Journal of Protein Chemistry, VoL 9, No. 4, 1990
Thyroxine Binding to Human Serum Albumin Immobilized on Sepharose and Effects of Nonprotein Albumin-Binding Plasma Constituents Keita Kamikubo, 1 S h i g e k i Sakata, 1'2 Shigenori N a k a m u r a , 1 Takashi Komaki, ~ and Kiyoshi M i u r a t
Received March 24 1990
125I-thyroxine (125I-T4) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied, t25I-T4 binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19 ± 0.53 x 10 -6 M and 2.69 ± 0.78 x 10 5 M, respectively. The number of binding sites of the high and the low affinity sites were 1.28 + 0.46 mol/mol and 23.5 ± 9.7 tool/tool of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of 125I-T4to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-l-naphthalene sulfonic acid (ANS) inhibited the high affinity T4 binding via reduction of the binding capacity. Unlabeled T4 showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity ~['4binding to HSA-Sepharose. KEY WORDS: Thyroxine; albumin; fatty acid; bilirubin; ANS; steroid.
1. I N T R O D U C T I O N 3
functions in subjects suspected to have thyroid disorders include bound and free hormones, it is important to examine the effects of various ligands on thyroid hormone binding to serum albumin. H u m a n serum albumin (HSA) covalently immobilized on agarose microparticle (Sepharose) has been reported to retain most of its ligand-binding characteristics (Stevenson et al., 1974; Lagercrantz et al., 1979, 1981). Such HSA preparations have been widely used for the study of HSA-ligand interaction, because they provide a simple method for the separation of bound and free ligands (Stevenson et al., 1974; Lagercrantz et al., 1979, 1981). In the present investigation, the physiological nature of ~2SI-T4binding to HSA, which had been covalently attached to CNBr-activated Sepharose (HSA-Sepharose), was characterized and the effects of fatty acids, bilirubin, and steroid hormones on the ~25I-T4 binding to HSA were studied. In addition, the effect of 8-anilino-1naphthalene sulfonic acid (ANS), which is a well-
Serum albumin is one of the three major thyroid hormone-binding proteins in human plasma. Approximately 10% o f thyroxine (T4) and 13% of t r i i o d o t h y r o n i n e (T3) are bound by serum albumin (Davis et aI., 1972). Because of its low affinity and high capacity for thyroid hormones, serum albumin plays a significant role as a hormone-binding protein only at high plasma concentrations of the hormones (Chopra, 1981). Serum albumin also binds many small molecules, such as fatty acids, bilirubin, and steroids. Since radioimmunoassay values of total serum thyroid hormone for the evaluation of thyroid 1The Third Department of Internal Medicine, Gifu University School of Medicine, Gifu 500, Japan. 2 To whom all correspondence should be addressed. 3 Abbreviations: "1"4,thyroxine; HSA, human serum albumin; ANS, 8-anilino-l-naphthalene sulfonic acid; TBG, thyroxine-binding globulin; TBPA, thyroxine-binding prealbumin.
461 0277-8033/90/0800-0461506.00/0 © 1990PlenumPublishingCorporation
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known inhibitor of thyroid hormone binding to thyroxine-binding globulin (TBG) (Green et al., 1972) and thyroxine binding-prealbumin (TBPA) (Branch et al., 1971; Ferguson et aL, 1975), on the binding of ~25I-T4 to HSA was studied. These studies have provided valuable information concerning the effects on the binding of thyroid hormones to HSA of some nonprotein physiological plasma constituents which are also known to bind to albumin. 2. MATERIALS AND METHODS 2.1. Preparation of HSA-Sepharose HSA (nondenaturated, No. 126654) was obtained from Calbiochem-Behring Corp (La Jolla, California). HSA was defatted by the acid-charcoal treatment (Chen, 1967) and was coupled onto CNBractivated Sepharose 4B as described elsewhere (Sakata and Atassi, 1979). The amount of HSA thus attached onto the adsorbent was 3.0-4.5/~mol/ml of Sepharose. 2.2. Interaction Between
1251-T4 and
Sepharose was determined by subtracting radioactivity bound to uncoupled Sepharose from those bound to HSA-Sepharose. The data represent specific binding, which accounts for more than 67% of the total binding. Unless otherwise indicated, means of duplicate determination that differed by less than 7% are shown. Binding isotherms were analyzed by the computerized nonlinear least-squares method (Kamikubo et al., 1986). Means and SEM of dissociation constants were calculated after logarithmic transformation of the values. 2.4. Effect of Various Concentrations of Unlabeled T4 on Binding of ANS to HSA Binding of ANS to HSA solution was measured fluorometrically at 25°C (Hitachi 650-10S Fluorescence Spectrophotometer), according to the method described by Danuel and Weber (1966). The reaction mixture contained 0.5/~M HSA, 100mM NaCI, 30 mM sodium phosphate (pH 7.4), and other additions. The excitation and emission wavelengths were 400 nm and 475 nm, respectively.
HSA-Sepharose
12SI-T4 (800 Ci/mmol)
was from New England Nuclear (Tokyo). T4, ANS, and steroids were from Sigma Chemicals (St Louis, Missouri). Experiments of ~25I-T4binding to HSA-Sepharose were performed at 37°C in a volume of 400/zl containing 70-140 nM HSA (amount of HSA coupled on to Sepharose), 100 mM NaC1, 30 mM sodium phosphate ( pH 7.4), and other additions, unless otherwise indicated. After 15 min incubation, the mixture was centrifuged by a temperature-controlled centrifuge and the ligand phase was aspirated off. 2.3. Effect of Fatty Acids, Bilirubin, ANS, and Steroids on Interaction Between 125I-T4 and HSA-Sepharose Bilirubin was from Wako Pure Chemicals (Osaka). Fatty acids, purchased from PL Biochemicals (Tokyo) were dissolved in ethanol and added to glass test tubes, followed by evaporation of ethanol. HSA-Sepharose suspension was added to the tubes and incubated for 30min at room temperature in order to allow to equilibrate the binding of HSA to fatty acids prior to the binding study with 125I-T4. Amounts of 125I-T4bound to HSA-Sepharose were determined by counting the radioactivity in a gamma counter. Specific binding of ~25I-T4 to HSA-
3. RESULTS 3.1. Interaction Between ~2SI-T4 and HSA-Sepharose and Effect of Fatty Acids, Bilirubin, ANS, and Steroids
125I-~T4binding to HSA-Sepharose was rapid and reached ~equilibrium after 3-min incubation (Fig. 1). The amount of bound ~25I-T4 was not changed thereafter for at least 4 h r (data not shown). Both Figs. 2 and 7 show representative Scatchard plots of
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Fig. 1. Time course studies of L25I-T4binding to HSA-Sepharose. The binding of 1251-T4(y-axis; pmol/tube) was measuredat 25°C. The concentrationof ~25I-T4and HSA-Sepharosewere 120 nM and 76 nM, respectively.
Thyroxine Binding to Human Serum Albumin
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[Fatty Acid]/[HSA] Fig. 2. Scatchard plot of 1251-T4 binding to HSA-Sepharose in the absence ((i)) or the presence of linoleic acid (A) or bilirubin (El). Straight lines represent fitted high- and low-affinity binding of 12SI-T4. The low-affinity binding in the presence of linoleic acid (2 # M ) or bilirubin (1 k~M), or in their absence, was not significantly different. The dissociation constants of the high-affinity sites in the absence or the presence of linoleic acid or bilirubin were 1.86 × 10 -6 M, 2.99 × 10 -6 M, and 1.70 × 10 5 M, respectively. The corresponding binding capacities were 1.08 tool/tool, 1.07 m o l / m o l , and 0.96 m o l / m o l , respectively.
125I-T4 binding to HSA-Sepharose. The binding isotherms could be appropriately analyzed by a twoclass sites model. The dissociation constants of the high-affinity and low-affinity sites (mean ± SEM of 8 experiments) were 2.19±0.53x t0-6M and 2.69± 0.78x 10-5 M, respectively. The binding capacities (mean ± SEM of 8 experiments) were 1.28± 0.46 tool/mot and 23.5±9.7 mol/mol of HSA for the high- and low-affinity sites, respectively. Patmitic acid and linoteic acid inhibited the 125IT4 binding to HSA in a dose-dependent manner (Fig. 3). The dependence of the inhibitory activity of saturated fatty acids on their chain length is shown in Fig. 4. Among saturated fatty acids studied, heptadecanoic acid was the most potent in inhibiting the 125I-T4 binding. Both shortening and elongation of the alkyl chain of the fatty acids reduced their inhibitory potency. As shown in Fig. 2, linoleic acid inhibited the 125I-T4 binding to the high-affinity sites without affecting the binding to the low-affinity sites. Linoleic acid increased the dissociation constant but
Fig. 3. Effects of palmitic acid and linoleic acid on 125I-T4 binding to HSA-Sepharose. The binding o f ~25I-T4 was measured at 140 m M 125I-T4 and 76 n M HSA-Sepharose in the presence of indicated concentrations of palmitic acid (©) or linoleic acid ( I ) .
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464
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[Bilirubin]/[HSA] Fig. 5. Effect of bilirubin on 125I-T4 to HSA-Sepharose. 1251-T4 binding was measured at 120nM '2SI-T4 and 70nM HSASepharose in the presence of the indicated amounts of bilirubin. d i d n o t a l t e r the b i n d i n g c a p a c i t y o f the high-affinity sites. B i l i r u b i n also r e d u c e d the 125I-T4 b i n d i n g (Fig. 5). It i n c r e a s e d the d i s s o c i a t i o n c o n s t a n t o f the highaffinity sites w i t h o u t i n f l u e n c i n g the b i n d i n g c a p a c i t y o f the sites. B i l i r u b i n h a d n o effect on the low-affinity sites (Fig. 2).
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Fig. 7. Scatchard plot of 125I-T4 binding to HSA-Sepharose in the absence or the presence of ANS. Straight lines represent fitted high- and low-affinity binding of '251-T4. The low-affinity binding in the presence of ANS was not significantly different from that in the absence of ANS. The dissociation constants of the highaffinity sites in the absence ((i)) or the presence of 10/zM ANS (O) or 1 mM ANS (11) were: 1.72× 10 -6 M, 1.26× 10 -6 M, and 0.71 x 10 -6 M, respectively. The corresponding binding capacities were 1.64 mol/mol, 0.73 mol/mol, and 0.11 mol/mol, respectively.
A N S also i n h i b i t e d t h e b i n d i n g o f 125I-T4 to H S A (Fig. 6). As s h o w n in Fig. 7, A N S i n h i b i t e d the '25I-T4 b i n d i n g to the high-affinity sites b u t h a d no effect o n the low-affinity sites. F r o m the S c a t c h a r d a n a l y s i s , it was s h o w n t h a t the i n h i b i t o r y effect o f A N S on the high-affinity sites was d u e to r e d u c t i o n o f b i n d i n g c a p a c i t y o f the sites. N o n e o f the s t e r o i d h o r m o n e s ( p r o g e s t e r o n e , c o r t i c o s t e r o n e , h y d r o c o r t i s o n e , cortisone, d e h y d r o e p i a n d r o s t e r o n e , a n d t e s t o s t e r o n e ) at t h e i r c o n c e n t r a t i o n s o f 1 0 / z M h a d a n y effect on the 12SI-T4 b i n d i n g to H S A - S e p h a r o s e ( d a t a n o t s h o w n ) .
3.2. Effect o f U n l a b e l e d T4 on Binding o f A N S to H S A
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Fig. 6. Effect of ANS on 1251-T4binding to HSA-Sepharose. The binding of ~251-T4 was measured at 250 nM ~251-T4 and 76 nM HSA-Sepharose in the presence of the indicated concentrations of ANS.
I n o r d e r to s t u d y the a c t i o n o f T4 o n the b i n d i n g o f A N S to H S A , the effect o f u n l a b e l e d T4 on the f l u o r e s c e n c e i n t e n s i t y o f A N S b o u n d to H S A s o l u t i o n was e x a m i n e d . T h e results are s h o w n in Fig. 8. T4, even at 37 m o l a r excess with r e s p e c t to H S A , h a d little o r n o effect o n t h e f l u o r e s c e n c e intensity of ANS.
Thyroxine Binding te Human Serum Albumin
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Fig. 8. Effect on T4 on ANS binding to HSA. ANS binding to HSA was measured fluorometrically at 9/zM ANS and 0.5 ~ M HSA in the presence of the indicated amount o f t 4. Similar results were obtained at 0.9 p~M ANS.
4. DISCUSSION HSA covalently attached to Sepharose was capable of binding 125I-T4. The dissociation constant and the binding capacity of HSA-Sepharose were comparable to those previously reported in HSA solution (Tritsch et al., 1961; Tabachnick, 1964a, 1967; Steiner et al., 1966. Therefore, it is reasonable to conclude that thyroid hormone-binding sites are not involved in the covalent linkage of HSA to CNBr-activated Sepharose. Alternatively, the local environment of binding sites was not affected by the procedure. In agreement with previous reports on HSA in solution, fatty acids and bilirubin inhibited 125I-T4 binding to HSA-Sepharose (Tabachnick 1964b; Tabachnick et al., 1965). These results also indicate that neither the binding sites nor the properties of thyroid hormone binding are affected after the covalent attachment of HSA to Sepharose. ANS is known to displace T4 competitively from TBG (Green et al., 1972) and TBPA (Branch et al., 1971; Ferguson et al., 1975). However, little has been known concerning the effects of ANS on the interaction of Y 4 with albumin. In the present study, the effects of ANS on ]25I-T4binding to HSA were characterized by employing HSA-Sepharose. Using this solid-phase technique, the inhibition by ANS of the binding of ~25I-T4 to HSA was shown. In contrast with the effects on T4-TBG and T4-TBPA interactions, the effects of ANS action on T4-HSA was "noncompetitive." It was shown that ANS reduced the binding capacity of the high-affinity sites without affecting the
binding affinity. These results imply that ANS may inhibit thyroid hormone binding to HSA through a heterotropic effect rather than a competitive manner. Since T4-induced quenching of ANS fluorescence intensity in TBG (Green et aL, 1972) and TBPA (Branch et aL, 1971), and this was not observed in HSA, it seems likely that there is no significant competitive interaction between T4 and ANS in the binding to HSA. Steroid hormones, which have been shown to bind to HSA-Sepharose (Lagercrantz et alo, 1979), did not show any effect on the 125I-T4 binding with HSA. These results indicate that there is no significant overlap between thyroid hormone-binding sites and the steroid-binding sites of HSA. Since HSA-Sepharose provides an obvious advantage in separation of bound and free ligands in binding experiments, the present results indicate that HSA-Sepharose affords a useful tool in the evaluation of the interaction of thyroid hormones with HSA. These studies have also permitted investigation of the effects of normal, physiological, nonprotein, HSAbinding plasma constituents on the binding of thyroid hormones to human serum albumin.
REFERENCES Branch, W. T., Edethoch, H., and Robbins, J. (1971). Z Cfin. Invest. 50, lla. Chen, R. F. (1967). J. BioL Chem. 242, 173-181. Chopra, I. J. (1981). In Triiodothyronine in Health and Disease (Chopra, I. J., ed.), Springer-Verlag, Berlin, pp. 8-14. Daniel, E., and Weber, G. (1966). Biochemistry 5, 1893-1907. Davis, P. J., Handwerger, B. S., and Gregerman, R. I. (1972). J. Clin. Invest. 51, 515-521. Ferguson, R. N., Edelhoch, H., Saroff, H. A., and Robbins, J. (1975). Biochemistry 14, 282-289. Green, A. M., Marshall, J. S., Pensky, J., and Stanbury, J~ B. (I 972). Science 175, 1378-1380. Kamikubo, K., Murase, H., Murayama, M., and Miura, K. (1986). Jpn. J. Pharmaeol. 40, 342-346. Lagercrantz, C., Larsson, T., and Karlsson, H. (1979). Anal. Biochem. 99, 352-364. Lagercrantz, C., Larsson, T., and Denfors, I. (1981). Comp. Biochem. Physiol. 69C, 375-378. Sakata, S., and Atassi, M. Z. (1979). Biochim. Biophys. Aeta. 576, 322-332. Steiner, R. F., Roth, J., and Robbins, J. (1966). J. Biol. Chem. 241, 560-567. Stevenson, A., Holmer, E., and Andersson, L-O. (1974). Biochim. Biophys. Acta. 342, 54-59. Tabachnick, M. (1964a). J. Biol. Chem. 239, 1242-1249. Tabachnick, M. (1964b). Arch. Biochem. Biophys. 106, 415-421. Tabachnick, M., Downs, F., and Giorgio, N. A., Jr. (1965). Proe. Soe. Exp. Biol. Med. 118, 1180-1182. Tabachnick, M. (1967). J. Biol. Chem. 242, 1646-1650. Tritsch, G. L., Rathke, C., Tritch, N., and Weiss, C. lvl. (1961). J. Biol. Chem. 236, 3163-3167.
Thyroxine Binding to Human Serum Albumin Immobilized on Sepharose and Effects of Nonprotein Albumin-Binding Plasma Constituents Keita Kamikubo, 1 S h i g e k i Sakata, 1'2 Shigenori N a k a m u r a , 1 Takashi Komaki, ~ and Kiyoshi M i u r a t
Received March 24 1990
125I-thyroxine (125I-T4) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied, t25I-T4 binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19 ± 0.53 x 10 -6 M and 2.69 ± 0.78 x 10 5 M, respectively. The number of binding sites of the high and the low affinity sites were 1.28 + 0.46 mol/mol and 23.5 ± 9.7 tool/tool of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of 125I-T4to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-l-naphthalene sulfonic acid (ANS) inhibited the high affinity T4 binding via reduction of the binding capacity. Unlabeled T4 showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity ~['4binding to HSA-Sepharose. KEY WORDS: Thyroxine; albumin; fatty acid; bilirubin; ANS; steroid.
1. I N T R O D U C T I O N 3
functions in subjects suspected to have thyroid disorders include bound and free hormones, it is important to examine the effects of various ligands on thyroid hormone binding to serum albumin. H u m a n serum albumin (HSA) covalently immobilized on agarose microparticle (Sepharose) has been reported to retain most of its ligand-binding characteristics (Stevenson et al., 1974; Lagercrantz et al., 1979, 1981). Such HSA preparations have been widely used for the study of HSA-ligand interaction, because they provide a simple method for the separation of bound and free ligands (Stevenson et al., 1974; Lagercrantz et al., 1979, 1981). In the present investigation, the physiological nature of ~2SI-T4binding to HSA, which had been covalently attached to CNBr-activated Sepharose (HSA-Sepharose), was characterized and the effects of fatty acids, bilirubin, and steroid hormones on the ~25I-T4 binding to HSA were studied. In addition, the effect of 8-anilino-1naphthalene sulfonic acid (ANS), which is a well-
Serum albumin is one of the three major thyroid hormone-binding proteins in human plasma. Approximately 10% o f thyroxine (T4) and 13% of t r i i o d o t h y r o n i n e (T3) are bound by serum albumin (Davis et aI., 1972). Because of its low affinity and high capacity for thyroid hormones, serum albumin plays a significant role as a hormone-binding protein only at high plasma concentrations of the hormones (Chopra, 1981). Serum albumin also binds many small molecules, such as fatty acids, bilirubin, and steroids. Since radioimmunoassay values of total serum thyroid hormone for the evaluation of thyroid 1The Third Department of Internal Medicine, Gifu University School of Medicine, Gifu 500, Japan. 2 To whom all correspondence should be addressed. 3 Abbreviations: "1"4,thyroxine; HSA, human serum albumin; ANS, 8-anilino-l-naphthalene sulfonic acid; TBG, thyroxine-binding globulin; TBPA, thyroxine-binding prealbumin.
461 0277-8033/90/0800-0461506.00/0 © 1990PlenumPublishingCorporation
462
Kamikubo et ai.
known inhibitor of thyroid hormone binding to thyroxine-binding globulin (TBG) (Green et al., 1972) and thyroxine binding-prealbumin (TBPA) (Branch et al., 1971; Ferguson et aL, 1975), on the binding of ~25I-T4 to HSA was studied. These studies have provided valuable information concerning the effects on the binding of thyroid hormones to HSA of some nonprotein physiological plasma constituents which are also known to bind to albumin. 2. MATERIALS AND METHODS 2.1. Preparation of HSA-Sepharose HSA (nondenaturated, No. 126654) was obtained from Calbiochem-Behring Corp (La Jolla, California). HSA was defatted by the acid-charcoal treatment (Chen, 1967) and was coupled onto CNBractivated Sepharose 4B as described elsewhere (Sakata and Atassi, 1979). The amount of HSA thus attached onto the adsorbent was 3.0-4.5/~mol/ml of Sepharose. 2.2. Interaction Between
1251-T4 and
Sepharose was determined by subtracting radioactivity bound to uncoupled Sepharose from those bound to HSA-Sepharose. The data represent specific binding, which accounts for more than 67% of the total binding. Unless otherwise indicated, means of duplicate determination that differed by less than 7% are shown. Binding isotherms were analyzed by the computerized nonlinear least-squares method (Kamikubo et al., 1986). Means and SEM of dissociation constants were calculated after logarithmic transformation of the values. 2.4. Effect of Various Concentrations of Unlabeled T4 on Binding of ANS to HSA Binding of ANS to HSA solution was measured fluorometrically at 25°C (Hitachi 650-10S Fluorescence Spectrophotometer), according to the method described by Danuel and Weber (1966). The reaction mixture contained 0.5/~M HSA, 100mM NaCI, 30 mM sodium phosphate (pH 7.4), and other additions. The excitation and emission wavelengths were 400 nm and 475 nm, respectively.
HSA-Sepharose
12SI-T4 (800 Ci/mmol)
was from New England Nuclear (Tokyo). T4, ANS, and steroids were from Sigma Chemicals (St Louis, Missouri). Experiments of ~25I-T4binding to HSA-Sepharose were performed at 37°C in a volume of 400/zl containing 70-140 nM HSA (amount of HSA coupled on to Sepharose), 100 mM NaC1, 30 mM sodium phosphate ( pH 7.4), and other additions, unless otherwise indicated. After 15 min incubation, the mixture was centrifuged by a temperature-controlled centrifuge and the ligand phase was aspirated off. 2.3. Effect of Fatty Acids, Bilirubin, ANS, and Steroids on Interaction Between 125I-T4 and HSA-Sepharose Bilirubin was from Wako Pure Chemicals (Osaka). Fatty acids, purchased from PL Biochemicals (Tokyo) were dissolved in ethanol and added to glass test tubes, followed by evaporation of ethanol. HSA-Sepharose suspension was added to the tubes and incubated for 30min at room temperature in order to allow to equilibrate the binding of HSA to fatty acids prior to the binding study with 125I-T4. Amounts of 125I-T4bound to HSA-Sepharose were determined by counting the radioactivity in a gamma counter. Specific binding of ~25I-T4 to HSA-
3. RESULTS 3.1. Interaction Between ~2SI-T4 and HSA-Sepharose and Effect of Fatty Acids, Bilirubin, ANS, and Steroids
125I-~T4binding to HSA-Sepharose was rapid and reached ~equilibrium after 3-min incubation (Fig. 1). The amount of bound ~25I-T4 was not changed thereafter for at least 4 h r (data not shown). Both Figs. 2 and 7 show representative Scatchard plots of
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Fig. 1. Time course studies of L25I-T4binding to HSA-Sepharose. The binding of 1251-T4(y-axis; pmol/tube) was measuredat 25°C. The concentrationof ~25I-T4and HSA-Sepharosewere 120 nM and 76 nM, respectively.
Thyroxine Binding to Human Serum Albumin
463
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2
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[Fatty Acid]/[HSA] Fig. 2. Scatchard plot of 1251-T4 binding to HSA-Sepharose in the absence ((i)) or the presence of linoleic acid (A) or bilirubin (El). Straight lines represent fitted high- and low-affinity binding of 12SI-T4. The low-affinity binding in the presence of linoleic acid (2 # M ) or bilirubin (1 k~M), or in their absence, was not significantly different. The dissociation constants of the high-affinity sites in the absence or the presence of linoleic acid or bilirubin were 1.86 × 10 -6 M, 2.99 × 10 -6 M, and 1.70 × 10 5 M, respectively. The corresponding binding capacities were 1.08 tool/tool, 1.07 m o l / m o l , and 0.96 m o l / m o l , respectively.
125I-T4 binding to HSA-Sepharose. The binding isotherms could be appropriately analyzed by a twoclass sites model. The dissociation constants of the high-affinity and low-affinity sites (mean ± SEM of 8 experiments) were 2.19±0.53x t0-6M and 2.69± 0.78x 10-5 M, respectively. The binding capacities (mean ± SEM of 8 experiments) were 1.28± 0.46 tool/mot and 23.5±9.7 mol/mol of HSA for the high- and low-affinity sites, respectively. Patmitic acid and linoteic acid inhibited the 125IT4 binding to HSA in a dose-dependent manner (Fig. 3). The dependence of the inhibitory activity of saturated fatty acids on their chain length is shown in Fig. 4. Among saturated fatty acids studied, heptadecanoic acid was the most potent in inhibiting the 125I-T4 binding. Both shortening and elongation of the alkyl chain of the fatty acids reduced their inhibitory potency. As shown in Fig. 2, linoleic acid inhibited the 125I-T4 binding to the high-affinity sites without affecting the binding to the low-affinity sites. Linoleic acid increased the dissociation constant but
Fig. 3. Effects of palmitic acid and linoleic acid on 125I-T4 binding to HSA-Sepharose. The binding o f ~25I-T4 was measured at 140 m M 125I-T4 and 76 n M HSA-Sepharose in the presence of indicated concentrations of palmitic acid (©) or linoleic acid ( I ) .
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(number of carbon atoms) Fig. 4. Effect of saturated fatty acids on ~25I-]"4 binding to HSASepharose. The binding of 125I-T4 was saturated at 140 nM 125I-T4 and 76 nM HSA-Sepharose in the presence of 7.6/~M of each of the saturated fatty acids,
464
K a m i k u b o et al.
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[Bilirubin]/[HSA] Fig. 5. Effect of bilirubin on 125I-T4 to HSA-Sepharose. 1251-T4 binding was measured at 120nM '2SI-T4 and 70nM HSASepharose in the presence of the indicated amounts of bilirubin. d i d n o t a l t e r the b i n d i n g c a p a c i t y o f the high-affinity sites. B i l i r u b i n also r e d u c e d the 125I-T4 b i n d i n g (Fig. 5). It i n c r e a s e d the d i s s o c i a t i o n c o n s t a n t o f the highaffinity sites w i t h o u t i n f l u e n c i n g the b i n d i n g c a p a c i t y o f the sites. B i l i r u b i n h a d n o effect on the low-affinity sites (Fig. 2).
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Fig. 7. Scatchard plot of 125I-T4 binding to HSA-Sepharose in the absence or the presence of ANS. Straight lines represent fitted high- and low-affinity binding of '251-T4. The low-affinity binding in the presence of ANS was not significantly different from that in the absence of ANS. The dissociation constants of the highaffinity sites in the absence ((i)) or the presence of 10/zM ANS (O) or 1 mM ANS (11) were: 1.72× 10 -6 M, 1.26× 10 -6 M, and 0.71 x 10 -6 M, respectively. The corresponding binding capacities were 1.64 mol/mol, 0.73 mol/mol, and 0.11 mol/mol, respectively.
A N S also i n h i b i t e d t h e b i n d i n g o f 125I-T4 to H S A (Fig. 6). As s h o w n in Fig. 7, A N S i n h i b i t e d the '25I-T4 b i n d i n g to the high-affinity sites b u t h a d no effect o n the low-affinity sites. F r o m the S c a t c h a r d a n a l y s i s , it was s h o w n t h a t the i n h i b i t o r y effect o f A N S on the high-affinity sites was d u e to r e d u c t i o n o f b i n d i n g c a p a c i t y o f the sites. N o n e o f the s t e r o i d h o r m o n e s ( p r o g e s t e r o n e , c o r t i c o s t e r o n e , h y d r o c o r t i s o n e , cortisone, d e h y d r o e p i a n d r o s t e r o n e , a n d t e s t o s t e r o n e ) at t h e i r c o n c e n t r a t i o n s o f 1 0 / z M h a d a n y effect on the 12SI-T4 b i n d i n g to H S A - S e p h a r o s e ( d a t a n o t s h o w n ) .
3.2. Effect o f U n l a b e l e d T4 on Binding o f A N S to H S A
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Fig. 6. Effect of ANS on 1251-T4binding to HSA-Sepharose. The binding of ~251-T4 was measured at 250 nM ~251-T4 and 76 nM HSA-Sepharose in the presence of the indicated concentrations of ANS.
I n o r d e r to s t u d y the a c t i o n o f T4 o n the b i n d i n g o f A N S to H S A , the effect o f u n l a b e l e d T4 on the f l u o r e s c e n c e i n t e n s i t y o f A N S b o u n d to H S A s o l u t i o n was e x a m i n e d . T h e results are s h o w n in Fig. 8. T4, even at 37 m o l a r excess with r e s p e c t to H S A , h a d little o r n o effect o n t h e f l u o r e s c e n c e intensity of ANS.
Thyroxine Binding te Human Serum Albumin
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Fig. 8. Effect on T4 on ANS binding to HSA. ANS binding to HSA was measured fluorometrically at 9/zM ANS and 0.5 ~ M HSA in the presence of the indicated amount o f t 4. Similar results were obtained at 0.9 p~M ANS.
4. DISCUSSION HSA covalently attached to Sepharose was capable of binding 125I-T4. The dissociation constant and the binding capacity of HSA-Sepharose were comparable to those previously reported in HSA solution (Tritsch et al., 1961; Tabachnick, 1964a, 1967; Steiner et al., 1966. Therefore, it is reasonable to conclude that thyroid hormone-binding sites are not involved in the covalent linkage of HSA to CNBr-activated Sepharose. Alternatively, the local environment of binding sites was not affected by the procedure. In agreement with previous reports on HSA in solution, fatty acids and bilirubin inhibited 125I-T4 binding to HSA-Sepharose (Tabachnick 1964b; Tabachnick et al., 1965). These results also indicate that neither the binding sites nor the properties of thyroid hormone binding are affected after the covalent attachment of HSA to Sepharose. ANS is known to displace T4 competitively from TBG (Green et al., 1972) and TBPA (Branch et al., 1971; Ferguson et al., 1975). However, little has been known concerning the effects of ANS on the interaction of Y 4 with albumin. In the present study, the effects of ANS on ]25I-T4binding to HSA were characterized by employing HSA-Sepharose. Using this solid-phase technique, the inhibition by ANS of the binding of ~25I-T4 to HSA was shown. In contrast with the effects on T4-TBG and T4-TBPA interactions, the effects of ANS action on T4-HSA was "noncompetitive." It was shown that ANS reduced the binding capacity of the high-affinity sites without affecting the
binding affinity. These results imply that ANS may inhibit thyroid hormone binding to HSA through a heterotropic effect rather than a competitive manner. Since T4-induced quenching of ANS fluorescence intensity in TBG (Green et aL, 1972) and TBPA (Branch et aL, 1971), and this was not observed in HSA, it seems likely that there is no significant competitive interaction between T4 and ANS in the binding to HSA. Steroid hormones, which have been shown to bind to HSA-Sepharose (Lagercrantz et alo, 1979), did not show any effect on the 125I-T4 binding with HSA. These results indicate that there is no significant overlap between thyroid hormone-binding sites and the steroid-binding sites of HSA. Since HSA-Sepharose provides an obvious advantage in separation of bound and free ligands in binding experiments, the present results indicate that HSA-Sepharose affords a useful tool in the evaluation of the interaction of thyroid hormones with HSA. These studies have also permitted investigation of the effects of normal, physiological, nonprotein, HSAbinding plasma constituents on the binding of thyroid hormones to human serum albumin.
REFERENCES Branch, W. T., Edethoch, H., and Robbins, J. (1971). Z Cfin. Invest. 50, lla. Chen, R. F. (1967). J. BioL Chem. 242, 173-181. Chopra, I. J. (1981). In Triiodothyronine in Health and Disease (Chopra, I. J., ed.), Springer-Verlag, Berlin, pp. 8-14. Daniel, E., and Weber, G. (1966). Biochemistry 5, 1893-1907. Davis, P. J., Handwerger, B. S., and Gregerman, R. I. (1972). J. Clin. Invest. 51, 515-521. Ferguson, R. N., Edelhoch, H., Saroff, H. A., and Robbins, J. (1975). Biochemistry 14, 282-289. Green, A. M., Marshall, J. S., Pensky, J., and Stanbury, J~ B. (I 972). Science 175, 1378-1380. Kamikubo, K., Murase, H., Murayama, M., and Miura, K. (1986). Jpn. J. Pharmaeol. 40, 342-346. Lagercrantz, C., Larsson, T., and Karlsson, H. (1979). Anal. Biochem. 99, 352-364. Lagercrantz, C., Larsson, T., and Denfors, I. (1981). Comp. Biochem. Physiol. 69C, 375-378. Sakata, S., and Atassi, M. Z. (1979). Biochim. Biophys. Aeta. 576, 322-332. Steiner, R. F., Roth, J., and Robbins, J. (1966). J. Biol. Chem. 241, 560-567. Stevenson, A., Holmer, E., and Andersson, L-O. (1974). Biochim. Biophys. Acta. 342, 54-59. Tabachnick, M. (1964a). J. Biol. Chem. 239, 1242-1249. Tabachnick, M. (1964b). Arch. Biochem. Biophys. 106, 415-421. Tabachnick, M., Downs, F., and Giorgio, N. A., Jr. (1965). Proe. Soe. Exp. Biol. Med. 118, 1180-1182. Tabachnick, M. (1967). J. Biol. Chem. 242, 1646-1650. Tritsch, G. L., Rathke, C., Tritch, N., and Weiss, C. lvl. (1961). J. Biol. Chem. 236, 3163-3167.
