Thyroxine-protein interactions

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

106, 415-421 (1964)

Thyroxine-Protein

Interactions

III. Effect of Fatty Acids, 2,4-Dinitrophenol Binding

of Thyroxine

and Other

Anionic

Compounds

on the

by Human Serum Albumin’

M. TABACHNICK From the Department of Biochemistry, New York Medical Flower and Fifth Avenue Hospitals, New York, New York (10029) Received

February

College,

10, 1964

The effect of various anionic compounds on the binding of thyroxine by human serum albumin has been studied. The results show that anions such as 2,4-dinitrophenol, salicylate, and fatty acids are capable of displacing thyroxine from binding sites on albumin. The compounds investigated exhibit the following order of effectiveness in reducing thyroxine binding by albumin: oleate > linoleate > dodecvl > laurate > octanoate > salicylate. _ sulfate > palmitate > 2,4-dinitrophenol

The binding of thyroxine by human serum albumin at pH 7.4 appears to involve in part the electrostatic interaction of negatively charged thyroxine molecules with cationic binding sites on the protein (1). In support of this view it was shown that an anionic azo dye (methyl orange) is capable of displacing thyroxine from binding sites on albumin (1). It seemed of interest to study other anionic compounds with regard to their ability to displace thyroxine from albumin. The present report describes an investigation of the effect of various compounds such as 2,4-dinitrophenol, salicylate, and fatty acid anions on the interaction of thyroxine with human albumin. MATERIALS

AND

METHODS

The human serum albumin used in the present binding study is identical with the preparation described previously (1) (Cohn Fraction V, Batch No. 1972, obtained from the American Red Cross through the courtesy of Dr. James H. Pert). The albumin was deionized by passage through a

mixed bed resin (Amberlite MB-l) and then dialyzed against glass-distilled water. For the experiments with defatted albumin, the fatty acids bound to albumin were removed by extraction with 5y0 glacial acetic acid in isooctane by the method of Goodman (2). 1’31.labeled-n-thyroxine was obtained from Abbott Laboratories, Oak Ridge, Tennessee. Nonradioactive thyroxine was purchased from the Aldrich Chemical Company, Milwaukee, Wisconsin. Equilibrium dialysis with Pal-n-thyroxine was used to study thyroxine binding by albumin as described previously (1, 3). All experiments were performed at pH 7.4 and 30°C (see Table I). When equilibrium was reached (after 6 hours), the extent of binding was determined by counting aliquots taken from both sides of the dialysis membrane (1, 3). The experimental data plotted in Figs. l-3 are the means of duplicate determinations. The fatty acid anions were made up in aqueous solution as described by Goodman (2). RESULTS

The reversible binding of a small molecule to several sets of binding sites on a protein with no significant interactions occurring among bound molecules can be described by Eq. 1 (4):

1 This work was supported in part by research grant AM-05344-03 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. 415

416

TABACHNICK

7

6 5

v 4 (A) xIO53 2

I I

2

:

v FIQ. 1. Effect of various anions on the binding of thyroxine by human serum albumin at pH 7.4 and 30”. -, Thyroxine binding, no anion added; 0, 3.1 moles of 2,4-dinitrophenol added per mole of albumin; A, 3.1 moles of dodecyl sulfate added per mole of albumin; A, 3.1 moles of salicylate added per mole of albumin; q ,7.7 moles of salicylate added per mole of albumin.

where a is the average number of moles of small molecule bound per mole of protein, (A) is the concentration of free small molecule, and na represents each set or group of binding sites (i = 1, 2, 3 . . .) with corresponding average apparent intrinsic association constants ki’. A plot of p/(A) versus B according to Eq. 1 gives a curve with intercepts c n&i (as B + 0) and c ni (as F/(A) + 0) (4). In a recent report (1) it was shown that the binding of thyroxine to human serum albumin at pH 7.4 and 30” can be described by assuming the existence of two groups of binding sites. Relatively strong binding takes place at the first group of sites, with nl = 2, and kl’ = 2.75 X lo6 M-l. A lower

FIG. 2. Effect of different fatty acid anions on the binding of thyroxine by albumin at pH 7.4 and 30’. --, Thyroxine binding, no anion added; A, binding in the presence of octanoate; q , binding in the presence of laurate; 0, binding in the presence of palmitate; 0, binding in the presence of linoleate; A, binding in the presence of oleate. The fatty acid concentration was kept constant at a ratio of 3.1 moles of fatty acid anion per mole of albumin.

affinity for thyroxine is exhibited by the second group of sites, with n2 = 6 and k2! = 2.5 X lo4 M-l. In the case of thyroxine, from Eq. 1

nlkl’(A) ’ = 1 + k{(A)

nzkz’(A) + 1 + kz’(A)

(2)

or (2)(2.75 X 10”>(A) ’ = 1 + (2.75 x 106)(A) (6)(2.5 X 104)(A) + 1 + (2.5 X 104)(A)

(3)

A plot of F/(A) versus P according to Eq. 3 gives the curve for thyroxine binding shown in Figs. l-3 with c niki’ = 7.0 X

EFFECT

OF ANIONS

ON THYROXINE

BINDING

BY ALBUMIN

417

at a ratio of 3.1 moles per mole of albumin, lowers thyroxine binding by about 50% as estimated from its effect on the c n,ici value of the thyroxine-binding curve. From previous results (1) it appears that methyl orange is somewhat more effective than 2,4-dinitrophenol in displacing thyroxine from albumin. Although salicylate has a relatively small effect on thyroxine binding at a molar ratio to albumin of 3.1/l, it can be seen from Fig. 1 that there is a definite reduction in binding in the presence of 7.7 moles of salicylate per mole of albumin. The data given in Fig. 1 demonstrate qualitatively that the anions investigated are capable of displacing thyroxine from binding sites on albumin. These data can be analyzed on a more quantitative basis by applying the method used by Klotz et ul. (5) to study competition betlveen two types of ion for the same locus on a protein molecule. For competition between thyroxine and another compound at the first group of sites (2)(2.75 X 10’)(A) ‘I = 1 + (2.75 X 105)(A) + k,(B) FIG. 3. Effect of palmitate on the binding of thyroxine by defatted albumin at pH 7.4 and 30”. 0, binding of thyroxine to defatted albumin, no palmitate added; l , 1 mole of palmitate added per mole of defatted albumin; A, 3.1 moles of palmitate added per mole of defatted albumin. The fatty acids bound to albumin were removed by extraction with 5y0 glacial acetic acid in isooctane (2). -----, Binding of thyroxine to unextracted albumin (this is the curve shown in Figs. 1 and 2)

lo5 for native albumin [x niki’ = 9.8 X lo5 for fatty acid-free albumin (Fig. 3)]. EFFECT OF ANIONS ON THYROXINE BINDING

The effect of 2,4-dinitrophenol, dodecyl sulfate, and salicylate on the binding of thyroxine by human albumin is shown in Fig. 1. It can be seen from Fig. 1 that the greatest reduction in thyroxine binding occurs in the presence of dodecyl sulfate, the order of effectiveness of the compounds investigated being: dodecyl sulfate > 2,4dinitrophenol > salicylate. DodecyI sulfate,

(4)

where ijl is the average number of moles of thyroxine bound per mole of albumin at the first group of sites, (A) is the concentration of free thyroxine, (3) is the concentration of free competing anion, and I?, is the relative binding or association constant of the competing anion at the first group of sites. At the relatively low concentrations of thyroxine used in this study (the molar ratio of thyroxine to albumin is varied from 0.31/l to 3.1/l), it is estimated that about 80% of the bound thyroxine would be complexed at the first group of binding sites. If the simplifying assumption is made that competition between the anion B and thyroxine takes place only at the first group of sites then the relation between the ohserved F and (A) is (2)(2.75 X 105)(A) Fobs’ = 1 + (2.75 x 105)(A) + Its(B) + (6)(2.5 X 10”)(A) 1 + (2.5 X 104)(A) where

(5)

418

TABACHNICK %ba. = g

Fi = i+ +

22

and 4 is the average number of moles of thyroxine bound at the second group of sites. The relation between thyroxine bound at the first group of sites ~~ and bound competing anion iis is (5) -R = $$ FB B

(ICI’ = 2.75 X 10”)

TABLE

(6)

1

CM-4XJL.4TION OF RELATIVE BINDINQ CONSTANT, kB, FOR 2,4-DINITROPNENOL FROM ABILITY TO DISPLACE THYROXINE FROM BINDING Srms ON ALBUMIN& hbs

64)

?I -----

h

(B)

kB

0.554 1.58 0.3260.2281.56 8.0X10’ x lo-en/I x IO-%l

(7) a The

free

2,4-dinitrophenol concentration, binding constant, kg, for 2,4-dinitrophenol were calculated by using Eqs. 5 and 7. Equilibrium dialysis was performed at 30” in 0.06 M potassium phosphate buffer (pH 7.4) in a total volume of 10 ml containing 6.8 lrmoles EDTA, 0.0725 pmole albumin (7.25 x 10e6 M), 0.056 pmole I Ia’-thyroxine (5.6 X 10-C M), and 0.224 pmole 2,4-dinitrophenol (22.4 x 10-O M). Five-ml volumes were used inside and outside the dialysis bag. For definitions of the parameters given in this table see the text.

(B), and the relative

A combination of Eqs. 5 and 7, a knowledge of the albumin concentration, the total amount of anion added, and the experimentally determined values for p&s. and (A) permits a calculation of &, (bound B), the free anion concentration (B), and ks(the relative binding constant of the competing anion for the first group of sites). A numerical example involving 2,4-dinitrophenol is given in Table I. The experimental data given in Fig. 1 for 2,4-dinitrophenol and dodecyl sulfate have thyroxine as estimated from its effect, on the value of the thyroxine-binding been analyzed by means of Eqs. 5 and 7, c r& curve (Fig. 2). and the values for Ice have been calculated The relative binding constants for the according to the example given in Table I. fatty acids anions (Fig. 2), calculated by The calculated k, values are listed in Table using Eqs. 5 and 7, are given in Table III. II. Under the given experimental conditions Since there are large variations in the k, the results indicate that dodecyl sulfate (average ka = 1.7 X 105; Table II) ex- values obtained for palm&ate, a comparison of relative binding affinities has been based hibits twice the binding affinity of 2,4dinitrophenol (average k, = 7.9 X 104; on the ke values derived for a single set, of conditions, i.e., at a thyroxine concentration Table II) for the first, group of thyroxineof 5.60 X 1O-6 M (a molar ratio of thyroxine binding sites on albumin. Salicylate (Fig. to albumin of 0.8/l). On this basis, from 1, Table II) appears to be bound at the first group of sites with about one-twentieth the Table III, oleate appears to exhibit an affinity about one-hundred times greater affinity of 2,4-dinitrophenol. than that of octanoate for the first group of EFFECT OF FATTY ACID ANIONS ON thyroxine-binding sites on albumin. THYROXINE BINDING Since most preparations of human serum albumin contain at least 1 mole of bound Fatty acid anions can displace thyroxine from binding sites on albumin, as demon- fatty acid (2), it was of interest to investistrated by the results given in Fig. 2. It can gate the binding behavior of defatted be seen from Fig. 2 that the greatest re- albumin. Figure 3 shows the effect of palmiductions in thyroxine binding take place in tate on the binding of thyroxine by albumin the presence of the fatty acids with the that had been extracted with 5 % glacial acetic acid in iso-octane. As shown by longer chain lengths, the order of effectiveGoodman (2), extraction with acetic acidness being: C18 (oleate) > Cl* (linoleate) > Cls (palmitate) > Crz (laurate) > CS (oct- iso-octane results in almost complete removal of all the fatty acid bound to alanoate). Oleate, at a molar ratio to albumin bumin. It can be seen from Fig. 3 that of 3.1/l (Fig. 2), causes a reduction of about defatted albumin has an enhanced affinity 70% in the ability of the protein to bind

EFFECT

OF ANIONS

TABLE

ON THYROXINE

was performed

as de-

-

Anion cont.

kg

calculated

‘)

2,4-Dinitrophenol (22.4 x 10-e M)

3.1/l

Dodecyl sulfate (22.4 X lo-6 M)

3.111

2.24 5.60 11.2 16.8 22.4

I I Salicylate (22.4 3.1/l X lo+ M) Salmylate (56.0 7.7/l x 10-o M)

7.5x104 8.0X104 8.8X104 9.4x10* 5.8X104

2.24 5.60 11.2 16.8 22.4

Avg. = 7.9X104 1 .6X105 1.9x105 2.0X105 1.2X105 1.6X105 -Avg. = 1.7X105

5.60 5.60

CALCULATED RELATIVE

-1

for thyroxine (c n&i’ = 9.8 X 105) compared to unextracted albumin (c niki’ = 7.0 X 105). This is similar to the result obtained previously (6) with a different preparation of human albumin. A reasonably good fit can be made of the experimental data by the use of Eq. 2 and assuming the existence of the following binding constants: nl = 2, kl' = 3.75 X 105; and n2 = 6, kz' = 4 X 104. As shown in Fig. 3, the addition of 1 mole of palmitate per mole of defatted albumin reduces thyroxine binding almost to the level of the original unextracted albumin. This indicates that the original unextracted albumin contained fatty acid equivalent to a little over 1 mole of palmitate. When the molar ratio of palmitate to defatted albumin is raised to 3.1/l, a further decrease in thyroxine binding is observed (Fig. 3). The data given in Fig. 3 have been ana-

III BINDING

CONSTANTS

FOR FATTY ACID ANIONS FROM ABILITY TO DISPLACE THYROXINE FROM

ALBUMIN

Equilibrium dialysis was performed as described in Table I. The concentration of fatty acid was 22.4 X 10e6M (a ratio of 3.1 moles of fatty acid to 1 mole of albumin). Thyroxine cont. 04 x 109

Fatty acid

Octanoate (C,) Laurate (Cl,) Palmitate (C16)

Linoleate

kr, Calculated

5.60 5.60 2.24 5.60 11.2 16.8 22.4 5.60 11.2 16.8 22.4 5.60

Oleate (Cl,)

(Cl,)

0.4x104 1.6X104

419

BY ALBUMIN TABLE

II

CALCULATED RELATIVE BINDING CONSTANTS, kg, FOR 2,4-DINITROPHENOL, DODECYL SULFATE, AND SALICYLATE FROM ABILITY TO DISPLACE THYROXINE FROM ALBUMIN

Equilibrium dialysis scribed in Table I.

BINDING

TABLE

1.5x104

3.9x104 1.0x105 1.6X105 1.8X105 1.9XlOj 3.9x105 1.2X106 1.0X106 1.8X106 1.3X106 5.6X105

IV

CALCULATED RELATIVE BINDING CONSTANT FOR PALMITATE FROM ABILITY TO DISPLACE ALBUMIN THYROXINE FROM DEFATTED

Equilibrium dialysis was performed as described in Table I. The concentration of defatted albumin was 7.1 X lOWeM. Palmitate cont.

Molar, ratio ‘P$t,~~$/

Thyroxine cont. 04 x 109

kr, Calculated (X 10-s)

7.1x10-e M

l/l

5.60 11.2 16.8 5.60 11.2 16.8

1.5 1.6 2.3 1.6 2.5 2.3

22.oX10-61M

3.1/l

lyzed by means of Eqs. 5 and 7, and the calculated k, values for palmitate are given in Table IV. A comparison of the kB values for palmitate given in Table IV with those in Table III indicates that removal of the fatty acids bound to albumin does not lead to a pronounced change in the relative ability of added palmitate to displace thyroxine from binding sites on the protein.

420

TABACHNICK DISCUSSION

The results demonstrate that various anions have the ability to displace thyroxine from binding sites on human serum albumin. The compounds investigated exhibit the following order of effectiveness in reducing thyroxine binding: oleate > linoleate > dodecyl sulfate > palmitate > 2,4-dinitrophenol > laurate > octanoate > salicylate. The calculated values for the relative binding constants for 2,4-dinitrophenol (kB = 7.9 X 104; Table II) and dodecyl sulfate (I%, = 1.7 X 105; Table II) are in good agreement with the constants reported in the literature. For the binding of dodecyl sulfate by bovine serum albumin, Karush and Sonenberg (7) report an intrinsic binding constant of 1.82 X 105, and from the data of Carsten and Eisen (8) an association constant of about 7 X lo4 can be estimated for the binding of 2,4-dinitrophenol. The close agreement between the calculated binding constants for these compounds and the constants determined by direct measurement indicate that among the multiple binding sites on albuiiun (9, 10) the group of sites that bind thyroxine the most strongly [i.e., the first group of sites (see Eqs. 2 and 3)] are also the sites most effective in binding 2,4-dinitrophenol and dodecyl sulfate. This is similar to the competitive binding shown to exist between dodecyl sulfate and the anionic azo dye, p-(hydroxy-5-methylphenylazo)-benzoic acid (10). The effect of the fatty acid anions on thyroxine binding (Fig. 2, Table III) is qualitatively consistent with the data reported by Goodman (2). As shown by Goodman the affinity of fatty acids for human albumin increases with increasing chain length. According to Goodman (2) the fatty acid anions appear to be bound to at least three groups (or classes) of sites on albumin (nl = 2, n2 = 5, and n3 = 20) with the following values for ICI’: oleate, 1.l X 10s; linoleate, 1.3 X 107; palmitate, 6.0 X 107; laurate, 1.6 X 106. The values for kz’ are: oleate, 4.0 X 106; linoleate, 2.5 X 106; palmitate, 3.0 X 106; and laurate, 2.4 X 105. The values for k3’ were of the order of 1 X 103. From Table III it can be seen that

the calculated ks values for the fatty acid anions are much lower than the values for kl’ obtained by direct measurement by Goodman (2). The calculated k, values, particularly for oleate and linoleate, are of the order of magnitude of the equilibrium constants given (2) for the binding of the fatty acids at the second group of sites (&I), and it may be that this group of fatty acid-binding sites is also the group interacting most strongly with thyroxine (nl = 2, A+ = 2.75 X 105). From the available data no simple explanation can be given for the relatively low lc, values obtained for palmitate (Table III). It may be noted, however, that of the 3.1 moles of palmitate added per mole of defatted albumin (Fig. 3, Table IV) about 2 moles are estimated to be bound to the first group of sites (for palmitate, nl = 2, kl’ = 6.0 X 107), and only 1 mole would be bound at the second group of binding sites (nz = 5, Icz’ = 3.0 X 10”). If the latter binding sites were the loci at which the competition with thyroxine was taking place, then only about one third of the added palmitate would be available for competition and the effect of the anion would be less than expected. The ability of the different anions to displace thyroxine from binding sites on albumin may be of some physiological significance. For example, it is known that the intake of either sodium salicylate or 2 ,Pdinitrophenol leads to a reduction in the level of protein-bound iodine (PBI) in human blood (11-13) and an accelerated rate of loss of thyroxine from the circulation (14, 15). A partial explanation for the increased rate of disappearance of thyroxine from blood is that salicylate and 2,4dinitrophenol interfere with the binding of thyroxine by serum proteins (16-18). Christensen (16) has shown that both salicylate and 2,4-dinitrophenol are capable of increasing the rate of dialysis of thyroxine from human serum in vitro. Recently, Osorio (18) has demonstrated by means of electrophoresis of human serum on paper at pH 7.4 that salicylate and 2,4-dinitrophenol interfere specifically with the binding of thyroxine by albumin. Electrophoretic studies have shown that salicylate and 2,4-

EFFECT

OF ANIONS

ON THYROXINE

dinitrophenol also interfere with the binding of thyroxine by thyroxine-binding globuhn at pH 7.4 (18) and with binding by thyroxine-binding prealbumin at pH 8.4 (17). With regard to the effect of the fatty acid anions on thyroxine binding by human albumin it may be noted that almost all of the fatty acids (unesterified) circulating in human blood appear to be bound to albumin (2). According to the analytical results reported by Dole et al. (19) the fatty acid anions used in the present investigation (i.e., oleate, palmitate, and linoleate) would comprise 70-8076 of the total fatty acids in normal human blood. Saifer and Goldman (20) determined the composition of the fatty acids bound to a human albumin preparation similar to the albumin used in this study (Cohn Fraction V, American Red Cross) and obtained the following percentage distribution: oleate, 33 o/o; linoleate, 20 5%; and palmitate, 25%. The normal level of unesterified fatty acids in human blood is such that about 1 mole of fatty acid is bound per mole of albumin (2). It is of interest to note that the level of unesterified fatty acids in blood is found to rise in hyperthyroidism to a ratio of about 2 moles of fatty acid per mole of albumin (21). The rise in the concentration of circulating fatty acids in thyrotoxicosis might lead to an increased rate of removal of thyroxine from binding sites on plasma albumin and thus may be one of the factors involved in producing the acceleration in the rate of disappearance of thyroxine from the circulation that has been observed in hyperthyroidism (22-24). ACKNOWLEDGMENT The technical assistance is eratefullv acknowledned.

of N. A. Giorgio,

Jr.

BINDING

BY

ALBUMIN

421

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