Studies on the Interaction Between Prealbumin, Retinol-binding … · RBP, still present in the...

THE JOURNAL OF B~LOGICAL CHEM~TRY Vol. 246, No. 1, Issue of January 10, pp, 44-49, 1971

Printed in U.S.A.

Studies on the Interaction Between Prealbumin, Retinol-binding Protein, and Vitamin A*

P. A. PETERSON

(Received for publication, August 12, 1970)

Frona the institute of Medical Chemistry, University of Uppsala, Uppsala, Sweden

SUMMARY The dissociation of the human vitamin A-transporting

protein complex into free prealbumin and free retinol-binding protein (RBP) was studied by gel chromatography under varying conditions of pH and ionic strength. The interaction between the two proteins was diminished at a low ionic strength and complete dissociation seemed to occur at an ionic strength of 0.002. Variations in pH did not appear to alter seriously the afhnity between prealbumin and RBP as the prealbumin-RBP complex was the predominant com- ponent in the pH range of 8 to 3.

varying values of ionic strength and pH. The behavior of free prealbumin and RBP at low ionic strength was also studied. Observations were made concerning the effect of antibodies on the stability of the prealbumin-RBP complex, and additional molecular properties of the prealbumin-RBP complex, prealbumin, and RBP were determined.

EXPERIMENTAL PROCEDURE

Materials

The release of vitamin A from free RBP and from prealbumin-RBP was studied at various values of pH and ionic strength. A facilitated release of retinol from RBP was found at conditions of low ionic strength, whereas variations in pH gave rise to only moderate changes in the release velocity of vitamin A. The binding of prealbumin to RRP seemed to strengthen the interaction between RBP and retinol.

These observations suggested a conformational change of one or both of the two proteins at low ionic strength. Sedi- mentation velocity analyses performed at various values of ionic strength indicated that RBP was amenable to conformational changes at a low ionic strength. Variations in ionic strength did not seem to alter the sedimentation behavior of prealbumin.

Proteins-Highly purified preparations of prealbumin-RBP, prealbumin, and retinol-binding protein were isolated from normal serum as described in a previous communication (2). RBP was also isolated from urine of patients with tubular pro- teinuria (3). A commercial preparation of prealbumin was purchased from Behringwerke AG, Mahrburg/Lahn (Germany). The following proteins were used as reference substances in analytical gel chromatography: human serum albumin (AB Kabi, Stockholm) ; light and heavy chains of a yG-myeloma protein (4) ; @, &, and o1 chains of haptoglobin (5) ; and &- microglobulin (6).

Immunochemical analyses showed that the prealbumin- RBP complex dissociated in the presence of antibodies directed against RBP, whereas antibodies directed against prealbumin did not seem to affect the interaction between prealbumin and RBP. These data suggest that RBP is more liable to conformational changes than is prealbumin.

Antisera-The rabbit anti-RBP serum has been described previously (2). An antiserum against prealbumin was obtained from Behringwerke AG (Mahrburg/Lahn, Germany).

Other Materials-Sephadex G-100 and G-200 were obtained from Pharmacia AB (Uppsala). Partially hydrolyzed starch was a product of Connaught Laboratories (Toronto, Canada). Guanidinium hydrochloride (Sigma) was treated with activated charcoal to remove material with absorbance in the ultraviolet region. For ultracentrifugations, specially purified guanidinium hydrochloride (British Drug Houses) was used. Urea (Mallinck- rodt), reagent grade, was deionized prior to use by passage over an Amberlit,e MB-1 column (Mallinckrodt) .

All other chemicals were of the highest quality available.

Vitamin -4 in the alcohol form (retinol) circulates in blood attached to its binding protein (1,Z). It was recently established that this retinol-binding protein is bound to prealbumin in normal plasma (1, 2). The complex between the two proteins dissociates at a low ionic strength (2).

An account is given in this paper of a qualitative investigation on the interaction between prealbumin, RBP’, and retinol at

Methods

* This work was supported by Swedish Medical Research Coun- cil Project 13X-512 and by grants from the Lennanders’ Research Foundation.

UZtracelztrifugationDeterminations of sedimentation velocities were performed in a Spinco model E analytical ultracentrifuge, equipped with a RTIC temperature unit. All meas- urements were conducted in a 12-mm standard double sector cell with sapphire windows. Recordings were made with the phase plate schlieren optics or with the photoelectric scanning system at 280 nm. The centrifuge was operated at 20’ at a speed of 59,780 rpm. Calculations were carried out according to Schachman (7).

1 The abbreviation used is: RBP, retinol-binding protein. Sedimentation equilibrium ultracentrifugations were per-

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Issue of January 10, 1971 P. A. Peterson 45

formed in 0.02 M Tris-HCl buffer, pH 8.0, containing 6 M guanidine hydrochloride and 0.15 M NaCI. Determinations were carried out according to the overspeeding-underspeeding technique of Richards, Teller, and Schachman (8). Recordings were made either with the Rayleigh interference optics or with the photoelectric scanning system, set at 280 nm. Equilibrium was assigned when no fringe shift could be observed on photo- graphs taken at intervals of least 3 hours. Calculations were performed according to the procedure of Nasarian (9).

Reduction and Alkylation of Proteins-Proteins were reduced with 0.1 M dithiotreitol (Calbiochem) for 2 to 3 hours and alkylated with 0.25 M iodoacetamide (Mann) in the dark for 0.5 hour. Alkylation, without prior reduction, was performed in the dark at 50 to 100 molar excess of iodoacetamide over free -SH groups in the protein. All reactions were carried out in 0.2 or 0.5 M Tris-HCl buffer, pH 8.0, containing 8 M urea (for starch gel electrophoresis) or 6 M guanidine hydrochloride (for gel chromatography).

Analytical Gel Chromatography-The dissociation of the prealbumin-RBP complex was studied by gel chromatography on Sephadex G-100 columns (100 x 1 cm) under conditions varying with respect to ionic strength and pH. Buffers varying in ionic strength consisted of 0.002 M Tris-HCl, pH 8.0, adjusted to the desired ionic strength with NaCI. Buffers varying in pH were as follows: 0.02 M Tris-HCl, pH 8.0, and 0.02 M

citrate-phosphate, pH 6, pH 5, pH 4, and pH 3. All of these latter buffers contained 0.2 M NaCI.

Gel Chromatography ilz Guanicline Hydrochloride-Molecular weights were also determined by gel chromatography in guanidinium hydrochloride. According to Tanford, Kawahara, and Lapanje (10) most proteins, extensively reduced and alkylated, behave as random coils in guanidine of high concentrations sedimentation and diffusion constants are directly related to the length of the polypeptide chain, and thus to the molecular weight. Therefore, Stokes’ molecular radius (r,), obtained by gel chromatography (II), should also be directly related to the molecular weight of a protein which behaves as a random coil. It thus seems justified to determine molecular weights of reduced and alkylated proteins by gel chromatography in guanidine hydrochloride (12).

Columns (100 x 1 cm) of Sephadex G-100 or G-200 were equilibrated with 0.02 M Tris-HCl buffer, pH 8.0, containing 5 or 6 M guanidine hydrochloride and 0.15 M NaCl. Elution was carried out with downward flow. The void volume (PO) and the total volume (vt) of the columns were determined as described elsewhere (13). Calculations of Stokes’ radii were carried out according to Laurent and Killander (11).

Starch Gel Electrophoresis-Starch gel electrophoresis was performed in 8 M urea-formate buffer, pH 3 (14), with the use of a thin layer technique (3).

Immurwchemical MethodsOuchterlony immunodiifusion analyses (15) and microimmunoelectrophoresis (16) were carried out as described elsewhere (6). A single radial immunodiffusion technique (17) was used as described earlier (18).

The prealbumin-RBP complex was separately precipitated with anti-prealbumin serum and anti-RBP serum. The amount of antiserum exceeded by approximately 20% the equivalent amount of antigen; the equivalence ratio was estimated by Ouchterlony immunodiffusion analyses. Mixtures of prealbumin-RBP complex (1 to 3 mg) and antiserum (3 to 6 ml) were left to precipitate for 2 hours at room temperature and

subsequently for about 24 hours at +4”. After the precipitate had been removed by centrifugation, another l-ml portion of antiserum was added, and incubation was continued for 24 hours at +4’. Usually, no further precipitate was observed, even after centrifugation. The amounts of prealbumin and RBP, still present in the supernatants, were quantitatively estimated by the single radial immunodiffusion technique.

F&min. A Extraction-The prealbumin-RBP complex and RBP were separately dissolved in aqueous solutions, varying with respect to pH or ionic strength, to which were then carefully added equal volumes of heptane (usually 1 ml). At various time intervals, the amounts of retinol in the two phases were estimated by measuring the absorbance at 330 nm or by deter- mining the fluorescence on an Aminco-Bowman spectrofluo- rometer. The samples were excited at 330 nm and emission was recorded at 470 nm. All experiments were carried out in vials of the same size, to ensure identical contact areas between the two phases.

Other Methods-Diluted protein solutions were concentrated by ultrafiltration (19) with the use of &inch Visking dialysis tubing (Union Carbide) as the ultrafiltration membrane (20). Protein concentrations were determined either by relating the absorbance at 280 nm to the relevant molar extinction coefficients (2) or by a single radial immunodiffusion technique (17).

RESULTS

Efect of Ionic Strength and pH on Stability of Prealbumin-RBP CompZez-It was earlier noted that the complex between prealbumin and RBP dissociated at a low ionic (2). A series of chromatographies on Sephadex G-100 at different ionic strengths was carried out to study this dissociation further. In 0.002 M

Tris-HCl buffer, pH 8.0, no dissociation occurred at the ionic strength of 0.20 (Fig. 1). Small amounts of free RBP appeared at an ionic strength of 0.02 (Fig. 2). Fig. 1 also shows that the dissociation was complete at an ionic strength of 0.002. When the separated prealbumin and RBP were recombined and re- &romatographed on Sephadex G-100 at an ionic strength of 0.2 all protein appeared in an elution position corresponding to that of the prealbumin-RBP complex.

The effect of pH on the stability of the prealbumin-RBP complex was examined as follows. Gel chromatographies on Sephadex G-100 were carried out at pH 8, 6, 5, 4, and 3. All buffers used had an ionic strength of 0.2. Dissociation of the prealbumin-RBP complex was not noted at pH 8 and 6. A small amount of unbound RBP (less than 15% of the total RBP) was found in the chromatogram obtained at pH 5. Further dissociation did not seem to occur at pH 4 or 3. These results suggest that molecular changes not consistent with complex formation between prealbumin and RBP take place at low ionic strength in a buffer of pH 8.0, whereas the conformations of the two proteins seem to be less affected by variations in pH within the range of pH 8 to 3.

Effect of Antibodies on Stability of Prealbumin-RBP Complex- Previous analyses by the Ouchterlony immunodiffusion technique showed a reaction of identity between free RBP and the prealbumin-RBP complex when tested against an anti-RBP serum (12). Free prealbumin and the prealbumin-RBP complex also gave a reaction of identity with an anti-prealbumin serum, Fig. 2 shows an Ouchterlony immunodiffusion analysis of free RBP, free prealbumin, and prealbumin-RBP complex against a mixture of anti-RBP and anti-prealbumin sera. The result


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46 Prealbumin, Retinol-binding Protein, and Vitamin A Interactions Vol. 246, No. 1

24

1.6

58

35 45 55 65 ELUTION VOLUME (ml)

FIG. 1. Chromatography on Sephadex G-100 of purified prealbumin-RBP complex at variations of ionic strength. Upper, 2.4 mg of prealbumin-RBP were applied to the column and eluted at an ionic strength of 0.20. Middle, 6.8 mg of prealbumin-RBP were applied to the column and eluted at an ionic strength of 0.02. Lower, 8.9 mg of prealbumin-RBP complex were applied to the column and eluted at an ionic strength of 0.002. RBP (A.... A) and prealbumin (A+. . .A), were estimated by a single radial immunodiffusion technique. The columns (100 X 1 cm) were equilibrated in 0.002 M Tris-HCl buffer, pH 8, adjusted to the desired ionic strength with NaCl.

FIG. 2. Ouchterlony immunodiffusion analysis of purified prealbumin-RBP (I), prealbumin (2), and RBP (3). The center well contained a mixture of antisera against RBP and prealbumin. The agar mixture had an ionic strength of 0.1.

TIME (hours)

FIG. 3. Release of vitamin A from RBP and prealbumin-RBP complex at various values of pH. Portions of RBP, 0.5 mg (C), and prealbumin-RBP complex, 2.0 mg (o), were separately submitted to aqueous phases (1 ml) overlaid by heptane (1 ml). The aqueous phase consisted of 0.01 M HCl, pH 2.0 (- - -), 0.02 M citrate-phosphate buffer, pH 6.0 (----), and 0.02 M Tris-HCl buffer, pH 8.0 (--- ). The plots for pH 6.0 and pH 8.0 coincide. All buffers were adjusted to an ionic strength of 0.2 with NaCI.

indicates that the prealbumin-RBP complex had dissociated into free prealbumin and free RBP since the precipitation lines of prealbumin and RBP did not coincide. This conclusion was corroborated by separate precipitation in solution of prealbumin- RBP complex with an anti-RBP serum and an anti-prealbumin serum, respectively. The results showed that antibodies directed against RBP displaced prealbumin from RBP, whereas antibodies directed against prealbumin did not seem to alter seriously the interaction between prealbumin and RBP. A detailed study of this phenomenon will be published elsewhere.

E$ect of Variations in pH and Ionic Strength on Release of Vitamin A-The sedimentation velocity analyses (see below) and the immunochemical results suggest that RBP is rather liable to molecular changes. Most conformational alterations would probably affect the interaction between RBP and vitamin A. This was explored by extractions of vitamin A from the prealbumin-RBP complex and RBP, under varying conditions of pH and ionic strength.

Vitamin A was released more slowly from the prealbumin- RBP complex than from RBP at pH 8, 6, and 2. Although retinol was extracted from the complex at a higher rate at pH 2 than at pH 8 or 6, the rate was consistently lower than that for free RBP (Fig. 3). Retinol bound to free RBP was extracted only somewhat more easily at pH 2 than at pH 8 or 6. Varia- tions in pH did not, however, seem to cause any dramatic changes in the ability of RBP to bind vitamin A. More remarkable differences in the vitamin A-binding property were noted when the ionic strength was varied at pH 8. As can be seen from Fig. 4, the release of retinol both from prealbumin-RBP and RBP was enhanced on lowering the ionic strength. It seemed as if the increase of release velocity for the prealbumin-RBP complex occurred at a somewhat lower ionic strength than that for the free RBP. At very low ionic strengths the release curves for the complex and RBP coincided.

Sedimentation Velocity Studies of Free RBP and Free Prealbumin at Varying Ionic Strengths--The behavior of free RBP and prealbumin was studied by sedimentation velocity analyses at varying ionic strengths. The sedimentation velocity of pre-


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; 60 2:

40

20

r

L

IONIC STRENGTH (x 103)

FIG. 4. Release velocity of vitamin A from RBP and prealbumin-RBP complex at variations of ionic strength. (- (E/d log t)) is expressed in arbitrary units.

The velocity C represents the

amount of retinol bound to the protein at time t. Portions of RBP, 0.5 mg (C), and prealbumin-RBP complex, 2.0 mg (0) were separately submitted to aqueous phases (1 ml) overlaid b$ heptane (1 ml). The aqueous phases consisted of 0.002 M Tris- HCI buffer, pH 8.0, adjusted to the desired ionic strength with NaCl.

4.6

3.8

IONIC STRENGTH (x 103)

~~FIG. 5. Analyses of the sedimentation velocities of RBP and prealbumin at variations of the ionic strength. RBP, 0.2 mg/ml (O)., and prealbumin, 0.5 mg/ml (O), were exhaustively dialyzed against 0.002 M Tris-HCl buffer, ionic strength with NaCI.

pH 8.0, adjusted to the desired

albumin showed no significant variation with ionic strength (sa~,~, 4.6 S), whereas RBP showed a decreasing sedimentation velocity with decreasing ionic strength, from 2.3 to 1.75 S (Fig. 5). These results should be interpreted with caution, as most proteins exhibit a nonideal behavior at a low ionic strength, as a result of the exposure of charged groups in the molecules. The observations, together with the findings of the immunochemical analyses, suggested, however, that the low ionic strength induced either a conformational change of the RBP or a dissociation of RBP into subunits. Sedimentation velocity experiments at low ionic strength, 0.002, conducted with a series of concentrations of RBP from 0.2 to 8 mg per ml, showed no con- centration dependence of the sedimentation coefficient, which together with evidence presented below indicates that the low sedimentation coefficient obtained is not due to a dissociation into subunits.

Molecular Weights of Reduced and Alkylated RBP and Pre- albumin in Dissociating Medium-Gel chromatographies were performed on Sephadex G-100 equilibrated with 6 M guanidine

25 -

I 2o IO

I I 1 I 13 16 19 22

MOLECULAR WEIGHT (JO-3)

;;,:-FIG. 6. Determination of the molecular weight of RBP by gel chromatography in 6 M guanidine hydrochloride. The Sephadex G-100 column (100 X 1 cm) was equilibrated with 0.02 M Tris-HCI buffer, pH 8.0, containing 6 M guanidine hydrochloride and 0.15 M

NaCI. The column was calibrated with a1 (1) and a!2 (3) chains of haptoglobin, &-microglobulin (Z), and light immunoglobuhn chains (4). The arrow indicates the value obtained for Stokes’ radius of RBP. All proteins were extensively reduced and alkylated as described under “Methods.”

-

FIG. 7. Starch gel electrophoresis in 8 M urea-formate buffer pH 3, of prealbumin: untreated (I), reduced and alkylated (2): of prealbumin-RBP complex: untreated (S), reduced and alkylated (4); and of RBP: untreated (6), reduced and alkylated (6). Re- duction and alkylation were carried out as described under “Methods.”

hydrochloride. The column was calibrated with the following proteins: albumin; heavy and light chains of a yG-myeloma protein; p, c?, and 0~~ chains of haptoglobin; and &-microglobulin. RBP emerged in the elution position between light chains of IgG and a? chains of haptoglobin. The estimated molecular weight for RBP was 20,500 (Fig. 6). Sedimentation equilibrium ultracentrifugations, performed in 6 M guanidine, gave a molecular weight of 20,300. The results indicate that RBP consists of a single polypeptide chain; the molecular weight in guanidine hydrochloride closely corresponds to that obtained at physiological conditions (2).

Prealbumin was chromatographed on a column of Sephadex G-200 equilibrated with 5 M guanidine hydrochloride. Calibra- tion was carried out with the proteins listed above. Pre- albumin was eluted in the same position as C? chains of hapto-


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48 Prealbumin, Retinol-binding Protein, and Vitamin A Interactions Vol. 246, Xo. 1

globin, that is, somewhat later than RBP. The elution position corresponded to a molecular weight of about 17,000. This observation suggests that prealbumin consists of subunits.

Starch gel electrophoresis in 8 M urea-formate buffer, pH 3, was carried out to explore further the suggested subunit struc- ture of prealbumin. Fig. 7 shows a typical experiment. The main zone of extensively reduced and alkylated prealbumin moved somewhat faster than the untreated protein. This result is in accord with a reduction in size after cleavage of disulfide bonds. Untreated and reduced and alkylated RBP had similar mobilities. This behavior of RBP on urea starch gel electrophoresis has been discussed elsewhere (3). The prealbumin- RBP complex behaved, as expected, like the sum of the single components.

DISCUSSION

The human retinol-binding protein was recently isolated and characterized (1, 2). In blood from normal individuals most of it is a,pparently bound to prealbumin. The linkage of RBP to prealbumin is probably of physiological importance. One conse- quence of the formation of a prealbumin-RBP complex should be prevention of excessive renal loss of the small RBP molecule. Free RBP seems to pass rapidly through the glomerular barrier (3).

Some characteristics of the interaction between RBP and prealbumin were studied in the present report. It was found that, at pH 8.0, the prealbumin-RBP complex dissociated readily at a low ionic strength, and that the dissociation was reversible. The binding of the two proteins seemed to be less susceptible to changes of pH, at least in the range from pH 8 to 3. Therefore, the linkage between prealbumin and RBP may primarily be of a hydrophobic nature. The exposure of charged groups in the proteins at a low ionic strength could then result in conformational changes, incompatible with a sustained interaction.

The sedimentation constant for RBP decreased rapidly at low ionic strengths, whereas the sedimentation constant for prealbumin seemed to be largely independent of variations in ionic strength. These results must be interpreted with caution as primary and secondary charge effects may exert marked influence on the sedimentation velocity. The difference in behavior between the two proteins would, however, suggest that the conformation of RBP is more easily changed than that of prealbumin at a low ionic strength. This view is supported by the results of the immunochemical analyses and by the observations on the reiease of vitamin A from RBP.

Antibodies against prealbumin completely precipitated the prealbumin-RBP complex, whereas antibodies against RBP precipitated only this protein. These results indicate that the binding of antibodies to the small RBP molecules induces conformational changes not consistent with a simultaneous binding to prealbumin. Prealbumin, which is considerably larger, could apparently bind both RBP and antibodies at the same time. These findings were somewhat unexpected, as it was noted earlier that not only RBP but also prealbumin gave reactions of immunological identity with the prealbumin-RBP complex, when examined by Ouchterlony immunodiffusion analyses (2). The latter observations may be due to the fact that the RBP binding site of the prealbumin is not an impor- tant part of the antigenic structures which react with the anti- prealbumin sera used.

The present results indicate that prealbumin consists of four polypeptide chains. Urea starch gel experiments suggested the presence of interchain disulfide bridges in the prealbumin molecules. It was earlier found that prealbumin contains only about 4 residues of half-cysteine (2). A maximum of two interchain disulfide bridges can therefore be present. Further structural work is needed to determine exactly the number of polypeptide chains in prealbumin and the mode of linkage between them.

Variations in the ionic strength influenced the vitamin A release both from the prealbumin-RBP complex and from RBP. The release velocity was increased but did not seem to be affected to the same extent by variations in pH. At all conditions examined RBP gave up retinol more easily than the prealbumin- RBP complex, except at a very low ionic strength. A tentative explanation would be that free RBP expands in the environment of low ionic strength, and that the retinol molecule becomes more exposed, so that it is removed more easily from the protein. The facilitated release of vitamin A from the complex at low ionic strength indicates that the dissociation between prealbumin and RBP has a marked influence on the release velocity. This is not unexpected, as the diffusion constants of free RBP and the prealbumin-RBP complex differ by a factor of about 2 (2). These diffusional differences should result in different rates of release of vitamin A from the water phase to the heptane phase. The expected difference in release velocity between RBP and prealbumin-RBP would thus be of similar size as the differences in diffusion constants, if size alone determined the release velocity. The difference obtained is, however, greater (cf. Fig. 4), which seems to indicate that prealbumin stabilizes the binding between RBP and retinol.

Acknowledgments-1 wish to thank Dr. I. Bergg%rd for many valuable discussions. Professor T. C. Laurent is gratefully acknowledged for placing an ultracentrifuge at my disposal and for critically reviewing the manuscript. I am indebted to Dr. U. Lindahl for helpful suggestions. Miss Elfi ohren provided skillful technical assistance.

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P. A. PetersonVitamin A

Studies on the Interaction Between Prealbumin, Retinol-binding Protein, and

1971, 246:44-49.J. Biol. Chem.

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