 2 4 7 - 2 5 7
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sedimentation coefficients of the S-carboxymethyl derivatives of two preparations of thyroglobulin (from goitrous glands) in the presence of O.Ol7 M sodium dodecyl sulfate are shown in Fig. 3a, t h a t of their diffusion coefficients in Fig. 3b. In these experiments
f i
0
i
1
i
2
i
3
I
4
i
5
[
6
I
7
I
I
I
i
8 9 1 2 Protein conch. (mg/rnD
I
3
i
4
i
5
i
6
i
7
I
8
i
9
f
Fig. 3. T h e d e p e n d e n c e of s e d i m e n t a t i o n velocity a n d diffusion on p r o t e i n c o n c e n t r a t i o n . Circles a n d s q u a r e s r e p r e s e n t two different p r e p a r a t i o n s of t h e r e d u c e d a n d c a r b o x y m e t h y l a t e d d e r i v a t i v e of t h y r o g l o b u l i n . All m e a s u r e m e n t s were carried o u t in o.o36 M glycine (pH 9.6), a n d in t h e presence of O.Ol 7 M s o d i u m dodecyl sulfate. Left, a; right, b.
the solutions with the highest protein concentration were diluted with O.Ol7 M sodium dodecyl sulfate in the p H 9.5 buffer for the determinations at the lower protein concentrations. Ratios of protein-sodium dodecyl sulfate complex to unbound sodium dodecyl sulfate in the solvent varied from 3.5 : I to 0.6 : I. This variation apparently did not affect the dissociation of the protein as a single symmetrical boundary was formed at all concentrations of protein and the values for each preparation fitted closely a straight line. In another experiment, protein to give a concentration at the lower level studied (2 mg/ml) was dissolved directly in the O.Ol7 M sodium dodecyl sulfate buffer solution and the s20, w was determined within 2 h. Again a single symmetrical boundary formed and the value of s20, w was in agreement with the data in Fig. 3a. This experiment appears to eliminate the possibility that hydrolysis of peptide or glycoside bonds occurred during experiments of longer duration. In the method described b y SCHACHMAN29 for determination of bound sodium dodecyl sulfate, the number of interference fringes contributed by the total amount of sodium dodecyl sulfate and the number of fringes attributed to bound sodium dodecyl sulfate sedimenting with the protein are measured. These data are given in Table I I for one of the preparations from a goitrous gland and show that the amount of sodium dodecyl sulfate bound in the hydrodynamic particles is 30 % by weight of the complex. A value of o.719 was calculated for the partial specific volume based on amino acid and carbohydrate composition. With the value of 0.885 for sodium dodecyl sulfate and correction made for the binding of sodium dodecyl sulfate, the partial specific volume of the complex is 0.769 . This together with the values of the sedimentation coefficients and diffusion coefficients of the glycoprotein-sodium dodecyl sulfate complex extrapolated to zero concentration gave molecular weights of Biochim. Biophys. Acta, i i i (1965) 247-257
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J.G. PIERCE 6t al.
TABLE II T H E B I N D I N G OF SODIUM D O D E C Y L S U L F A T E B Y S - C A R B O X Y M E T H Y L A T E D H U M A N T H Y R O G L O B U L I N *
Material
Method
Concn. in fringes
Conch. (mg/ml)
Rev./min
Thyroglobulin Sodium dodecyl sulfate Thyroglobulin plus sodium dodecyl sulfate Thyroglobulin plus sodium dodecyl sulfate Thyroglobulin plus b o u n d sodium dodecyl sulfate
Synthetic b o u n d a r y cell Synthetic b o u n d a r y cell Calculated
23.66 12.oo 35.66
5.8 4.5
6569 6569
Synthetic b o u n d a r y cell
36.oo
Double sector cell
29.35"*
6569 42 o4o
* All m e a s u r e m e n t s were carried o u t ill a Spinco Model E analytical ultracentrifuge, equipped w i t h interference optics. ** Therefore 6.65 of the 12 available sodium dodecyl sulfate fringes sedimented with the protein or 55 % of the sodium dodecyl sulfate was bound. We therefore assume t h a t °.55 × 4.5 X IOO 5.8 + (0.55 x 4-5) or 3° % of the weight of the sedimenting species is due to b o u n d sodium dodecyl sulfate.
113 ooo and 123ooo for the two preparations (corrected for bound sodium dodecyl sulfate) *. There have been few reports concerning the determination of the molecular weights of proteins by measurement of sedimentation and diffusion in the presence of sodium dodecyl sulfate, fl-Galactosidase has been studied by WALLENFELS gt al. 31 and HOFFMAN AND HARRISONa2 have reported values for the molecular weights of the subunits of apoferritin based on physical measurements in the presence of sodium dodecyl sulfate. In the latter case the data agreed well with data obtained by other methods. Following completion of the observations on thyroglobulin given herein, determinations of the molecular weight of a well characterized protein derivative, S-carboxymethyl ribonuclease, were carried out. Sedimentation, diffusion and determination of bound sodium dodecyl sulfate were done as described above. Extrapolation of the data to zero protein concentration together with correction for binding of sodium dodecyl sulfate (30 %) gave a value of 14400 for the molecular weight of the S-carboxymethyl ribonuclease (in good agreement with the actual value of 1416o). In these experiments a ratio of protein-sodium dodecyl sulfate complex to unbound sodium dodecyl sulfate of 0.8 : I was needed to give values of s20, w and D which yielded the correct molecular weight. When native thyroglobulin from goitrous glands was subjected to treatment with O.Ol7 M sodium dodecyl sulfate at p H 9.5, 9 ° % of the material was converted to * A f u r t h e r dissociation was suggested on two occasions w h e n a p r e p a r a t i o n of the S-carboxym e t h y l derivative gave sedimentation and diffusion d a t a (s~o,w = 2.2; D = 2.76 ) indicating a mol. wt. of a b o u t 5 ° ooo-6o ooo b u t these d a t a could n o t be obtained consistently. The d a t a leading to the values of 113 ooo-123 ooo were obtained with two different p r e p a r a t i o n s of the S-carboxym e t h y l derivative of " g o i t r o u s " thyroglobulin and similar values were obtained from two preparations of derivative from the thyroglobulin of n o r m a l glands. I t should also be noted t h a t storage at --2o ° for 3 m o n t h s of a S - c a r b o x y m e t h y l derivative of " n o r m a l " thyroglobulin led to deterioration in t h a t the material no longer yielded a single peak in the ultracentrifuge as it had s h o r t l y after its preparation.
Biochim, Biophys. Acta, I I i (1965) 247-257
PROPERTIES OF HUMAN THYROGLOBULIN
255
a species with an s20, w of 5.3 (protein concentration, o.5 %). This result is consistent with previous findings ~. Due to the presence of lO% faster sedimenting material, however, molecular weights could not be determined with any c e r t i n t y by measurements of sedimentation and diffusion. DISCUSSION
The present investigation shows that the thyroglobulin isolated from goitrous glands is identical by several criteria to the protein from normal glands even though the amount synthesized is many times greater. The amino acid compositions of the two types of human thyroglobulins are identical within experimental error but differ somewhat from those of the two other species studied, particularly in their contents of arginine, proline, alanine and leucine and half cystine. The latter appears to be all in disulfide form and the molecule therefore must be extensively cross linked. It is noteworthy that the amount of carbohydrate in the thyroglobulin from goitrous glands is equal to that found in normal thyroglobulin. This finding suggests that the biosynthesis or linking of the carbohydrate moieties is governed by the amount of the protein component present. The amounts of neutral sugar in these experiments are somewhat higher than those recently reported for normal human material by SPIRO AND SPIRO33. As expected the iodine content of the normal material is the greater. Similar electrophoretic patterns in starch gel were found for all preparations with observation of the three-banded patterns attributed by SPIRO14 to the presence of 6-S and 27-S components in addition to the I9-S material. The 6-S and 27-S fractions are diffficult to remove from the I9-S component and their amounts are increased by dialysis and freeze drying 14. The results of the immunoelectrophoresis in starch gel show these three components to be immunologically identical. This provides evidence that most, if not all, the 27-S component of the preparations is an aggregated form of thyroglobulin rather than another species of protein, a conclusion further supported by the sedimentation patterns of the S-carboxymethyl derivative in o.o17 M sodium dodecyl sulfate (see Fig. 2c) where only a single peak was observed. The peptide mapping studies of BOUCHILLOUXet al. ~° provide additional evidence for this conclusion. The number of subunits or peptide c h i n s constituting thyroglobulin is of great importance if detailed structural studies are to be undertaken. Thyroglobulin has been defined as the iodine-contining, saline-extractable protein component of the thyroid gland with a sedimentation coeËficient of 19 S. The molecular weight of the native protein from several species has been estimated to be between 660000 and 690000. Some heterogeneity has been found in I9-S material by chromatographic studies but is presumably due to the existence of a family of proteins which vary only in their iodine and sialic acid content 1°, 34,35. While careful studies of its physical parameters, notably by EDELHOCH and colleagues, have shown that the molecule undergoes complex conformational changes as well as dissociation and aggregation phenomena, the number of peptide c h i n s constituting the molecule is not known. Of particular interest have been studies in the presence of sodium dodecyl sulfate 7 where dissociation readily occurs yielding two components whose sedimentation coefficients have values of approx. 3 and 4 S when extrapolated to zero ionic strength. The persistence of two Biochim. Biophys. Acta, I i i (1965)247-257
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J.G. PIERCE et al.
components when native thyroglobulin has been placed in sodium dodecyl sulfate and in several other systems which promote dissociation has hitherto prevented estimation of molecular weight of subunits from centrifugal data 5. Polarization of fluorescence of derivatives of thyroglobulin m also suggests subunits of relatively low molecular weight but interpretation of the data in terms of molecular size alone is complicated b y probable losses in internal rigidity of the molecule. Results of end-group determinations have been variable and also have not given clear information about the number of peptide chains. Difficulties have arisen because of contamination of thyroglobulin preparations with proteolytic enzymes of the thyroid 37. The most recent results (with hog thyroglobulin) 37 have indicated the presence of 2 moles of aspartic acid or asparagine and one mole of glycine per molecular weight of 660 ooo*. Thus in the present investigation it was encouraging to find that the S-carboxymethyl derivatives of both the "goitrous" and "normal" human thyroglobulins were completely soluble in sodium glycinate at p H 9.6 and that the addition of O.Ol7 M sodium dodecyl sulfate caused dissociation to a species which appears homogeneous in the ultracentrifuge. This result enabled measurement of physical parameters with most consistent data obtained with two samples of the carboxymethyl derivative prepared from goitrous material. These two preparations showed sedimentation coefficients which gave a straight line plot over a 4-fold range of protein concentration and extrapolated values for s°20,w of 3.42 and 3.12 S. Values for several samples of the S-carboxymethyl derivative from the normal human thyroglobulin were all within the range of those shown in Fig. 3 although more scatter of points was present, perhaps due in this instance to deterioration of the derivative during storage. I t should also be noted that with the Scarboxymethyl derivative the slope of the plot of sedimentation constant against protein concentration is far less than that reported by EDELI-IOCHAND LIPPOLDT2 for the slower moving of two components seen when native calf thyroglobulin is placed in sodium dodecyl sulfate. The change in slope is most likely due to creation of a more flexible molecule by reduction of the disulfide bridges. The calculated molecular weight of the S-carboxymethyl derivative in the presence of O.Ol7 M sodium dodecyl sulfate is in the range 110000-125000. The determination of molecular weight in the presence of sodium dodecyl sulfate requires a correction for the amount of bound sodium dodecyl sulfate both in the buoyancy factor (partial specific volume) and the determination of the percent by weight of the hydrodynamic particle which is the glycoprotein. The molecular weight values thus m a y be subiect to greater experimental error than if it had been possible to obtain the subunits in the absence of sodium dodecyl sulfate. The most reasonable interpretation of our data, thus far, is that human thyroglobulin after reduction of its disulfide bonds can dissociate to 5 or 6 physically identical subunits. That the observed subunits m a y not represent the total number of peptide chains in thyroglobulin is suggested by the fact that on two occasions the observed values of the sedimentation and diffusion coefficients of material giving symmetrical peaks in the ultracentrifuge indicated a further dissociation. This possibility was also suggested b y the data of STEINER AND EDELHOCH~ concerning the polarization of fluorescence of derivatives of * Preliminary end group determinations carried out in this laboratory by the method of STARK AND S M Y T H 38 gave values of approx. 2 moles of aspartic acid or asparagine per 660000 tool. wt. together with lesser amounts of several other amino acids for the human preparations from both normal and goitrous glands. Biochim. Biophys. Acta, iii (1965) 247-257
PROPERTIES OF HUMAN THYROGLOBULIN
257
thyroglobulin and further work is required to finally identify the number and types of peptide chains in thyroglobulin. ACKNOWLEDGEMENTS
This investigation was supported in part by a U.S. Public Health Service grant C-229o by Cancer Research Funds of the University of California and by the National Health and Medical Research Council, Canberra. The authors express their appreciation for stimulating advice from Dr. A. N. GLAZER and for the capable assistance of Miss T. REIMO. The authors are indebted to Dr. A. LORBER,Wadsworth Veterans Administration Hospital, West Los Angeles, Calif., for the amperometric analyses.
REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 i8 19 2o 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38
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