THE <JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 21, Issue of November IO. pp. 8554-8563, 1975

Printed in U.S.A.

Studies on Thyroid Hormone-binding Proteins II. BINDING OF THYROID HORMONES, RETINOL-BINDING PROTEIN, AND FLUORESCENT PROBES

TO PREALBUMIN AND EFFECTS OF THYROXINE ON PREALBUMIN SUBUNIT SELF- ASSOCIATION*

(Received for publication, June 26, 1975)

STEN F. NILSSON, LARS RASK, AND PER A. PETERSON

From The Institute of Medical Chemistry, Biomedical Centre, University of Uppsala, Uppsala, Sweden

Vitamin A in human plasma is transported by its specific carrier protein, the retinol-binding protein. Under physiological conditions the protein forms a stable protein-protein complex with the tetrameric plasma protein, the thyroxine-binding prealbumin. Human prealbumin was shown to interact with the fluorescent probes l,%anilinonaphthalene sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS). ANS bound to the protein at two independent sites with the apparent association constant 3 x 10’ M-l, whereas TNS interacted with a single site with the binding constant 5 x 10’ M-l. The fluorescent yield of protein-bound ANS was 0.95, a more than 200.fold enhancement compared with that of ANS in aqueous solutions. TNS enhanced its quantum yield nearly 500-fold to 0.37.

On addition of thyroid hormones the fluorescent probes could be quantitatively displaced from the protein. This finding suggested that triiodothyronine, thyroxine, and the probes bound to a common site in prealbumin? which is likely to have a strongly hydrophobic character.

The association constants for the interaction between prealbumin and the thyroid hormones could be calculated by using the hormones as competitive inhibitors in the TNS-prealbumin titrations. The data from the competition experiments together with those obtained from equilibrium dialysis revealed one major hormone binding site on the protein. The calculated association constants were 9 x lo6 Mm’

and 1 x 10’ M- ’ for triiodothyronine and thyroxine, respectively. Prealbumin monomers were bound to Sepharose by covalent attachment, and their properties were

examined. Evidence was obtained demonstrating that the retinol-binding protein could interact with a single subunit of prealbumin. The estimated apparent association constant for the interaction of the protein and the matrix-bound monomeric prealbumin was 3 x 10’ M-l, approximately 250-fold lower than that measured for protein and matrix-bound tetrameric prealbumin. The data, however, strongly suggest that there are four retinol-binding protein sites per prealbumin molecule.

Using the technique of sedimentation equilibrium ultracentrifugation the prealbumin subunit self- association has been studied. The energy of the interaction for the prealbumin subunits is very high, and various concentrations of guanidine hydrochloride had to be used to perturb the equilibrium. All experiments indicated that prealbumin dissociates directly into monomers without the presence of intermediate forms. Thyroxine perturbed the chemical equilibrium of the prealbumin monomer-t’,Cramer system by strengthening the interaction between the subunits.

The fat-soluble vitamin A is transported from its main storage site in the liver to its target cells by a specific carrier plasma protein, the retinol-binding protein (l-3). It is a small protein, molecular weight 21,000, and should according to its size be rapidly eliminated from plasma due to glomerular filtration in the kidney (4, 5). However, under physiological conditions the protein is firmly bound to the thyroxine-binding prealbumin (l-3). The resulting protein complex, constituted by 1 molecule each of the two proteins, is of a size large enough

*This work was supported by the Swedish Medical Research Council (Projects B-74-13X-3531 and B74-03X.4-1OB) and by Svenska LivfdrsZkringsbolagens nHmnd fdr Medicinsk Forskning.

to restrict its glomerular filtration (6). Furthermore, the interaction between the protein and prealbumin enhances the affinity for retinol (7, 8), and the vitamin is more shielded from the aqueous medium on complex formation (9).

Recent results have clearly established that prealbumin is composed of four noncovalently linked, apparently identical, subunits, each of a molecular weight of approximately 15,000 (10-15). X-ray crystallographic examination of prealbumin has indicated that the subunits are arranged in a tetrahedral fashion (13). Therefore, except for the central cavity all structures in prealbumin occur with at least 2-fold symmetry (13).

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The present study was initiated to explore further the ligand fluorescent prohes to prealhumin was made fluorometrically with use

and protein interactions pertaining to the prealbumin-protein of three different methods. In the first method (18) the equation

complex. The mode of dissociation of the tetrameric preal- nPl(c, + K) = x + [K/cc, + K)] (x” + x3 t .) (1) bumin in the absence and presence of thyroxine was investi- gated by sedimentation-equilibrium ultracentrifugation, and the binding properties of the prealbumin subunit were investi- gated. The thyroid hormone binding site of prealbumin was also studied with use of fluorescent probes. The probes 1,8-anilinonaphthalene sulfonate and 2-p-toluidinylnaph- thalene&sulfonate were shown to bind to this site, and the quantum yields, the association constants, and the stoichiome- tries for the protein-probe interactions were calculated.

EXPERIMENTAL PROCEDURE

was used, where P is the total protein concentration, n the number of fluorophore binding sites on the protein molecule, and K the equilih- rium constant of dissociation. x is the fraction offluorophore molecules hound, F/F,, where F is the observed fluorescence intensity and F, the intensity when all of the fluorophore at the concentration C, is bound to the protein. The value F, is obtained by determining the fluores- cence intensity of a solution of AN’S or TNS at increasing protein concentrations, by graphing l/F versus l/P and determining the extrapolated value at infinitely high protein concentrations. For values of x small in comparison with unity, x2 and higher powers of x can be considered negligible, and in the plot n versus P the initial slope equals (n/C, + K). C, + K can be replaced by C, if C, >> K, and conse- quently the initial slope of the plot equals n/C,.

Proteins

Prealbumin was isolated from outdated plasma as previously described (11). Retinol-binding protein was prepared from urine of patients with tubular proteinuria according to an earlier published procedure (16). Peak 1 and Peak 2 materials’ were obtained by separating the protein on a prealhumin-coupled Sepharose column (17).

Fluorescent Probes

In the second method (23) the binding constant and the stoichiome- try for the ANS-prealhumin interaction and the corresponding interac- tion between TN’S and prealhumin were determined by using the equation of Scatchard

Y/C = K(n i) (2)

where in is the molar ratio of bound fluorophore to prealhumin; c, the concentration of free ligand; K, the apparent association constant; and n, the number of binding sites on the protein. The mole fraction of

The magnesium salt of 1,8-anilinonaphthalene sulfonate and 2-p. ANS or TNS bound at .each probe concentration was obtained by

toluidinylnaphthalene-6.sulfonate were obtained from Pierce Chemi- dividing the fluorescence intensity (F) by the corresponding F, value

cai Company, Rockford, Ill. The concentration of ANS* and TNS were which was obtained as described above.

determined hy measuring the ahsorhance at 350 and 317 nm, respec- In the third method employed to study the binding of TNS to

tively. The molar extinction coefficients used at the indicated wave- prealhumin, Equation 3 of the preceding paper was used (24).

lengths were 4.95 x lo3 M 1 cm -‘forANS (18) and 1.89 x 10’~~‘cm-’ Triiodothyronine and thyroxine were used as inhibitors in the fluores-

for TNS (19). cent probe-prealbumin titrations, and the binding constants for the hormones could he determined by using Equation 4 of the preceding

Thyroid Hormones

Nonradioactive sodium salts of L-triiodothyronine and I.-thyroxine were obtained from Sigma. The hormones were purified by gel chromatography on Sephadex LH-20 according to a previously de- scribed procedure (20). The concentration of triiodothyronine and thyroxine was determined by measuring the absorbance at 320 and 325 nm, respectively. The molar extinction coefficient used for triiodo- thyronine is 4.66 x lo3 M ’ cm-’ at pH 10 (21). The corresponding extinction coefficient used for thyroxine at pH 11 is 6.18 x 103 Mm’

cm ’ (22). Radioactive L-triiodothyronine and L-thyroxine of known specific activities were purchased from Amersham/Searle and purified as described above.

Other Materials

Sephadex G-25 and G200, Sepharose 4B, and Sephadex LH-20, products of Pharmacia Fine Chemicals (Uppsala, Sweden), were used according to instructions supplied by the manufacturer. Guanidine hydrochloride was purchased from Heico (Watergap, Delaware). All other chemicals were of the highest quality available.

Methods

Fluorescence Measurements-Most fluorescence experiments were carried out using an Aminco Bowman spectrofluorometer, but some emission spectra were recorded with a Zeiss ZFM 4C spectrofluorome- ter. All measurements were performed with cells of l-cm light path that were placed in a thermostatically controlled sample compart- ment. The temperature was kept at 22 r 1 O. The monochromator slit widths were never more than 1 mm, and complete spectra were recorded at each observation. Characterization of the binding of

‘Peak 1 and Peak 2 materials are denoted in order of their appearance on a column of prealhumin-coupled Sepharose. Detailed studies of Peak 1 and Peak 2 retinol-hindin:: protein have revealed that they have different conformations and that Peak 2 protein, but not Peak 1 protein, is able to form a complex with prealbumin. The differences encountered for the two forms of binding protein most probably derive from the different COOH-terminal amino acid se- quences (cf. Ref. 32).

‘The abbreviations used are: ANS, 1,8-anilinonaphthalene sulfonic acid; TNS, 2.p-toluidinylnaphthalene-6.sulfonate.

article (24). Determination of Quantum Yield-To correct for instrumental

variations of sensitivity of wavelength, a rhodamine B solution in ethylene glycol was used as a quantum counter (XI). The validity of the correction factors thus obtained was tested hy correcting the excita- tion and emission spectra earlier reported (26). Excellent agreement was obtained for both excitation and emission. Quinine sulphate, with a quantum yield of 0.55 in 0.1 N H,SO, (27), was used as a reference in all determinations. Samples used for fluorescence meas- urements always had an optical density below 0.05 at the exciting wavelength.

The buffer used in all fluorescence experiments was 0.05 M sodium phosphate, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA. All determinations were made in triplicate, and the intensities of fluores- cence were corrected for self-absorption (28).

Equilibrium Dialysis-The interaction of prealhumin with triiodo- thyronine and thyroxine was studied by means of equilibrium dialysis. The data were graphed according to the Scatchard form of the mass action equation (23). Details of the experimental setup are given in the preceding article (24).

Protein Labeling-Prealhumin was labeled with “‘1 (Amersham/ Searle) according to the chloramine T method of Greenwood et al. (29).

Preparation of Prealbumin Monomer-coupled Sepharose-Preal- humin was coupled to CNBr-activated Sepharose as previously de- scribed (17) using the following modifications as outlined by Chan (30). The amount of CNBr was greatly reduced, 2.5 mg/ml of’ packed Sepharose, to minimize the possibility of attachment of a prealhumin molecule to the gel uia more than one subunit. For the same reason the prealhumin concentration was kept below 1 ma/ml. After extensive washing with sodium phosphate buffer (0.05 M), pH 7.2, remaining activated groups in the Sepharose were allowed to react with 0.1 M glycine. By this procedure only about 20 to 30’4 of the added protein was covalently attached to the matrix.

For the preparation of Sepharose-hound subunit prealbumin, the above derivative was washed in a column at room temperature with 15 volumes of 6 M guanidine hydrochloride, adjusted to pH 4.8 with acetic acid, and then dialyzed against 0.05 M ‘Iris-HCI buffer, pH 7.4, containing 0.15 M NaCl to renature the remaining Sepharose-hound prealhumin. Glycine was coupled to Sepharose according to the procedure described above.

Measurement of the Affinity between Prealbumin Coupled to Sepharose and Retinal-binding Protein-Two-milliliter portions of

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prealbumin coupled to Sepharose suspended in 0.05 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl were added to small glass vials. Varying amounts of an equimolar mixture of Peak 1 and Peak 2 retinol-binding protein were added to the tubes, which were then tightly capped and allowed to rotate slowly during 48 hours at 4”. Subsequently, the tubes were centrifuged in a clinical centrifuge to remove the gel. Samples, removed from the clear top phase, were taken for separate determinations of Peak 1 and Peak 2 protein concentra- tions in the supernatant. The concentration of Peak 1 protein was calculated by subtracting the Peak 2 concentration, estimated by fluorescence measurements, from the total protein concentration, determined by a single radial immunodiffusion technique (31).

Calculations-It has been adequately demonstrated that of the two forms of retinol-binding protein only Peak 2 binds to prealbumin (32). It was, however, noted in the present experiments that due to the low protein concentrations employed, small amounts of Peak 2 protein were bound unspecifically to the Sepharose, i.e. to glycine-coupled Sepharose. This unspecific binding was found to be identical for Peak 1 and Peak 2 material. Accordingly, to distinguish between specific binding to prealbumin and unspecific binding to the matrix, an equal mixture of Peak 1 and Peak 2 protein was used. Since both forms are of identical size no correction for differences in distribution volume within the gel phase had to be employed.

The concentration of protein in the supernatant, a, is the sum of the concentrations of Peak 1 and Peak 2 retinol-binding protein. Accord- ingly,

a=p+q (3)

where p and q are the free concentrations of Peak 1 and Peak 2 protein, respectively. The added amounts of Peak 1 and Peak 2 protein, p and q, respectively consist of

P = P, + P, (4a)

Q= Q,+Q,+R (4b)

where f denotes free, b unspecifically bound, and R the amount of Peak 2 protein bound to prealbumin. Since in these experiments P equals Q and P, equals Q,

R = P, - Q, (ia)

All components in the reaction mixture occur in the same volume, V. Accordingly,

and thus

RIV=p-q (5b)

RJS = (p - c)/s (SC)

where S is the amount of matrix-bound prealbumin and s its concentration, S/V.

It is apparent that R/S equals V, the number of moles of ligand bound per mole of prealbumin, in the Scatchard version of the law of mass action (Equation 2). Accordingly, a plot of q (which equals c in Equation 2) uerau~ (p ~ q)/qs will yield a straight line, provided the reaction has a single association constant.

Ultracentrifu~ations-For the ultracentrifuge experiments with prealbumin in guanidine hydrochloride, the protein (0.1 to 1.0 mg/ml) was dissolved in guanidine of appropriate concentration and equili- brated by dialysis against solvent for at least 48 hours. Final adjustment to the desired protein concentration was accomplished by dilution with the solvent.

The dissociation of prealbumin was investigated using a Spinco model E ultracentrifuge equipped with a slit-beam photoelectric scanning absorption optical system (33). Sedimentation equilibrium studies were conducted at 20” using the high speed meniscus depletion technique of Yphantis (34) or a modification thereof (35). The time to reach equilibrium was reduced by using an overspeeding-under- speeding technique (36). The centrifuge runs were carried out at 24,000 to 32,000 rpm. At low initial concentrations recordings were made at 280 nm with use of the scanning system. When thyroxine was present the 280.nm absorbance was corrected for contributions by thyroxine. This was accomplished by separately measuring the thyroxine distribu- tion estimated as the absorbance at 330 nm throughout the cell. Point average molecular weights (M,,, and M,,,) were calculated as sug- gested by Yphantis (34). The assumptions used in the evaluation of the parameters for the prealbumin self-association are (a) that the partial

specific volumes of all species are the same in the presence of thyroxine; (6) that the refractive index increments of all species are equal; (cl that the system undergoes no volume change on chemical reaction.

Calculations of apparent M,,,, apparent M,,,, f,, and K were accomplished as described by Chun and Kim (37) and Chun et al. (38). The symbols used have the same meaning as in these two publications.

Errors in the estimation of the true prealbumin binding constant arise from errors in determining M, as a function of activity. In the present study such errors may be due to the presence in the protein solution of molecules of molecular weight less than M,, the generation of nonequilibrating, denatured monomer, error in the partial specific volume, sensitivity to base-line error of the absorption measurements in the analytical ultracentrifuge, and nonideal behavior. At very low 280 nm absorbances we sometimes obtained anomalously low M, apparent values which resulted in peculiar S-shaped plots. These values could be due to a contaminant of low molecular weight. but extensive tests for such heterogeneity in our preparation of prealbumin have been negative. It seemed more likely that uncertainty at low absorbances arose from the sensitivity of the method to base-line error. For this reason, we have not used values of apparent &f, obtained at low protein concentrations when S-shaped plots were apparent.

In this work we depended on a value for the partial specific volume obtained from amino acid analysis of prealbumin (31. Rigorously, U should be determined under all of the varied conditions employed in this study for both monomer and polymer. However, it is felt that the degree of departure from the conditions under which U is known to be valid is unlikely to introduce serious error in the calculations.

Nonideality can introduce serious errors for estimations of K apparent as well as in determining the mode of dissociation (39). The calculations employed in the present study, however, take this into account and permit the actual determination of the magnitude of HM, the nonideality term.

Amino Acid Analysis-Quantitative amino acid analyses were carried out as described by Spackman et al. (40). Matrix-bound prealbumin (0.2 to 0.8 mg) was hydrolyzed in 6 N HCl at 110’ for 24 hours (41). Chromatography was carried out on a Biochrom automatic amino acid analyzer (Biocal, Munich).

Other Methods-Radioactivity was measured in a gamma spec- trometer (Tri-Carb 3003, Packard Co., Chicago, Ill.).

Peak 2 retinol-binding protein was quantitatively estimated by measuring the retinol fluorescence. The measurements were carried out using an Aminco-Bowman spectrofluorometer. The exciting wave- length was 340 nm and the emitted light was recorded at 470 nm. No corrections for variation in the sensitivity of the detector system at different wavelengths were made in these measurements. Total con- centrations of Peak 1 and Peak 2 protein were estimated by a single radial immunodiffusion technique (31) employing a specific antiserum. Highly purified solutions of’ prealbumin or binding protein wzere quantitatively estimated by measuring the absorbance at 280 nm and relating the observed values to the relevant molar extinction co- efficient (3).

The circular dichroism spectrum of prealbumin was measured in a Jasco model 5-20 spectropolarimeter as outlined previously (421. The reduced mean ellipticity [0’] was calculated using a mean residue weight of 110.

RESULTS

Characterization of the Binding of ANS and TNS to Prealbumin-A striking enhancement of fluorescence, concom-

itant with a hypsochromic shift of emitted light from green to

blue accompanied the binding of the fluorescent probes ANS

and TNS to human thyroxine-binding prealbumin. ANS has a

maximum of emission at 515 nm in aqueous solutions and a

quantum yield of 0.004. When bound to prealbumin the

emitted light shifted its maximum to 470 nm, and the

quantum yield increased more than 200.fold to 0.95. TNS,

upon binding to the protein, exhibited a maximum of fluores-

cence at 415 nm, a hypsochromic shift of 85 nm compared with

the emission maximum in aqueous solutions. The correspond-

ing quantum yield of fluorescence was markedly enhanced

from 0.0008 (43) to 0.37, i.e. nearly 500-fold. In a previous

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paper (44) it was shown that prealbumin when excited at 280 nm gives rise to a strong fluorescence with its maximum at 350 nm. This is characteristic for a protein that contains trypto- phan. Addition of the fluorescent probes to prealbumin mark- edly quenched the tryptophan fluorescence emission concomi- tant with the appearance of the characteristic fluorescence of ANS and TNS at 470 and 415 nm, respectively. This quenching of the protein fluorescence is a result of energy transfer from the aromatic amino acid residues in prealbumin to the bound fluorescent probe. Moreover, in absence of energy transfer the excitation spectrum for the ANS and TNS fluorescence should coincide with the absorption spectra for these two ligands. An additional peak at 280 nm in the excitation spectra for ANS and TNS in the presence of prealbumin was, however, ob- tained for the two fluorescent probes studied, giving support to the energy transfer shown above.

The stoichiometry for the binding of ANS and TNS to prealbumin was determined using Equation 1 under “Experi- mental Procedure,” and the data obtained were plotted in a graph of x versus P. The concentration of fluorescent probe was never less than 5 x lo-” M in these experiments, and consequently the initial slope (x < 0.1) of the plot approx- imated n/c,. The concentration of fluorescent probe ranged between 5 x lo-’ M and 1 x lo-’ M. A typical experiment from the studies on the ANS-prealbumin interaction is shown in Fig. 1. The value of n was found to be 1.9, whereas the correspond- ing value for the TNS-prealbumin interaction, shown in Fig. 1, was 0.9.

The number of ANS and TNS binding sites on the preal- bumin molecule and the association constants were investi- gated using the Scatchard equation (Equation 2). The concen- tration of protein used in these experiments ranged between 5 x lo-’ M and 8 x lo-’ M. Extrapolated F, values for 15 concentrations of ANS and TNS (ranging in concentration from 5 x lo-’ M to 5 x lo-‘M) were achieved at infinitely high protein concentration, i.e. at a concentration where all probe molecules are bound, from the graph l/F versus l/P (see “Experimental Procedure”). The FO values thus obtained for the various concentrations of probe were plotted in the standard graph F, versus probe concentration. Fig. 2 shows representative Scatchard plots with ANS and TNS, respec- tively. It is evident from Fig. 2 that the plot extrapolates to a value of 1.9 on the abscissa. The graph does not deviate from a straight line, and consequently prealbumin is considered to have two equal independent ANS binding sites with the binding constant 3.2 x 10’ M-‘. In contrast, experiments with TNS revealed only a single binding site on prealbumin with the apparent association constant 5.1 x 10’ M-l.

The association constant for the TNS-prealbumin reaction was also determined by experiments using Equation 3 of the preceding paper (24). In these experiments the protein concen- tration was held constant at 5 x 1OM6 M whereas that of the probe was varied by dilution with the appropriate protein solution. The result from a typical experiment is shown in Fig. 3. The binding constant obtained from these titrations, 5.3 x 106 M-‘, is in excellent agreement with that found by the other experiment in this study (see above). Table I sum- marizes the results obtained from the studies on the interac- tions between prealbumin and the fluorescent probes.

Determination of the Binding Constants in Thyroid Hor- mone-Prealbumin Interactions by the Use of TNS and of

Equilibrium Dialysis-It was noted that the binding of triiodo-

1 X107M-')

FIG. 1 (left). Solutions of ANS (O-O, 5 x lo-’ M) and TNS (O-O, 5 x 10mL M) titrated with prealbumin. The buffer used in these experiments was 0.05 M sodium phosphate, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA. The exciting wavelengths for ANS and TNS were 370 nm and 360 nm, respectively, whereas the analyzing wavelengths were 470 nm and 415 nm, respectively.

FIG. 2 (right). Scatchard plots on the interaction between ANS (0) and TNS (O), respectively, and prealbumin. The concentration of nrotein used was 1.0 x lo-’ M. Buffer: 0.05 M sodium phosphate, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA. Exciting and analyzing wavelengths were 370 nm and 470 nm, respectively, for ANS and 360 nm and 415 nm, respectively, for TNS.

by the presence of TNS. Results from fluorescent probe-protein titrations depicted in Fig. 3 revealed that the two thyroid hormones acted like competitive inhibitors since the plots always intersected the ordinary at a common point. Equation 4 of the accompanying paper (24), which is analogous to the “inhibitor form” of the Lineweaver-Burk equation, was used to treat the data obtained in the competition experiment. The binding constants thus obtained were 8.2 x 10” M-’ and 1.1 x 10’ M-’ for triiodothyronine and thyroxine, respectively.

The interaction of the thyroid hormones and prealbumin was further characterized by means of equilibrium dialysis. The data obtained from these studies were treated according to the Scatchard equation (Equation 2 under “Experimental Proce- dure”). Typical results are depicted in Fig. 4. It can be seen from the figure that the graphs for the two hormones are compatible with one major hormone binding site on the prealbumin molecule (i is 0.95). The association constant obtained from the slope is 9.6 x 10’ M-’ for the triiodothyro- nine-prealbumin interaction, whereas the corresponding con- stant for thyroxine is 9.3 x 10’ M-l.

However, a slight curvature in the plots was always noted. This finding is most easily explained by the assumption that there exist additional weak thyroid hormone binding sites (44).

Properties of Prealbumin Monomer Bound to Sepharose- Prealbumin was covalently bound to Sepharose employing low concentrations of the activating CNBr to minimize the number of prealbumin molecules attached to more than one site on the matrix. The prealbumin-coupled Sepharose was washed with 6 M guanidine hydrochloride. This treatment should eliminate all prealbumin subunits from the gel which were not covalently attached to it. Table II summarizes the result of analyses performed on the prealbumin-coupled Sepharose to estimate the amount of bound protein prior to and after washing with the guanidine hydrochloride. Approximately one-fourth of the initially bound prealbumin remained on the gel after washing, indicating that only one subunit per prealbumin molecule was covalently attached to the Sepharose. Furthermore, when the

thyronine and thyroxine to prealbumin was markedly impeded prealbumin monomer-coupled Sepharose was mixed with an

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excess of prealbumin in 6 M guanidine hydrochloride and subsequently the denaturant was removed by. dialysis, it was found that the gel contained approximately four times as much prealbumin. This demonstrated that the monomeric preal- bumin could bind free subunits to reform the tetrameric molecule (cf. Table II). The guanidine hydrochloride washed gel, dialyzed against 0.05 MTris-HCl buffer, pH 7.4, containing 0.15 M NaCl, thus represented matrix-bound prealbumin monomers.

Prealbumin coupled Sepharose binds retinol-binding protein which can be eluted from the gel by lowering the ionic strength (17). To test the ability of the prealbumin monomer to bind the

v FIG. 3 (left). Representative fluorescence titrations of prealbumin

(1.0 x 10m7) with TNS (04). Titrations of the protein with TNS in the presence of triiodothyronine (8.0 x 1Om8 M), (O----O) and thyroxine (5 x 1Oe8 M), (A-A). All titrations were performed in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.15 M NaCl and 1 mM

EDTA. The exciting and analyzing wavelengths used were 370 nm and 420 nm, respectively.

FIG. 4 (right). Scatchard plots of the interaction between triiodo- thyronine and prealbumin (upper) and thyroxine and prealbumin (lower) measured by equilibrium dialysis. The prealbumin concentra- tion was in the triiodothyronine experiment 2.0 x 10m7 M, and in the thyroxine experiment 1.0 x 1Om8 M. The buffer used was composed of 50 mM sodium phosphate, pH 7.5, 0.15 M NaCl, and 1 mM EDTA.

TABLE I

Properties and protein content of matrix-bound prealbumin

derivatives

Association constants, number of binding sites, and quantum yields for All data given are the mean of three experiments. The values are the fluorescent probe-prealbumin interactions expressed per ml of packed Sepharose gel.

Probe Use of

equation number”

Association constant

Number of binding

sites

Quantum yield of

fluorescence

ANS 1 1.9 2 3.2 x 10’ 1.9

0.95 TNS 1 0.9

2 5.1 x lo6 1.1 3b 5.3 x 105

0.37

u See “Experimental Procedure.” b Refers to Equation 3 of the preceding paper (24).

protein, the gel containing monomeric prealbumin was packed into a column and retinol-binding protein was subjected to chromatography on the column. The retinol-binding protein preparation employed consisted of an equimolar mixture of Peak 1 which does not bind to prealbumin and Peak 2 material. It can be seen in Fig. 5 that no retinol-binding protein bound to the column with an affinity high enough to be retained. However, of the eluted material Peak 1 protein appeared earlier (demonstrated by the absorbance curve at 280 nm) than Peak 2 (estimated from the appearance of the specific retinol fluorescence). The ratio of protein to retinol was accordingly high in the frontal part of the eluted peak and low, close to unity, in the rear part. A control experiment employing glycine-coupled Sepharose demonstrated that no separation occurred between Peak 1 and Peak 2 retinol-binding protein on such a column (Fig. 5). This result therefore shows that monomeric prealbumin retained its affinity for Peak 8 protein, although to a diminished degree.

Measurements of the Affinity between Retinal-binding Pro-

tein and Monomeric Prealbumin-The experiments depicted in Fig. 5 clearly indicated that the prealbumin monomer was able to interact with retinol-binding protein. The affinity for the protein was, however, evidently diminished. An experiment was therefore devised to estimate the apparent association constant. The rationale of the procedure and the experimental details are given in “Experimental Procedure.” Fig. 6 depicts a Scatchard plot of the data obtained for monomeric as well as tetrameric prealbumin-coupled Sepharose. The apparent association constants differed approximately 250-fold (Kapp was 8 x lo6 Mm1 and 3 x 10’ M-l, respectively). The intercept on the abscissa was approximately 0.45 for both types of gels, indicating that less than 50% of the prealbumin was accessible to retinol-binding protein.

Mode of Dissociation of the Prealbumin Tetramer-Preal- bumin on sedimentation-equilibrium analytical ultracentrifu- gation exhibits molecular weight distributions such as shown in Fig. 7. The figure depicts the weight average molecular weight data as a function of concentration. Because the molecular weights decrease at low concentration, the tetra- merit molecule does not seem to be the only species present in the solution. However, at very low concentration, errors in molecular weight measurements are difficult to avoid for experimental reasons. It is therefore hard to establish unequiv- ocally whether such a dissociation is characteristic of an

TABLE II

Protein Retinol-binding protein bound

Prealbumin tetramer Prealbumin monomer Renatured prealbumin

of’ PMb w WC 9od 13.6 13.8 100 1.50 44

3.42 3.56 25.7 1.58 45 12.0 12.1 88.4 1.47 49

a Determined by amino acid analysis. bDetermined by estimations of the amount of matrix-bound “‘I-

labeled prealbumin. ‘ Determined by extrapolation of Scatchard plots (cf. Fig. 7). d Percentage occupied ligand binding sites in prealbumin assuming

one site per tetramer and monomer, respectively.

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EFFLUENT VOLUME (ml)

FIG. 5 (left). Affinity chromatography of retinol-binding protein on Sepharose. The column (1.5 x 3.4 cm) contained the following: A, 13.5 nmol of monomeric prealbumin/ml of packed gel, prepared as de- scribed in the text; B, 13.7 nmol of tetrameric prealbumin/ml of packed gel; and C, 20 nmol of glycine/ml of packed gel. An equimolar mixture of Peak 1 and Peak 2 protein (total, 130 nmol of binding protein) was applied to the columns in a volume of 6.0 ml. The buffer used was 0.02 M Tris-HCl, pH 7.4, containing 0.15 M NaCl. The arrow indicates the start of the elution with 2 mM Tris-HCl buffer, pH 8.0.

artifact of computer techniques, contamination of the prepara- tion by small material, or a true chemical equilibrium. Dissociation of prealbumin at higher protein concentrations can, however, be achieved only in solutions of guanidine hydrochloride. Thus, a series of experiments was carried out in solvents containing this compound.

It was noted that prealbumin dissociated poorly at pH 8.0, even in the presence of high concentrations of guanidine hydrochloride (47), but lowering the pH to 4.8 prompted a rapid dissociation of prealbumin into its constituent polypep- tide chains in 6 M guanidine hydrochloride. At this pH, in the presence of various concentrations of guanidine hydrochloride, prealbumin exhibited a considerably changed circular dichro- ism spectrum, the most notable feature of which was the diminished ellipticity at about 212 nm. Since experiments in the ultracentrifuge revealed that a great proportion of the prealbumin molecules displayed molecular weights smaller than the tetramer even at concentrations of guanidine hydro- chloride as low as 1 M if the pH was kept around 4.8, all experiments were performed at this pH.

In order to establish that a solute system is in chemical equilibrium it is necessary to demonstrate that point-by-point molecular weight moments are functions only of concentration (45). Fig. 8 shows a high speed sedimentation equilibrium experiment in 2 M guanidine hydrochloride with prealbumin at initial concentrations of 0.2, 0.6, and 1.0 mg/ml.

Fig. 8A depicts the number average molecular weights and demonstrates that they are reasonably superimposable. Also M,,, data (Fig. 8B) in three channels appear in a manner expected for a system in chemical equilibrium. Tests like these

I I I 1 1

01 0.2 0.3 0.4 0.5 CONCENTRATION (g/l)

FIG. 6 (center). Scatchard plots of the interaction between binding protein and monomeric (A) and tetrameric (B) prealbumin-coupled Sepharose, respectively. The buffer used was 0.05 M Tris-HCl, pH 7.4, containing 0.15 M NaCl.

FIG. 7 (right). M, data for prealbumin as a function of concentra- tion. The experiment was conducted at 24,000 rpm in 0.02 M Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl. The initial prealbumin concentrations were 0.2 g/liter (O), 0.6 g/liter (O), and 1.0 g/liter (0). Data were recorded using the photoelectric scanning system.

1 I I I I 1

50 A

"?o 50

x

,' LO

30

20 1 1 4 , I I I

02 0.L 0.6 0.8 IO 12

CONCENTRATION (g/l)

FIG. 8. M, (A) and M, (B) data for prealbumin in 2.0 M guanidine hydrochloride at pH 4.8. The experiment was conducted at 30,000 rpm at initial protein concentrations of 0.3 g/liter (O), 0.6 g/liter (0), and 1.0 g/liter (W), respectively, in the three channels.

were performed at all guanidine hydrochloride concentrations, since the possibility of irreversible dissociation of prealbumin had to be investigated. All the data were compatible with the view that chemical equilibrium was achieved and no signs of higher molecular weight material than the prealbumin tetra-

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mer or irreversibly denatured prealbumin monomers were apparent. In addition, prealbumin in 4 M guanidine hydrochlo- ride (the highest concentration of guanidine hydrochloride employed) was diluted with water to a final concentration of 2 M, and the resulting solution exhibited molecular weight distributions indistinguishable from those obtained with preal- bumin directly dissolved in 2 M guanidine hydrochloride.

A second criterion of chemical equilibrium was offered by experiments in 30-mm centerpieces which theoretically should yield the same dissociation constant as those performed with 12-mm centerpieces. This was the case as demonstrated in Table III.

To establish the dissociation reaction mechanism, use was made of the graphical procedure developed by Chun and Kim (37). Provided only two species are involved in chemical equilibrium, the resulting values for M,, l/M,, and f1 will lie along one of the lines of the standard graphs (Fig. 9). It can be seen in Fig. 9 that all data computed from the prealbumin molecular weight distribution in 2 M guanidine hydrochloride corresponded closely to the l-4 line, establishing that a monomer-tetramer equilibrium is operating in the prealbumin system. Data obtained at guanidine hydrochloride concentra- tions lower than 2 M corroborated the finding in Fig. 9. At higher guanidine hydrochloride concentrations the data de- viated from the 1-4 line. Since we had previously established that a chemical equilibrium most probably was valid also at high guanidine hydrochloride concentrations it was likely that nonideality parameters were introduced. Accordingly, the data were computed according to the procedure of Chun et al. (38) taking nonideality into account. Fig. 10 which is a standard graph of Chun et al. shows that the second virial coefficient was non-negligible and that the data in this plot appear along the l-4 line. It, therefore, seems reasonable to conclude that prealbumin in guanidine hydrochloride (0.5 M to 4 M) appears to be in a monomer-tetramer equilibrium.

Evaluation of Association Constants for the Monomer- Tetramer Equilibrium-Estimation of the apparent associa- tion constant governing the monomer-tetramer equilibrium was performed according to Chun and Kim (37) assuming an ideal system or according to Chun et al. (38) when nonideality was apparent. A representative plot of (1 - fI)/fl uersus (cfJ”-’ is shown in Fig. 11. The value adopted for n is four, since all data were compatible with the operation of a monomer-tet- ramer equilibrium for prealbumin. In most individual runs the standard deviation of K,,, was low (usually less than 15%) whereas in identical experiments differing only in prealbumin preparations employed, the K,,, differed in extreme cases as much as 5-fold. This discrepancy was most probably not the result of variations in the degree of purity, but reflected uncontrollable parameters like the age of the starting material for the purification, etc. Similar discrepancies have been reported for other proteins (36). Accordingly, the same preal- bumin preparation was used for all studies. The apparent association constants obtained at various concentrations of guanidine hydrochloride are summarized in Table III.

Effect of Thyroxine on the Prealbumin Self-association-As described above, thyroid hormones appear to have one strong binding site in prealbumin. For reasons of symmetry it is likely that the thyroid hormones bind to. the central cavity in prealbumin, which is formed by the four subunits (13). Moreover, this would suggest that 1 thyroxine molecule might interact with more than one subunit at a time. It is conceivable

TABLE III

Apparent association constants for the prealbumin monomer-tetramer equilibrium in the presence and absence of

thyroxine

All data are the mean i S.E.M. of’ four separate experiments. Both

M, and M, data are included in the table.

Guanidme Prealbumin

hydro- to K 81’1’

chloride thyroxine

ratio

B

0.5 M 2.1 * 0.4 x 108

1:2 4.9 * 1.1 x 108

1:6 6.8 * 2.1 x 106

1.0 M 2.8 + 1.6 x 10’

1:2 4.0 i 3.1 x 108

1:6 6.1 i 2.8 x 10’

1.3 M 7.2 + 4.2 x lo5

1:2 2.5 * 1.8 x 106

1:6 5.0 * 3.1 x 106

2.0 M 4.9 i 1.6 x 10’

1:2 1.6 I 1.1 x 10’

1:6 2.9 + 1.6 x lo6

3.0 M 7.5 + 4.8 x 10’ 1.2 x 10m6

1:2 1.2 * 0.4 x 103 1.6 x lo-’

1:6 5.2 * 1.2 x 103 0.8 x lOme

4.0 M 1.1 * 0.2 x 10 3.1 x 10-S

1:2 4.2 + 1.8 x 10 2.3 x lo-’

1:6 8.2 i 2.4 x 10 3.8 x 1om5

that such a mode of binding might increase the apparent association constant for the subunit-subunit interaction. To test this idea, sedimentation-equilibrium ultracentrifugations were performed in the presence of thyroxine, since experiments have shown that prealbumin retained its affinity for thyroxine even in 4 M guanidine hydrochloride.

At all guanidine hydrochloride concentrations employed, standard graphs according to Chun and Kim (37) demon- strated unequivocally that the prealbumin mode of dissocia- tion was tetramer to monomer without the presence of interme- diary species to any measurable extent. It thus seems reasona- ble to conclude that prealbumin exhibited a monomer-tet- ramer chemical equilibrium also in the presence of thyroxine.

Evaluation of the apparent association constants for the prealbumin monomer-tetramer equilibrium at various concen- trations of guanidine hydrochloride and thyroxine was per- formed as described above. The data obtained are summarized in Table III. It is apparent from the table that at each guanidine hydrochloride concentration examined, thyroxine shifted the equilibrium in favor of the tetramer. This result is thus compatible with the notions that thyroxine binds to structures engaging more than one subunit and that the binding site is close to the center of the protein.

DISCUSSION

The intricate problem of the protein-protein and ligand- protein interactions pertaining to the prealbumin-binding protein complex is far from understood. The protein complex involves five polypeptide chains and two ligands, thyroxine and retinol, and it is evident that this poses certain problems for reasons of symmetry. A number of investigators have been able to find the binding of only a single molecule of thyroxine and binding protein to prealbumin. To shed further light on the interaction between the proteins and ligands this study was performed.

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I 05 10

f 1

2.8 1-L

2.4 i \

2.0 -

r\ 1.6 -

1.2 -

0.8 -

I I I I I

02 0.4 0.6 0.8 1 0

5

FIG. 9 (left). Standard graphs according to Chun and Kim (37) demonstrating the theoretical lines for various discrete chemical equilibria. The dots represent data obtained for prealbumin in 2.0 M guanidine hydrochloride. The numbers in the figures indicate the mode of dissociation.

FIG. 10 (center). Standard graph of Chun et al. (38) demonstrating

Fluorescent probes have been found useful in a number of studies concerned with the exploration of ligand binding sites (46). These probes often exhibit enhanced fluorescence on their binding to hydrophobic sites in proteins. It is well established that prealbumin binds thyroid hormones which by their hydrophobic nature are supposed to interact with nonpolar sites in the protein. It thus appeared likely that ANS and TNS might bind to structures in prealbumin involved in the thyroid hormone binding. The results presented in this paper show indeed that this was the case. ANS and TNS bound to prealbumin as measured by fluorescence enhancement, by a hypsochromic shift of the emitted fluorescence, and by energy transfer of the protein tryptophyl fluorescence to the ligands.

The quantum yield of ANS increased to 0.97 on binding to prealbumin. This very high fluorescence enhancement indi- cates that ANS bound to very hydrophobic structures in the protein. The data are consistent with the idea that ANS binds independently at two sites with an apparent association constant of 3 x 10’ M-l. Since crystallographic work on prealbumin has shown that this molecule has 2-fold symmetry (13) it may be anticipated that the ANS binding site in prealbumin consists of structures contributed by two subunits.

The TNS fluorescence showed a considerable enhancement when the probe was found to prealbumin, giving a quantum yield of 0.37. TNS, in contrast to ANS, however, bound only to a single site in the prealbumin molecule. It appears likely, however, that both probes bound to sites in. close proximity. This can be inferred from the fact that ANS as well as TNS were displaced from the prealbumin molecule by triiodothyro- nine and thyroxine (see below).

the theoretical line for a monomer-tetramer equilibrium when noni- deality is operative. The dots in the figure are data obtained for prealbumin in 4.0 M guanidine hydrochloride.

FIG. 11 (right). Graph for evaluation of the association constant for the prealbumin monomer-tetramer equilibrium in 4.0 M guanidine hydrochloride. Filled symbols are MU-data and open symbols M, data.

It has earlier been found that 2 mol of 4-hydroxy-3,5-diiodo- benzaldehyde bind to 1 mol of prealbumin (47). It thus appears likely that there are two equal and independent sites in the prealbumin molecule that can bind ANS and 4-hydroxy-3,5- diiodobenzaldehyde. Other ligands like TNS, triiodothyronine, and thyroxine which obviously give only binary complexes with prealbumin may bind to the same structures in the protein. Due to their size or orientation or due to other factors these ligands when bound to one site may sterically exclude binding to the other identical site.

By means of the triiodothyronine and thyroxine competition with ANS and TNS for binding to prealbumin it was possible to evaluate the strength of interaction between the thyroid hormones and the protein. Triiodothyronine and thyroxine bound to single sites with apparent association constants of 9 x lo6 M-’ and 1 x lo6 M-‘, respectively, which is in excellent agreement with the results of Pages et al. (48). Equilibrium dialysis studies corroborated these findings and gave evidence for a single thyroid hormone binding site in prealbumin. The rather low association constants obtained in a previous study (44) can possibly be explained by the indirect methods used. In the earlier publication it was also suggested that there exist additional thyroid hormone binding sites of much lower affinity. The idea was therefore advanced that the thyroid hormones might possibly bind to prealbumin in a cooperative fashion. The presently described experiments corroborate that indeed there is more than a single thyroid hormone binding site in prealbumin. It now appears unlikely, however, that this implies the existence of more than one physiological thyroid hormone binding site, since ANS and TNS only compete with

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the binding of hormone to the high affinity site. Prealbumin exhibits in addition to its thyroxine-binding

ability a specific interaction with the retinol-binding protein (l-3). For reasons of symmetry it would be anticipated that either thyroxine or retinol-binding protein binds to the center of prealbumin. A way to examine this possibility is afforded by sedimentation-equilibrium ultracentrifugations. It is, hence, possible to estimate directly the subunit interaction energies for the liganded and unliganded protein, respectively. The determination of equilibrium constants in highly associated systems by sedimentation equilibrium leads, however, to large errors in the calculated equilibrium constants (36). As the observed molecular weight moments approach the molecular weight of the tetramer, the value of the apparent association constant becomes very large. In this study we have attempted to circumvent this difficulty by the calculation of point-aver- age association constants together with an averaging system to obtain the best constants (37). This seems to have been effective, since experiments performed in 12-mm and 30-mm cells yielded very similar association constants. However, even this approach would have been unsuccessful if guanidine hydrochloride had been excluded from the solvent.

In spite of the numerous parameters which may affect the estimation of the true prealbumin self-association (see “Exper- imental Procedure”) it seems possible to conclude from the present data that prealbumin dissociates directly to monomers without the presence of intermediary forms. Determinations of the apparent binding constant for the monomer-tetramer equilibrium were heavily dependent on the guanidine hydro- chloride concentration. All data are, however, compatible with thyroxine being a specific perturbant of the chemical equilib- rium. The energy of subunit interactions was, thus, reproduci- bly higher in the presence of thyroxine and regardless of the accuracy of absolute parameters, this observation is compati- ble with the thyroid hormone binding site of prealbumin being situated in the center of the molecule. X-ray analysis has shown that there is a central cavity, lined by the subunits, which may harbor the binding site (13). Moreover, the present data are consistent with the fact that more than one subunit is engaged in binding a single molecule of the thyroid hormones.

It is, however, difficult to characterize the exact mechanism of the interactions using centrifuge data alone. All that can be said at present is that interaction with thyroxine leads to increased subunit binding. Some additional approach is neces- sary to ascertain exactly how thyroxine alters the monomer- tetramer equilibrium.

In previous studies, it has been shown that prealbumin and binding protein occur physiologically in a complex exhibiting one to one stoichiometry (l-3). In view of the fact that thyroxine occupies the center of the prealbumin molecule, a reinvestigation of the interaction between prealbumin and retinol-binding protein was warranted. Since prealbumin is composed of four identical polypeptide chains there should possibly exist more than one protein binding site. Following the method devised by Chan (30) matrix-bound monomeric prealbumin was prepared. Such a derivative would enable di- rect studies of the interaction of retinol-binding protein and the prealbumin subunit. A 75% reduction of the amount of preal- bumin coupled to Sepharose would be.expected on the basis of the change from the tetrameric to the monomeric state. The actual values (25.1 and 26.2% measured by independent methods) suggested that a small portion of the protein might

have been bound to the matrix uia more than one subunit. It ap- pears unlikely, however, that this excess amount of subunits (less than 1.2%) would be responsible for the protein binding. Apart from the protein content, several other observations suggest the presence of monomers in this derivative, notably the renaturation experiment. Thus, renaturation resulted in association of additional subunits to the monomeric preal- bumin-coupled Sepharose. Furthermore, the properties of the renaturated matrix-bound protein were very similar to those of the original derivative.

Although all data are compatible with the preparation of matrix-bound monomeric prealbumin, measurements on the interaction of ligands or proteins with the derivative have to be interpreted with caution. The first difficulty arises from the fact that the protein is immobilized onto a matrix. The possible binding sites for binding protein are probably strictly defined. That means that they may be inaccessible due to steric hindrance by the matrix. Furthermore, the matrix itself may exhibit affinity for the ligand or the protein. Another problem, which is harder to assess, is given by the conforma- tional state of the monomer. Assuming that the guanidine hydrochloride treatment does not irreversibly derange the subunit structure, the conformation of the free subunit is still obscure. The mere fact that the protein occurs in a monomeric form would most likely implicate a conformational change since polypeptide stretches, many of which are hydrophobic, normally buried by the neighboring subunits, would be fully exposed to the aqueous medium. It would thus seem likely that charged residues normally present on the surface of the monomer should be redistributed. Theoretically, such changes could have profound effects on any specific interaction. How- ever, according to this view, new unspecific binding sites could arise as well, especially involving hydrophobic ligands. It was therefore impossible to evaluate the binding of thyroxine to the matrix-bound monomer. The circumstances that retinol-bind- ing protein occurs in two physiological forms only differing in vitamin A content and in the COOH-terminal amino acid se- quence (32), however, renders control experiments feasible. Assuming that the two forms of binding protein behave identically with respect to unspecific binding to the matrix or to newly exposed prealbumin sites, appropriate corrections could be performed. The validity of this assumption gained support from direct measurements.

It was evident that monomeric prealbumin had a considera- bly diminished affinity for retinol-binding protein compared to the tetrameric protein. Chromatography of retinol-binding pro- tein on the derivatized Sepharose gel did not result in the protein being retained, but merely retarded. Direct estimation of the affinity constant of the interaction of retinol-binding protein and monomeric prealbumin corroborated this result. It was thus found that the apparent association constant was decreased approximately 250.fold compared to the value ob- tained from the interaction of the protein and matrix-bound tetrameric prealbumin. The reaction stoichiometry obtained (0.45 mol of protein/m01 of prealbumin) was identical for the two prealbumin derivatives, It seems inevitable from this figure that retinol-binding protein, indeed, was specifically bound to the monomeric prealbumin. The results obtained are thus fully compatible with the view that prealbumin has multiple protein binding sites, probably four, since the subunits are identical. The diminished affinity between retinol-binding protein and monomeric prealbumin may be

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explained on the assumption that monomeric prealbumin has a somewhat different conformation when free than when inter- acting with its neighboring subunits.

One conspicuous result is the reaction stoichiometry of retinol-binding protein and prealbumin-coupled Sepharose. The value 0.45 obtained both for the monomeric and tetrameric protein can probably be explained by retinol-binding protein being sterically hindered by the gel matrix to interact with all possible binding sites. Despite this, it is astonishing that tetrameric prealbumin did not bind several binding protein molecules. After completion of this work van Jaarsveld et al.

(49) showed that 1 molecule of prealbumin in solution could bind 4 binding protein molecules. The reason for the dis- crepancy noted between these data and the present is not immediately apparent.

Acknowledgments-We wish to express our sincere gratitude to Professor T. C. Laurent for generously placing an ultracen- trifuge at our disposal. Miss Yvonne Fernstedt provided excellent technical assistance.

1. Kanai, M., Raz, A., and Goodman, D. S. (1968) J. Clin. Inuest. 47, 2025-2044

2. Peterson, P. A. (1969) Abstracts of Uppsala Dissertations in 37. Medicine 75, l-12 38.

3.

4.

5.

Peterson, P. A. (1971) J. Biol. Chem. 46, 34-43 Peterson, P. A. (1971) Eur. J. Clin. Inuest. 1,437-444 Smith, F. R., and Goodman, D. S. (1971) J. Clin. Inuest. 50,

2426-2436

6. Vahlquist, A., Peterson, P. A., and Wibell, L. (1973) Eur. J. Clin. Inuest. 3, 352-362

7. 8. 9.

10.

Peterson, P. A. (1971) J. Biol. Chem. 246, 44-49 Goodman, D. S., and Raz, A. (1972) J. Lipid. Res. 13, 338-347 Peterson, P. A., and Rask, L. (1971) J. Biol. Chem. 246,7544-7550 Alper, C. A., Robin, N. J., and Refetoff, S. (1969) Proc. N&l. Acad.

Sci. U. S. A. 63, 775-781 11.

12.

Rask, L., Peterson, P. A., and Nilsson, S. F. (1971) J. Biol. Chem. 246, 6087-6097

Branch, W. T., Jr., Robbins, J., and Edelhoch, H. (1971) J. Biol.

Chem. 246, 6011-6018 13.

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Blake, C. C. F., Swan, I. D. A., Rerat, C., Berthou, J., Laurent, A., and Rerat, B. (1971) J. Mol. Biol. 61, 217-224

Gonzalez, G., and Offord, R. E. (1971) Biochem. J. 125, 309-317 Morgan, F. J., Canfield, R. E., and Goodman, D. S. (1971)

Riochim. Biophys. Acta 236, 798801 Peterson, P. A., and Berrggdrd, I. (1971) J. Biol. Chem. 246, 25-33 Vahlquist, A., Nilsson, S. F., and Peterson, P. A. (1971) Eur. J.

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S F Nilsson, L Rask and P A Petersonthyroxine on prealbumin subunit self association.

retinol-binding protein, and fluorescent probes to prealbumin and effects of Studies on thyroid hormone-binding proteins. II. Binding of thyroid hormones,

1975, 250:8554-8563.J. Biol. Chem. 

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