Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin.

The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.     

Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin.

The structure of cleaved thyroxine-binding globulin (TBG) has been modelled on the crystal structure of cleaved alpha 1-antitrypsin (a member of the serine proteinase inhibitor, serpin, superfamily) based on the high sequence homology exhibited by the two proteins. Particular attention was paid to the identification and modelled characteristics of the thyroxine binding site. The primary aim of the study was to compare the site qualitatively with the crystallographically determined binding site of transthyretin, the other major transporter of thyroxine, in an attempt to explain the higher binding affinity of the site compared with the known thyroxine binding site in transthyretin (10(10) versus 10(8) M-1). The proposed binding site shares some similar characteristics with the transthyretin binding site but also includes a cluster of aromatic residues which are entirely absent in transthyretin. It is proposed that this might account for the substantial difference in binding affinities.     

Effect of long-chain fatty acids on the binding of thyroxine and triiodothyronine to human thyroxine-binding globulin

The effect of long-chain fatty acids on the binding of thyroxine to highly purified human thyroxine-binding globulin has been studied by equilibrium dialysis performed at pH 7.4 and 37 degrees C. At a fixed molar ratio of 2000:1 of fatty acid to thyroxine-binding globulin, the degree of binding inhibition based on the percent change in nK value relative to the control as determined from Scatchard plots was: palmitic, 0%; stearic, 0%; oleic, 76%; linoleic, 69%; and linolenic, 61%. At a 500:1 molar ratio of oleic acid to thyroxine-binding globulin, equivalent to 0.125 mM free fatty acid in serum, thyroxine binding was inhibited by 18%, increasing to 93% at a 4500:1 molar ratio. At molar ratios of oleic acid to thyroxine-binding globulin of 1000:1, 2000:1 and 4000:1, the degree of inhibition of triiodothyronine binding was 24%, 41% and 76%, respectively. The results indicate that the unsaturated long-chain fatty acids are potent inhibitors of thyroxine binding to thyroxine-binding globulin, whereas the saturated fatty acids have little or no effect on thyroxine binding.     

Study of the binding of thyroxine by thyroxine-binding prealbumin by a dialysis method using a fixed free concentration of ligand (author's transl)]

In order to study ligand-protein binding in solution, a dialysis method was used in which the free concentration of ligand can be controlled. The method has certain advantages and was applied to the binding of thyroxine by thyroxine-binding prealbumin, a system about which the results found in the literature are not in good agreement. From the isotherm drawn according to the Scatchard plot, it was found that thyroxine-binding prealbumin only presents a single binding site for thyroxine per molecule, the association constant being 1.7 . 10(8) M-1.     

Binding activities of thyroxine binding globulin versus thyroxine binding prealbumin in rat sera: differential modulation by thyroid hormone ligands, oleic acid and pharmacological drugs

We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.     

Vitamin A and thyroxine carrier proteins in chicken plasma: steady-state control of the plasma level of free retinol-binding protein and free thyroxine.

1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occurring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. The thyroxine-binding proteins were found to be of two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz., prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.     

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken.

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and that prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool. Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the 'buffering' action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintenance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.     

A spin label study of the thyroid hormone-binding sites in human plasma thyroxine transport proteins

The binding site topographies of the three thyroid hormone-transporting proteins in human serum--prealbumin, thyroxine binding globulin, human serum albumin--have been studied with the aid of five spin-labeled analogs of L-thyroxine in which the distance between the phenolic hydroxyl and the nitroxide nitrogen ranged from 17 to 23 A. In the presence of prealbumin, the electron spin resonance spectrum of 3-([alpha-carboxy-4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenethyl]-carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolinl-yloxy-ethyl ester revealed the presence of a highly immobilized spin label. As the chain length between the thyroxyl moiety and the pyrroline ring was increased, the mobility of the nitroxide group in the prealbumin-bound labels increased. If the spin labels bind in an extended conformation, the thyroxine-binding site was estimated to be approximately 21 A in depth. This finding is consistent with the known crystal structure of prealbumin and suggests that the solution and crystal conformations of the protein are very similar. In contrast to prealbumin, the thyroxine-binding site on thyroxine-binding globulin was found to be more open and possibly deeper. Human serum albumin has two binding sites for thyroxine, one of which has a higher affinity and is deep enough to accommodate a molecule that is 23 A in length. The lower affinity site is somewhat shallower and probably wider, as thyroxine spin labels bound to this site exhibited greater mobility.     

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and the prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool.Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the ‘buffering’ action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintainance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.     

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.     

Binding of thyroid hormones and their analogues to thyroxine-binding globulin in human serum.

The present study was undertaken to study the binding of several thyroid hormones and structurally related compounds to human serum thyroxine-binding alpha-globulin (TBG). The source of TBG was normal human serum diluted 1:100 in 0.035 M barbital buffer, pH 7.4. In the binding assays, 125I-thyroxine, unlabeled thyroxine, and diluted serum were incubated for 20 h at 37 degrees in Plexiglas equilibrium dialysis units. Two orders of binding sites were discerned: a high affinity, low capacity binding site with an affinity constant of approximately 2.5 X 10(9) M-1, and a low affinity, very high capacity binding site with an affinity constant of less than 10(6) M-1. Studies with purified TBG, serum deficient in TBG, and purified human serum albumin indicated that the high affinity site represented binding to TBG and the low affinity site represented binging to albumin. The ability of several groups of thyroid hormone analogues to bind to TBG was then investigated. As a result of these studies, the following structural features of thyroid hormones were found to be important for optimal binding activity: (a) the L-alanine side chain conformation, (b) the presence of a 4'-hydroxyl group, (c) the presence of two substituents in the inner and outer rings (positions 3, 5, 3', and 5'), and (d) the presence of either bromines or iodines in the inner ring and iodines in the outer ring. Of lesser importance was the presence of an oxygen atom in the ether position.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr(342) of the reactive loop and Tyr(241) of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.     

Binding characteristics of a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine, to bovine serum albumin as measured by fluorescence

The interaction between a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine and bovine serum albumin was investigated by fluorescence measurements. The apparent binding constants were obtained at various pHs assuming the equivalence and independence of the interaction sites on the protein from the fluorescence titration curves. The maximum binding was attained at pH 8.0, and the apparent binding constant was (5.28 +/- 0.13).10(5) M-1 with one binding site per albumin molecule. Thermodynamic parameters were also determined from the van't Hoff plot of the apparent binding constants at pH 7.5. The free energy change, enthalpy change and entropy change were -7.70 +/- 0.09 kcal.mol-1, -4.59 kcal.mol-1 and 10.2 e.u., respectively.     

Reduced serum free thyroxine concentration in postmenopausal women receiving oestrogen treatment.

Thyroid hormone state was assessed in a group of postmenopausal women who had received long term treatment with oestrogen. Serum concentrations of total thyroxine, triiodothyronine, and thyroxine binding globulin were raised compared with those in a control group given placebo; serum concentrations of thyroid stimulating hormone did not differ between the groups. Oestrogen treatment resulted in a significant decrease in the serum free thyroxine concentration and in the ratio of thyroxine to thyroxine binding globulin, which supports the view that oestrogen is the causative factor of the physiological reduction in free thyroid hormone during pregnancy.     

Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site

Ligand binding characteristics of rat liver microsomal type I iodothyronine deiodinase were evaluated by measuring dose-response inhibition and apparent Michaelis-Menten or inhibitor constants of iodothyronine analogues to compete as substrates or inhibitors for the natural substrate L-thyroxine. These data show strong correlations with the binding requirements of hormone analogues to serum thyroxine-binding prealbumin since iodothyronine analogues with a negatively charged side chain, a negative charge or hydrogen bonding function in the 4'-position, tetraiodo ring substitution, and a skewed hormone conformation are structural features shared in common which markedly affect enzyme activity and protein binding affinity. 3,3',5'-Triiodo-L-thyronine is the most potent natural substrate (IC50 = 0.3 microM) and tetraiodothyroacetic acid is the most potent inhibitor (IC50 = 0.2 microM). Both thyroxine (T4)-5'- and T4-5-deiodination pathways are inhibited by these potent analogues, providing further evidence for a single enzyme catalyzing the rat liver microsomal deiodination reactions. These data also show that L-hormone analogues are preferentially deiodinated via the T4-5'-deiodination pathway, whereas D-analogues produce products via the T4-5-deiodination pathway. The thyroxine-binding prealbumin complex was used to model the interaction of thyroid hormones with the deiodinase active site. Computer graphic modeling of the prealbumin complex showed that only those analogues which are potent deiodinase inhibitors or substrates can be accommodated in the hormone binding site. This model suggests the design of functionally specific ligands which can modulate peripheral thyroid hormone metabolism and act as antithyroidal drugs.     

Rapid simultaneous radioimmunoassay for triiodothyronine and thyroxine in unextracted serum

A three hour radioimmunoassay for the simultaneous measurement of triiodothyronine and thyroxine in 25λ of unextracted serum has been developed. 8-anilino-1-napthalene sulfonic acid has been used to inhibit binding of the two hormones to thyroxine binding globulin. Comparisons of thyroxine with those obtained by competitive protein binding assay and triiodothyronine with those determined by our previously developed radioimmunoassay afford excellent agreement. The speed, accuracy, sensitivity and specificity of this assay renders it highly attractive for routine clinical determinations and for research applications as well.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.