Binding of 3,5-diiodotyrosine to serum proteins

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Clinica Chimica Acta, 69 (1976) 417-422 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

CCA 7756

BINDING

OF 3,5-DIIODOTYROSINE

JANINE BISMUTH, MATHILDE

CASTAY

TO SERUM PROTEINS

and SERGE LISSITZKY

*

Laboratoire de Biochimie Me’dicale et U 38 de l’lnstitut National de la Sante’ et de la Recherche Mkdicale, Facultk de Mkdecine, 27 Bd. Jean-Moulin, 13385 Marseille Ckdex 4 (France) (Received

January 15, 1976)

summary The reversible binding of 3,5diiodotyrosine (DIT) to human and bovine serum protein and to.purified human serum prealbumin and human and bovine albumin has been studied by equilibrium dialysis. Maximum binding occurred at pH 8.6-9.0. Human serum bound DIT less than did bovine serum. Adult ox and fetal calf sera showed similar binding. The main DIT-binding protein of human serum was prealbumin. It showed a single affinity site with a K, of 0.85 X lo6 M-’ at pH 8.6 and 0.40 X lo6M-’ at pH 7.4. The affinity constant of serum albumin for DIT was 2.8 X lo3M-’ at pH 8.6. The elevated binding of DIT to bovine serum is essentially .due to albumin whose affinity constant for DIT is 16-times higher than that of human serum albumin. Fetuin was not responsible for any noticeable DIT binding in fetal calf serum.

Introduction There are conflicting results on the binding of the iodolyrosines (3-iodotyrosine and 3,5-diiodotyrosine) to serum proteins (see ref. 1 for a review). In blood from mammals, DIT might be bound to albumin, prealbumin or a fastmoving y-globulin fraction but not to thyroxine-binding globulin. Using Sephadex gel filtration or adsorption on charcoal to separate free from proteinbound DIT, we have previously demonstrated that human serum was able to bind DIT [2]. In our experiments, and in contrast to other studies [3], attempts to characterize the DIT-binding protein(s) by electrophoresis on several supports (paper, cellulose acetate, polyacrylamide-agarose gels) have failed. In this paper, we describe studies concerned with the binding of DIT to serum proteins, albumin and prealbumin using equilibrium dialysis. * To

whom

to address

correspondence.

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Material and methods Human prealbumin was obtained from Behringwerke (Marburg-Lahn, G.F.R.) and used without further purification. It gave a single band in agar and polyacrylamide gel electrophoresis. Human serum was a pool of sera with normal PBI and adult ox serum was obtained from blood collected at the slaughter house. Human and bovine serum albumin (twice crystallized) were obtained from Sigma (St. Louis, MO., U.S.A.), bovine fetuin from Gibco (Grand Island, N.Y., U.S.A.) or Calbiochem (Lucerne, Switzerland) and calf embryonic serum from Sorga (Paris, France). Stable DIT (3,5-diiodo-L-tyrosine * 2H20) was obtained from Calbiochem (Los Angeles, Calif., U.S.A.). ‘251-labeled DIT and MIT were purchased from the C.E.A. (Saclay, France). Their specific activities were from 42 to 50 mCi/mg. They were purified by Sephadex filtration in 0.2 M ammonium acetate pH 5.8 according to Bismuth et al. [Z]. The purified preparations contained less than 2% iodide which was the only cont~in~t. Equilibrium dialysis was carried out in stoppered, 19-ml, round-bottomed flasks and S/32 visking dialysis tubing. Before use, the tubes were soaked for 24 h in 0.1 N HN03, for 48 h in 0.01 N HN03 and then thoroughly washed in quartz twice-distilled water [4]. The dialysis bags, filled with 1.5 ml of protein solution (diluted human serum or protein in appropriate buffers) were introduced in the flasks containing 10 ml buffer enriched with about 15 000 cpm/ml [‘251]DIT, i.e. 4 ng/ml, and increasing content of stable DIT. The flasks were stoppered and shaken in a Gallenkamp shaker for 16-17 h at 37°C. Preliminary experiments showed that complete equilibrium was achieved under these conditions. The buffers used were: 50 mM Tris/HCl pH 7.4 or 8.6 (or 50 mM ammonium acetate pH 5.8) containing 100 mM NaCl and 1 mM Na2-EDTA. After dialysis l-ml aliquots were taken from both the inside and the outside of the dialysis bag and were assayed for radioactivity in a Packard Auto-Gamma spectrometer. The fraction of DIT bound was obtained as follows: Concentration outside)

of bound

DIT = R X (cpm of 1 ml inside) - R X (cpm in 1 ml

where R is the ratio of total moles of DIT added/total cpm. The average number of DIT molecules bound per prealbumin or albumin molecule, r, was then obtained by dividing the concentration of bound DIT by the protein concentration. For these calculations, molecular weights of 50 000 and 66 000 were taken for prealbumin and albumin, respectively. The data were analyzed by means of the equation of Scatchard [5] : r/c=K,-I&.

where c is the molar concentration of free DIT, K the average apparent association constant for each binding site and rz the average maximal number of sites with association constant K. This analysis gives the values for the y-intercept (& ) and the slope of the line (-K). No significant DIT loss due to binding to the dialysis bag or to the walls of the flasks was observed. Possible deiodination occurring during incubation was controlled by paper chromato~aphy in ~-bu~ol/acetic acid/wa~r (78 : 5 : 17, v/v) followed by determination of radioactivity distribution along the chro-

419

matogram on l-cm wide sections. Deiodination was either nil.or never higher than 1%; appropriate corrections were made, assuming that there was no appreciable iodide binding to proteins. Results 1. Diiodotyrosine binding to serum proteins Fig. 1 shows that maximum binding to human total serum proteins, serum prealbumin or serum albumin occurred at pH 9. No binding was observed at pH 11 for all proteins and at pH 6.0 for prealbumin. A very small amount of DIT was still bound to total serum protein and albumin at pH 4.0 to 6.0. Binding to albumin and prealbumin was additive but when the dialysis bags contained an amount of both proteins roughly corresponding to that present in total serum only about 56% of the DIT binding to the latter was found (Fig. 1). This suggests that serum proteins other than albumin and prealbumin are able to bind DIT. 2. Diidotyrosine binding to prealbumin and albumin At pH 5.80 in 0.2 M ammonium acetate buffer, no binding of DIT to prealbumin was observed, for DIT concentrations up to 100 pg/ml. At pH 7.4 and 8.6 in 50 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, the Scatchard plots of the data showed a straight line with an intercept of the r axis close to 1 indicating that at both pH values, human prealbumin contains a single binding site for DIT (Fig. 2). From the slope of the curve the association constants were computed as 0.85 X lo6 M-’ at pH 8.6 and 0.40 X lo6 M-’ at pH 7.4.

0.5 PH

r

1

diluted serum (1 : 10); Fig. 1. pH dependence of kl* 5 I] DIT binding to human serum proteins. 0 -3, serum albumin (5 mg/ml). O-0. prealbumin (0.05 .-, prealbumin (0.05 mg/ml). A-A, mg/ml) and serum albumin (5 mg/ml). In the conditions described in Methods, [ l2 5 11 DIT was added at concentration of 15 000 cpm (or 0.4 ng/ml)/ml. Fig. 2. Scatchard plots of DIT binding to prealbumin at PH 7.4 (a) and 8.6 (0). Equilibrium dialysis was performed in 50 mM Tris/HCl. 100 mM NaCl and 1 mM Naz-EDTA at 37’C. Protein concentration, 0.5 mg/ml.

420

0.5

0.1

r

Fig. 3. Scatchard plots of DIT binding to bovine serum albumin at pH 7.4 (C) and 8.6 (0). Equilibrium dialysis was performed in 50 mM TrisfHCl, 100 mM N&l and 1 mM Na2-EDTA at 37°C. Protein concentration, 4 n&ml for experiments performed at pH 7.4: 2 or 4 mg/ml for those performed at pH 8.6.

No binding of MIT at pH 7.4 or 8.6 was observed. In contrast to the data obtained with human pre~bumin, the relation between r/c and r for bovine albumin was curvilinear (Fig. 3). One site with an affinity constant of 0.45 X 10’ M-’ was observed at pH 8.6 and 1.7 X lo4 M-l at pH 7.4. Surprisingly, a fraction of a site (0.05) was also found with K = 1.3 X lo7 M-’ at pH 8.6 and 3 X lo6 M-’ at pH j.4.

0.25

0.50

r Fig. 4. DIT binding by bovine serum (o), embryonic or adult serum (0) at 1 : 10 dilution. Seatchard plots were computed assuming that only serum albumin binds DIT. r values were calculated on the basis of serum albumin contents of 3.3 mg/ml (0) and 2.2 mg/ml (0). Equilibrium dialysis was performed in 50 mM Txis/HCl pH 8.6, 100 mM NaCl, 1 mM Na2-EDTA. Concentrations of serum albumin were determined by planimetry of densitometer tracings obtained after electrophoresis on cellogel of diluted sera and staining with Ponceau red.

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Scatchard plots of DIT binding to human serum albumin at pH 8.6 also showed one binding site with K = 2.8 X lo3 M-’ and 0.01 site with K = 5.6 X 10’ M-‘. This was done using concentrations of human serum albumin of 4 mg/ml. No significant results were obtained at pH 7.4 even using a protein concentration of 20 mg/ml. Comparison of DIT binding to adult or fetal bovine serum at pH 8.6 is shown in Fig. 4. 3. Binding of DIT to fetuin It was recently reported [6] that fetuin in ovine and bovine fetal sera was able to bind thyroid hormones with a high affinity. We have studied this protein for DIT binding by equilibrium dialysis. At pH 8.6 and at concentrations of protein of 0.5 and 4 mg/ml no significant binding was observed, whatever the DIT concentration. Discussion Binding of DIT to serum protein as studied by equilibrium dialysis is pH dependent and maximum at pH 8.6. Since zone electrophoresis was unable to permit the characterization of the DIT-binding proteins, we have chosen to study those serum proteins known to bind thyroid hormones for obvious structural analogies between the latter and iodotyrosines. Serum thyroxine-binding globulin having no affinity for DIT [7], we have investigated the DIT-binding properties of the commercially available human prealbumin. This protein is formed of 4 identical subunits, but possesses only 1 site of high affinity for T4 [ 8-111 and low-affinity sites whose number differs (3 or 8) according to the methods used for the determination [9-lo]. Reported K values for the T4 high-affinity site measured at pH 7.4 varied from 1.1 X 10’ to 1.3 X lo8 M-l. Similar variations were observed for the single high-affinity site for T3 : 2.6 X 10’ to 1.2 X 10’ M-l. In addition, human prealbumin seems to contain 2 binding sites for iodotyrosine analogs [ll]. Using equilibrium dialysis, we have found 1 site for T, and 2 for T3. However, with the same batch of prealbumin, we detected only one site for DIT, with an affinity constant of 0.4 X lo6 M-’ at pH 7.4. Anomalies in the thyroid hormonebinding properties of prealbumin have been already reported suggesting heterogeneity of this protein [lo]. Genetic polymorphism of the rhesus monkey prealbumin was also found [ 121. Binding of DIT to human serum albumin was not measurable at pH 7.4. At pH 8.6 one high-capacity site with an affinity constant of 2.8 X 10e3 M was detected. Thus, it is likely that both prealbumin and albumin are involved in the serum binding of DIT. However, equilibrium dialysis using total serum showed that these two proteins did not account for total DIT binding (see Fig. l), suggesting that other serum proteins bound DIT. Since prealbumin showed a rather high affinity for DIT and since fetal calf serum did not contain prealbumin, we hoped to find a decreased binding of DIT to this serum. In fact, the affinity of fetal calf serum for DIT is higher than that of adult human serum. This is not explained by the presence of fetuin in fetal calf serum since we were unable to show noticeable DIT binding to purified calf fetuin from

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two commercial sources. This is in contrast with the results of Fisher and Lam [6] who showed by competition experiments that on a molar ratio, calf fetuin bound DIT only 140-times less strongly than T,. A binding constant of calf fetuin for T4 of 1.2 X 10’ M-’ was found by these investigators. Additional evidence allowed fetuin to be excluded in accounting for the higher capacity of calf serum to bind DIT. Fetal calf and adult bovine sera at the same protein concentration showed nearly identical DIT binding as shown by equilibrium dialysis. From the experiments reported in this paper, bovine serum albumin was shown to exhibit at pH 8.6 an affinity constant for DIT 16-times higher than that of the human protein. Such a difference in the behavior of bovine and human serum albumin was not found for T4 binding [13,14]. Radioimmunological estimations of serum DIT gave a mean value of 156 ng/lOO ml in normal humans [ 151, close to that of serum T3 (120 to I50 ng/ 100 ml). The presence of active iodotyrosine dehalogenase in the body tissues, the absence of binding of DIT to thyroxine-binding globulin and the much lower affinity constants of serum prealbumin and albumin for DIT explain its short biological half-life (1 to 1.5 h) as compared to T3 (half-life of about 1 day). In contrast to T3 and Tq, labeled iodotyrosines were not detected in the venous thyroid effluent of unstimulated normal rats prelabeled in vivo with radioiodide [ 161. Small amounts were detectable only when the gland was strongly stimulated by thyrotropin. It is therefore unlikely that thyroid secretion of DIT in amounts much higher than those of T3 accounts for blood DIT levels. Such considerations suggest an extrathyroid~ source for circulating DIT. Some possibilities are: (1) formation of DIT as an intermediate in thyroid hormone catabolism; this is highly improbable with respect to the metabolic stability of the diphenyl ether linkage of thyroid hormones [17,18]; (2) synthesis of DIT from iodide by non-thyroid tissues; (3) dietary origin. Additional studies are necessary to test whether blood DIT originates from the latter two possible sources. References 1 2 3 4 5 6 7 8 9 10 11 12

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