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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 8, Issue of April 25, pp. 1903-1912, 1970

Printed in U.S.A.

Studies on the Protein-Protein and Protein-Ligand Interactions Involved in Retinol Transport in Plasma*

(Received for publication, October 2, 1969)

AMIHAM RAZ,$ TATSUJI SHIRATORI, AND DEWITT S. GOODMAN~

From the Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032

SUMMARY

Studies were conducted to examine the chemical and physical properties of human plasma retinol-binding protein (RBP) and prealbumin (PA), and the factors affecting their interaction with each other and with the ligands retinol and thyroxine, respectively. The effects of changes in temperature, pH, and urea concentration and the effects of reduction and alkylation of disulfide bonds and of iodination were assessed. Analytical ultracentrifuge studies established the formation of a 1: 1 molar complex between the two proteins. The estimated molecular weight of the complex (approximately 70,000) was almost exactly the sum of the molecular weights of the individual proteins (PA, approximately 49,000 and RBP, 21,000). The complex was stable in the temperature range 442.5” and in the pH range 5.8 to 7.5. Considera- ble dissociation of the complex occurred as the pH was raised from 8.6 to 10.3. Addition of 6 M urea completely disrupted the RBP-PA complex, markedly reduced the affinity of PA for thyroxine, but did not interfere with the association of retinol with RBP. Only one of the two disulfide bonds of PA was readily accessible to reduction and alkylation. Re- duction of this one disulfide bond did not affect the binding of either RBP or thyroxine to PA. In contrast, both disulfide bonds of RBP were highly resistant to reduction. When the disulfide bonds of RBP were reduced and alkylated, however, the retinol-RBP complex was disrupted, and retinol was completely lost from the protein. Iodination of RBP up to the level of 4.3 atoms of iodine per molecule selectively interfered with the interaction of RBP with PA, without disrupting the retinol-RBP complex. These studies demonstrate that it is possible to affect selectively the different interactions involved in the RBP-PA system responsible for retinol transport in plasma.

Previous work from this laboratory has demonstrated that retinol circulates in human plasma bound to a specific protein,

* This work was supported by Grant AM-05968 from the Na- tional Institutes of Health.

$ Trainee under Grant Tl-AM-05397 from the National Insti- tutes of Health.

$ Career Scientist of the Health Research Council of the City of New York under Contract I-399.

retinol-binding protein (1). This protein has a molecular weight of approximately 21,000 and one binding site for 1 molecule of retinal. In plasma, RBPl circulates in the form of a protein-protein complex, together with plasma prealbumin. The PA molecule also binds thyroxine and has been designated by some earlier workers as thyroxine-binding PA. We have recently reported the results of quantitative studies of the interaction of thyroxine with PA and with the PA-RBP complex (2).

The studies reported here were undertaken in order to obtain more information about the chemical and physicochemical properties of isolated RBP and PA and about the factors affecting their interaction with each other and with the ligands retinol and thyroxine, respectively. To this end, the influence of a number of parameters, including temperature, pH, urea addition, and structural modification by reduction and alkylation of disulfide bonds and by iodination were studied. The effects of perturbations in each of the parameters on the various interactions were examined.

METHODS

Isolation of Prealbuumin and Retinal-binding Protein-Whole human plasma (usually obtained as outdated plasma from the blood bank) was chromatographed directly on DEAE-Sephadex as previously described (2). Further purification of both PA and RBP was achieved by a sequence of procedures, including gel filtration on Sephadex G-200, repeat DEAE-Sephadex chromatography, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. These procedures have been described in detail in our previous publications (1,2).

Sulfl~ydryl Assay-The method adopted was a modification of the assay developed by Janatova, Fuller, and Hunter (3). To minimize the amount of protein needed for analysis, we scaled down the reaction mixture proport,ionally as follows. Proteins t,o be analyzed were dissolved in 0.084 M potassium phosphate buffer, pH 7.50, either containing or devoid of 8 M

urea; 0.75 ml of the protein solution was mixed with 0.2 ml of 0.01 M DTNB in 0.037 M potassium phosphate buffer, I&I 8.0, and with 0.05 ml of 0.025 M EDTA in 0.0074 M potassium phosphate buffer, pH 8.1. The pH of the final mixture was 7.1. The mixture was allowed to stand for 45 min and the absorbance

1 The abbreviations used are: RBP, retinal-binding prot.ein; PA, prealbumin; HSA, human serum albumin; DTNB, 5,5’- dithiobis(Z-nitrobenzoic acid).

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was measured at 412 rnp. Human serum albumin was used as standard for each set of assays; the -SH content of the HSA was consistently found by this method to be 0.54 residue per mole. A molar extinction coefficient of 1.36 x lo4 was used to calculate the monothiol content from the absorbance at 412

w. DisulfLde Assay-The method of Zahler and Cleland (4) was

used with some modification as follows. Protein to be analyzed was dissolved either in water or in 0.05 M phosphate buffer containing 8 M urea, pH 8.9. To 0.3 ml of the protein solution, 0.05 ml of 0.1 M Tris-HCl buffer, pH 9.0, and 0.05 ml of 6 IIIM 1,4- dithioerythritol in water were added. The final pH of the reduction mixture was 9.0 in all cases. Reduction time was usually 20 min, but in some cases it was as long as 90 to 120 min. Following reduction, the resulting sulfhydryl groups were assayed with DTNB as described by Zahler and Cleland (4). With cystine as a standard, a molar extinction coefficient of 1.34 x

lo4 was observed; this value is very close to the value of 1.36 x lo4 reported previously (4). The latter value was used for the calculation of the number of monothiols produced by reduction.

Carbohydrate Analysis-The method of Dubois et al. (5) was used; a mixture of n-mannose and n-galactose in a 2: 1 ratio was used as a standard (6).

Analytical Ultracentrifugation-Sedimentation velocity and equilibrium analyses were kindly carried out by Drs. W. Poillon and P. Feigelson of the Columbia University College of Physi- cians and Surgeons in a Spinco model E ultracentrifuge equipped with a monochromator and photoelectric scanner. The absorbance of the cell contents at 280 rnp was determined at intervals as a function of distance from the center of rotation. For samples containing RBP, after analysis the cell contents were mixed and the analysis was repeated under identical conditions but scanning for absorbance at 330 mp. Sedimentation velocity studies were carried out on protein solutions of 0.43 to 0.68 mg per ml in 0.1 M sodium phosphate buffer, pH 7.0. Sedimen-

TUBE NUMBER

FIG. 1. Separation of the PA-BBP complex from RBP by gel filtration on a column of Sephadex G-100. A mixture of RBP and PA in a molar ratio of approximately 2: 1 was used. The PA-RBP complex (tubes 12 to 21, combined to yield Pool 1) and free RBP (tubes 22 to 32, Pool 2) were clearly separated, as shown.

tation equilibrium analyses used protein solutions of 0.054 to 0.085 mg per ml in the same buffer. The conditions for sedimentation velocity and equilibrium analyses were identical with those previously reported (2). Molecular weights were calculated as reported previously (2), with the method of Yphantis (7).

Assay of Prealbumin-Retirwl-binding Protein Complex For- mation by Chromatography on Xephadex G-100-Mixtures of PA and RBP (either untreated or modified) were chromatographed on columns of Sephadex G-100 (Pharmacia) previously equilibrated with 0.02 M potassium phosphate buffer containing 0.2 RI NaCl, pH 7.5 (designated NaCl-phosphate buffer), and eluted with the same buffer. Columns were 1.7 cm (inner diameter) x 55 to 60 cm, and the elution rate was generally 5 to 7 ml per hour. The effluent stream from the column was continuously monitored for absorption at 280 rnl.c with a Uvicord II spectrometer (LKB Instruments, Inc., Rockville, Maryland). An example of a typical elution pattern is given in Fig. 1. The PA- RBP complex (mol wt about 70,000) and free RBP (mol wt 21,000) were effectively separated by this procedure. The relative amounts of RBP in the complexed and free form were routinely determined by (a) the relative distribution in the two pools of absorption at 330 mp (due to the presence of protein- bound retinol in RBP (I)) and (b) the relative content of emitted fluorescence at 462 rnp in the two pools.* In addition, in experiments in which RBP was labeled with either 14C or 1311, the radioactivity distribution between the two pools served to determine the percentage of free and complexed RBP. When testing the binding capacity of PA for apo-RBP, the percentage of free and complexed apo-RBP was estimated by the distribution of absorbance at 280 rnp between the free and complex pools, together with disc gel electrophoretic analysis of the two pools.

Prealbumin-Thyroxine Studies-The binding of thyroxine to untreated and modified PA preparations was studied by the method of equilibrium dialysis as described in detail previously (2). The data were analyzed by the equation F/C = kn - kit, where J is the moles of thyroxine bound per mole of PA; k is the apparent association constant between PA and thyroxine; and n is the number of binding sites with the same association constant k. Since PA was shown (2) to possess a single binding site for thyroxine (i.e. n = I), the equation becomes B/C = k - kn. Plotting F/C against sj yields a straight line, whose intercept on the F/C axis (3 = 0) is equal to k. Binding studies were done in either potassium phosphate buffer, 0.05 M, pH 7.5, or in potassium phosphate, 0.05 M, containing 6 M urea, pH 7.5.

Reductive Alkylation of Disuljide Bonds in Prealbumin and Retinol-binding Protein-The method of Margolis and Langdon (8) was adapted with some modification as follows. The lyophilized protein (4 to 30 mg) was dissolved in 0.5 ml of 0.2 M Tris- acetate buffer, pH 8.6, or in the same buffer containing 6 M urea, and the vessel (a 13-ml centrifuge tube) was flushed with nitro- gen for 10 min and sealed with a thin rubber stopper. Ethane- thiol (approximately loo-fold excess, e.g. 6 ~1 for 10 mg of PA or for 4 mg of RBP) was injected through the rubber stopper into the vessel. Reduction was then allowed to proceed at room temperature for 4 hours (for PA) or 16 hours (for RBP). Fol- lowing reduction the solution was frozen in a Dry Ice-acetone

2 RBP is strongly fluorescent, because of the prot,ein-bound retinol, with peak uncorrected excitation and emission frequencies of 332 rnp and 462 rnp, respectively (unpublished observations).


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bath and lyophilized to complete dryness. The lyophilized reduced protein was dissolved in 0.5 ml of 1 M Tris-acetate buffer, pH 8.6, containing a lo-fold excess of 2.14C-iocloacetic acid, and the alkylation was allowed to proceed for 15 min (for PA) or 1 hour (for RBP) at room temperature with occasional shaking. Following alkylation, 2-mercaptoethanol was added in an amount equimolar to that of the iocloacetic acid added previously. The alkylated protein was separated from excess reagents by gel filtration on a column of Sephaclex G-25 (1.4 cm (inner diameter) X 25 cm) with 0.1 M Tris-acetate, pH 8.6, as the eluting buffer. The protein was then dialyzed exhaustively against NaCl-phosphat’e buffer before use.

TABLE I

Monothiol and dithiol analyses of prealbumin and retinal-binding protein

Protein H;y;;-.;ine phosphate’ Ph;rw;$e P+osphaW Ph$$te

residues/mole mole -SH/mole firotein mole S-S/mole firocein

‘* 4c 0.05 0.07 1.0 0.9 RBP 5-6d 0.01 0.02 0.1 0.4

a Assay in potassium-phosphate buffer. b Assay in potassium-phosphate buffer containing 6 M urea. c Value from Reference 2.

IO&nation of Prealbumin and Retinol-binding Protein--The d Value from Reference 1. ICI method of McFarlane (9) was used. Stock ICl solution was diluted as desired, and a tracer amount of 1311 (carrier free) ”

r

dissolved in dilute ICl was added, followed by the addition of 1 2 3 4,.-‘j the protein. The protein-IClPI mixture was vigorously shaken

i* * \ ,: : _ \, ,’ -

for 5 to 10 set and immediately applied to a Sephadex G-50 column (1.4 cm (inner diameter) X 35 cm) previously equilibrated with borate buffer, pH 8.0. The distribution of radioactivity in the effluent from this column indicated complete

separation of the iodinated protein from unreacted ICl and I-. The ioclinated protein was dialyzed overnight against NaCl- phosphate buffer and stored at 4”. All experiments with iodinated PA or RBP preparations were completed within a week after iodination. The number of microgram atoms of iodine incorporated per pmole of protein was calculated for each preparation from the specific radioactivity of the iodine in the reaction mixture and from the incorporation of radioactivity per kmole of protein.

Other Methods-Radioassay for l4C was carried out with a Packard Tri-Carb scintillation spectrometer, with the solution described by Bray (10) as scintillation solvent and with a count- ing efficiency of 80%. Samples containing Is11 and lz51 were assayed for radioactivity in a Packard autogamma spectrometer. Fluorescence measurements were made with an Aminco-E,owman spectrophotofluorometer (American Instrument Company, Silver Spring, Maryland). Preparative and analytical (disc) poly- acrylamicle gel electrophoresis, immunodiffusion, ultraviolet spectral studies, and the determination of protein concentration were carried out as reported previously (1, 2).

MATERIALS

HSA was obtained from Eehringwerke (Hoechst Pharmaceu- tical Company, Kansas City, Missouri); n-mannose and n-galactose, from Mann; DTNB and cystine from Calbiochem; 1,4- dithioerythritol from Cycle Chemical Company. Ethanethiol (ethyl mercaptan), 2-mercaptoethanol, and iodoacetic acid were obtained from Eastman. 2J4C-Iodoacetic acid, specific activity 15.5 mCi per mmole, obtained from Nuclear-Chicago, was diluted approximately 40 times with unlabeled iodoacetic acid. The specific activity of the diluted preparation (used for protein alkylation) was 8.9 x lo5 cpm per pmole. The sources of other materials were described in our previous publications (I, 2).

RESULTS

Carbohydrate Content of Retinol-binding Protein-Analysis of RBP for its carbohydrate content yielded a value of 0.9 mole of hexose per mole of RBP, indicating that RBP probably contains 1 mole of hexose per mole of protein.

Monothiol and Dithiol Analyses of Prealbumin and Retinol- Microheterogeneity of Retinal-binding Protein on Acrylamide binding Protein-Samples of PA and of RBP were analyzed for Gel Electrophoresis-RBP, isolated as described under their detectable -SH and S-S content as described under “Methods,” was found to contain three bands on disc gel elec- “Methods.” The results of these analyses together with the trophoresis (Fig. 2, Gel 1). The faster moving band (labeled half-cystine contents from the amino acid analyses previously the A band) was not fluorescent under ultraviolet light and did

FIG. 2. Disc gel electrophoretic patterns of RBP and RBP subfractions. Gel 1 shows the pattern for purified RBP. Gels 2, S, and 4 show the patterns for the A, H-I, and H-2 peaks isolated after preparative gel electrophoresis of whole RBP as illustrated in Fig. 3.

reported (1, 2) are given in Table I. Free -SH groups were not detected in either protein, even when the DTNB reaction was carried out in the presence of 6 M urea. If we assume that this means that the half-cystine residues in the proteins were all present in -S-S- form, then these data indicate that PA contains two disulfide bonds and RBP contains either three disulfide bonds or two &sulfide bonds and one inaccessible monothiol. RBP was quite resistant to reduction of disulfide bonds by 1,4-dithioerythritol. Even after prolonged reduction time (90 to 120 min), an apparent disulfide content of only 0.1 mole per mole of RBP was detected. Reduction in the presence of 6 M urea caused a significant but small rise in the reactivity of the disulfide bonds toward reduction by 1,4-dithioerythritol, yielding an apparent, disulfide cont’ent of 0.4 mole per mole of RBP. In the case of PA, only one of the two disulfide bonds presumed present was reduced by 1,4&thioerythritol, in both the presence and the absence of 6 M urea in the reduction mixture.


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1906 Interactions hvolved in Retinol Transport Vol. 245, l\:o. 8

not contain bound retinol (as monitored by absorption at 330 w.4. The two other bands (labeled H-l and H-2) were both fluorescent and contained bound retinol. The relative amounts of these t,hree bands in a given isolated RBP preparation were shown, from a large number of such isolations, to depend on the relative freshness of the plasma sample used and on the rapidity of the purification and the extent of handling during the purification procedure. Thus, when freshly drawn plasma was rapidly processed, the isolated D preparation (PA-RBP complex prior to dissociation of the complex by gel electrophoresis, see Reference 1 for details) showed on disc gel electrophoresis only one fluorescent band, corresponding to H-2. However, the same preparation, after storage in solution for 2 weeks at 4”, showed, in addition to the major H-2 band, a significant (albeit lesser) fluorescent H-1 band. Similarly, when purified RBP which had been isolated by rapid purification from freshly drawn plasma was analyzed by gel electrophoresis, the three bands were found in a ratio of approximately 0.5:1:‘8 (il.-H-l :H-2). In contrast, Rlil’ isolated from outdated blood bank plasma usually contained the three bands in a ratio of about 1:4:4 (A:&1 :H-2); see Fig. 2, Gel 1.

Separation of the three bands of RBP (Fig. 2, Gel 1) was achieved by preparative polyacrylamide gel electrophoresis as showu in Fig. 3. The fractions corresponding to each of the three separated peaks in Fig. 3 were pooled, and the three pools were analyzed by disc gel electrophoresis. The results are shown in Fig. 2, Gels d to .J. Only the first pool, corresponding to the A band, was found to be nearly homogeneous (Gel 2). The second pool, corresponding to the H-l band, was found to also contain a considerable a.mount of the ‘4 band (Gel S), and the third pool, corresponding to the H-2 band, contained a mixture of all three bands. These findings are consistent with the conclusion that during the processes of purification and analysis of RBP the naturally occurring H-2 form is gradually converted to the more rapidly migrating H-l form and, in turn, to the retinol-free A form.

Chromatographic Behavior of Prealbumin, Retinal-binding Protein, and Prealbumin-Retinol-binding Protein Complex (D Preparation) during DEA E-Sephadex Column Chromatography-

We have previously shown that RBP isolated from whole human plasma is obt,ained in a form of a protein-protein complex together with plasma P.4. A study was conduct.ed to compare purified RBP, PA, the RBP-PA complex (in the form of the D preparation), and I-ISA, with regard to their relative binding affinity for DEAE-Sephadex. For this purpose, portions of

purified PA, RBl’, and of the D preparation were each mixed

20 25 30 35 40

TUBE NUMBER

FIG. 3. Preparative polyacryalmide gel electrophoresis of RBP. Approximately 25 mg of the RBP preparation shown in Fig. 2, Gel I, was subjected to electrophoresis. The three separated peah correspond to the three bands seen in Fig. 2, Gel 1.

with the same amount of HSA and chromatographed on DEAE- Sephadex. All three runs were done under identical conditions, and the elution patterns obtained are shown in Fig. 4. As can be seen, both PA (Fig. 4C) and the RBP-PA complex (Fig. 4A) were well separated from HSA, eluting at higher salt concentrations than those required to elute HSA. Purified RBP, on the other hand, was eluted together with HSA (Fig. 4B). It should be noted that RBP and HSA were previously shown (1) not to form a complex with each other, since the two proteins separate completely during gel filtration on Sephadex G-100. These results show that RBP alone has a lesser affinity for DEAE-Sephadex than does the RBP-PA complex. The chromatographic behavior of the RBP-PA complex on DEAE-Sepha- dex was similar to that of PA alone.

Analytical Ultracentrifugal studies-Sedimentation velocity and equilibrium studies were performed on samples of pure RBP and PA, and on three different mixtures of RBP and P-4.

E 0.3”

2 . B

f 0.2 -

2

8 0.1 - cu

, 2 m 0.3

E m 4: 0.2

0.1

C

d \ 0.

0 O-0 250 300 350 400

EFFLUENT VOLUME ( ml 1

FIG. 4. Chromatography of PA, RBP, the D preparation, and HSA on DEAE-Sephadex. A column of size 1.6 cm (inner diameter) X 59 cm, was used for the three rulls shown. Elution was carried out with Tris-HCI buffer, 0.05 M, pH 7.5, and a linear gradient of NaCl from 0 to 0.6 M, at a flow rate of about 10 ml per hour. The samples which were chromatographed consisted of the following mixtures: A, 4.8 mg of HSA + 4.8 mg of the D preparation (PA-RBP complex); B, 4.8 mg of HSA + 2.0 mg of RBP; C, 4.8 mg of HSA + 4.0 mg of PA. In all three runs the HSA peak was eluted at an effluent volume of 290 to 300 ml; in A and C the PA peak was eluted at a peak of 340 to 350 ml. The position of the RBP peak is shown by t,he curve for 330 rnp absorbance (protein- bound retinol).


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TABLE II

Analytical uliracentrifugal analyses of retinal-binding protein,

prealbumin, and relinol-bincling protein-prealbumin mixtures

Samplea

s*o.lx Molecular weight

“,c,Kl;t Scan at Scan at Scan at 330 mp 280 nip 330 Inp

RBP 2.13 2.13 21,300 21,400 PA 3.70 b 49 ) 400 b RBP-PA, l:l.lc 4.57 4.57 d 70,200 RBP-PA, 4: 1~ 2.4, 4.8 2.0, 4.7 b b RBP-PA, 1:2.3c d 4.7 b b

a The protein concentrations for sedimentation velocity analyses were (in milligrams per ml): RBP, 0.43; PA, 0.68; RBP-PA, l:l.l, 0.60; RBP-PA, 4:1,0.63; RBP-PA, 1:2.3,0.51. Thesamples were diluted 1 in 8 for the sedimentation equilibrium analyses.

b The analysis was not carried out. (PA was not studied at 330 rnp because of its lack of 330 rnp absorbing chromophore; sedimentation equilibrium studies were not performed on the heterogeneous RBP-PA, 4: 1 and 1:2.3 mixtures.)

c The values listed are the estimated molar ratios of the two proteins. The mass ratios (RBP:PA) were 1:3, 4:3, and 1:6 for the three samples, respectively.

d See the text.

Since RBP possesses a characteristic chromophore absorbing at 330 rnp, samples containing RBP were analyzed both at 280 rnp and at 330 rnp. The results are shown in Table II. RBP and PA, when analyzed separately, each appeared to be a single homogeneous component in both the sedimentation velocity and the equilibrium analyses. Identical values for the sedimentation coefficient (2.13) and molecular weight (approximately 21,300) were obtained when RBP was analyzed at the two different wave lengths. When RBP and PA were mixed in nearly equimolar amounts (estimated molar ratio RBP: PA of 1: 1.1) the proteins migrated together and at both wave lengths in the sedimentation velocity analysis appeared to be a single homogeneous component with ~20,~ of 4.57. Fig. 5 shows the results of the sedimentation equilibrium analyses of RBP and of the RBP-PA equimolar mixture. When scanned at 330 rnp, the RUl’-PA mixture appeared to be homogeneous (see Fig. 5), with an estimated molecular weight of 70,200. When the same solution was analyzed, however, with a 280 rnp scan, the solution showed inhomogeneity, as evidenced by the deviation of the innermost data points from a straight line. This finding was consistem with the fact that the mixture contained a slight molar excess of PA as compared to RBP. The results obtained with the nearly equimolar RBPPA mixture demonstrate the formation of a I:1 molar complex between the two proteins, with an apparent molecular weight equal to the sum of that of RBP plus PA.

Sedimentation velocity analyses were also carried out on RBP-PA mixtures containing either RBP or PA in amounts in excess of a 1: 1 molar ratio (see Table II). The mixture of RBP and PA in a molar ratio of 4: 1 showed two components when scanned at eibher 280 or 330 mw. The slower moving component had an ~20 ,W of 2.0 to 2.4 and the faster component, of 4.7 to 4.8. It should be noted that the calculation of the sedimentation coefficients of the two components is less precise when a mixture is present compared to a single component. The results indicate the presence in this mixture of both free RBP

1.7 -

1.6 -

1.5 -

1.4 -

1.3 -

no I.2-

“b s

I.1 -

q 1.0 -

8 0.9-

0.6 -

g J 0.7’-

0.6 -

- REP. 260 mu - RBP 330 rnfl E----D REP-PA 330mu

y 1 I I I I I I k, , , , ,

49 .2 .4 .6 .6 so .2 .4 .6 .6 IJ .2 A .6

r2 ( ctd2) FIG. 5. Sedimentation equilibrium analyses of BBP and of the

RBP-PA complex. See the text for details. The figure shows the logarithm of 100 times the absorbance at either 280 or 330 rnE.c versus the square of the radial distance, within the rotor, from the center of rotation.

and a complex of RBP and PA in a 1: 1 molar ratio. Analysis of the mixture of RBP and PA in a molar ratio of 1: 2.3 showed a single component when scanned at 330 rnp with an sao,m (4.7) corresponding to that of the REP-PA complex. However, when this mixture was scanned at 280 rnp, the mixture was not clearly resolved into two components, but rather appeared to sediment as a broad peak with an s20,W of approximately 4.1. This value for the sedimentation coefficient is intermediate between that of PA (3.7) and the RBP-PA complex (4.6), the two components present in this mixture. Since the 330 rnp data establish the presence of the RBP-PA complex in this mixture, it is apparent that the analytical ultracentrifuge was unable to adequately resolve the two components under the conditions used.

Interaction of Prealbumin with Apo-retinol-binding Protein- Studies on the extraction of retinol from RBP (to be reported in detail elsewhere) have revealed that as much as 80 to 9O70 of the retinol can be removed from holo-RBP, without apparent damage to the protein, to yield apo-RBP. An experiment was conducted to compare the ability of whole RBP and apo-RBP to form a complex with PA. Mixtures of PA with either RBP or apo-RBP (approximately 1: 1 molar ratios) were chromatographed on Sephadex G-100, and the percentage of uncomplexed RBP or apo-RBP was estimated (see “Methods” for details). The results revealed that following Sephadex G-100 chromatography almost all of the detectable holo-RBP was found to form a complex with PA. In contrast only about 60% of the apo-RBP formed a complex with PA. The remaining 40 y0 may represent apo-RBP molecules which underwent structural changes during retinol extraction, rendering them unable to form a complex with PA. It is also possible that the affinity of apo-RBP for PA is reduced as compared to holo-RBP. It is clear, however, that the presence of retinol on RBP is not essential for the ability of RBP to form a complex with PA.


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E;ti’ects of Temperature and pll on Dissociation of Prealbumin- Retino-binding Protein complex-The stability of the PA-RBP complex at several temperatures was evaluated. Equal amounts of the D preparation were chromatographed on Sephadex G-100 columns previously equilibrated with 0.1 M potassium phosphate buffer, pH 7.5, at either 4”, 37”, or 42.5”. The extent of dissociation was determined by quantitatively measuring the amount of free RBP present after gel filtration. There was no significant dissociation of the PA-RBP complex when the temperature was raised from 4” to 37” to 42.5”. The stability of the PA-RBP complex in alkaline pH was tested on a Sephadex G-100 column equilibrated with 0.1 M carbonate-bicarbonate buffer, pH 10. Under these conditions, approximately 60 to 70% of the RBP in the D preparation became dissociated from PA, indicating a marked pH effect on the PA-RBP interaction. A more detailed study which used purified PA and RBP was then carried out to examine the effect of pH on the interaction of RBP and PA. An equimolar mixture of PA and RBP was prepared and divided into several equal portions. To each portion, a particular buffer was added to a final concentration of 0.05 M, and the solution was chromatographed at 4” on a Sephadex G-100 column equilibrated with the same buffer. Table III gives the percentage of free RBP at various pH values as estimated from the relative distribution of the 330 rnp absorption and of the fluorescence intensity between the PA-RBP complex peak and the free RBP peak. The complex appeared to be most stable in the lower portion of the pH range tested. A gradual but small increase in the amount of dissociation of the complex was observed as the pH was increased from 5.8 to 8.65. A marked increase in dissociation occurred between pH 8.65 and 10.3 (from approximately 17 to 90%).

Effect of 6 A{ Urea on Prealbumin, Retinal-binding Protein, and Prealbumin-Retinol-binding Protein Interactions-h order to gain some information about the molecular forces involved in the interaction of PA with RBP, the PA-RBP complex was exposed to 6 M urea. PA and RBP in a 1: 1 molar ratio were dissolved in 0.3 ml of 0.05 M potassium phosphate buffer, pH 7.7; 0.9 ml of 0.05 M potassium phosphate buffer containing 8 M

urea, pH 7.7, was added, and the mixture was allowed to stand at 4” for 20 min. The mixture was then chromatographed on a Sephadex G-100 column equilibrated with 0.05 M potassium phosphate buffer containing 6 M urea, pH 7.7. The elution pattern obtained showed two distinctly separated peaks with elution volumes corresponding to PA and RBP, respectively.

TABLE III Dissociation of the retinal-binding protein-prealbumin complex at

various pH values

Potassium-phosphate 5.80 Potassium-phosphate 7.60 Tris-HCI 8.20 Tris-HCl 8.65 Tris-HCI 10.30

FreeRBP&

0.7 5.4

13 18 87

% 0.1 5.2

11 15 93

0.4 5.3

12 17 90

0 As evaluated from either absorbance at 330 mp or fluorescence intensitv at 462 mu (see “Methods”). .

FIG. 6. Effect of 6 M urea on the interaction of PA wit.h t.hy- roxine. The estimated values for the apparent association constants (k, values) were as follows: untreated PA and PA exposed to 6 M urea and then dialyzed to remove the urea, both approximately 1.5 X 101; PA in 6 M urea, 0.1 X 107.

Analysis of these peaks by disc gel electrophoresis and by in- munodiffusion confirmed that the first peak contained only PA and the second only RBP. Portions from the two peaks were mixed and dialyzed against 0.05 M potassium phosphate buffer, pH 7.7, to remove urea, and the mixture was then chromatographed on a Sephadex G-100 column equilibrated with the same phosphate buffer. The mixture was eluted as a single peak containing both PA and RBP, i.e. as a complex of PA with RBP. The results demonstrate that the presence of 6 M urea caused complete dissociation of the PA-RBP complex and that the effect of urea was reversible since the two proteins reassoci- ated fully upon removal of the urea.

The ultraviolet absorption spectra of PA and RBP in phosphate buffer containing 6 M urea were approximately the same as their respective spectra in the same buffer devoid of 6 M

urea. RBP showed the same two peaks of absorbance (at 330 and 280 mp) and the same AZSC,:AX,O ratio (0.80) in the presence and absence of urea. Thus, 6 M urea had no apparent effect on the retinol bound to RBP, i.e. did not disrupt the retinol-RBP complex.

Effect of 6 M Urea on Prealbumin-Thyroxine Interaction-The binding of L-thyroxine to PA was studied with several different PA preparations. In the first experiment, PA was dissolved in 0.05 M potassium phosphate buffer containing 6 M urea and binding studies with thyroxine were carried out in this buffer. In the second experiment, PA was dissolved in the same phosphate-urea buffer and after 20 min at 4” the PA was dialyzed exhaustively against 0.05 M potassium phosphate buffer, pH 7.5, to remove urea. Thyroxine-binding studies were carried out with this PA preparation in 0.05 M potassium phosphate, pH 7.5. The same buffer was also used for thyroxine-binding studies with PA that was not exposed to 6 M urea. The results of the three experiments are shown in Fig. 6. The binding affinity of

1.6

h 0 UNTREATED

fl UREA + DIALYSIS 1.4 A 6M UREA

0 0.2 0.4 0.6 0s 1.0


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Pa4 for thyroxine was reduced more than lo-fold in the presence of 6 M urea (k approximately 1 X lo6 as compared to 1.5 X lo7 in t,he same buffer but devoid of urea). The effect of urea was reversible since removal of urea by dialysis completely restored t,he binding affinity of PA for thyroxine. Urea therefore changes the structure of PA so as to reduce its affinity for thyroxine; these structural changes are, however, completely reversible.

Reductive Alkylation of Disul$de Bonds of Prealbumin-Studies were carried out to assess the effect of reduction of the disulfide bonds of PA on its ability to interact with RRP and with thyroxine. The method used for reduction and alkylation was adopt,ed from that used by Margolis and Langdon for the reductive alkylation of p lipoproteins (8). PA was reduced with ethanethiol and then alkylated with 2-‘SC-iodoacetic acid. From the amount of radioactivity incorporated, the PA was calculated to contain an average of 1.41 moles of acetic acid residues per mole of PA. This value suggests that approximately 70% of the PA molecules underwent reductive alkylation of one disulfide bond and hence contained 2 moles of acetic acid per mole of protein. The remaining 30% of the PA molecules presumably remained unreacted. Disulfide assay of the alkylated P,4 indicated the presence of 0.25 mole of detectable S-S per mole of PA. These results are all consistent with the dat.a shown in Table I, which indicate that only one of the disulfide bonds of PA can be detected by the disulfide assay of Zahler and Cleland (4).

The capacity of this alkylated PA preparation to form a complex with RR1 was not significantly modified from that of untreated PA. Gel filtration on Sephadex G-100 of a mixture of alkylated PA and untreated RBP, in a molar ratio of approximately 1.1:1, revealed that more than 95% of the RBP was eluted as a complex with the alkylated PA. Furthermore, thyroxine-binding studies with alkylated and untreated PA revealed both preparations to have essentially the same affinity for thyroxine, with L, approximately 1.6 X 107. Finally, a mixture of alkylated and untreated PA was analyzed by disc gel electrophoresis. Only one protein band, with PA mobility, was observed. The gel was cut into several slices, each slice was extracted with NaCl-phosphate buffer, and the extracts were assayed for W. More than 95% of the recovered radioactivity was located in the slice containing the PA band, indicating that both PA preparations had the same electrophoretic mobility.

Reductive A lkylation of Disuljide Bonds of Retinol-binding Protein-REP assayed for disulfide content by the method of Zahler and Cleland (4) was found to be resistant to reduction by 1,4-dithioerythritol under normal assay conditions and to be slightly more suscelnible to reduction in the presence of 6 M urea (see Table I). We therefore decided to extend the reduction time from 4 to 16 hours and also to carry out the reduction and alkylation in both the presence and the absence of 6 M urea. From the amounts of radioactivity incorporated it was then calculated that RIIP, reduced and alkylated in the presence of 6 M urea, contained an average of 3.6 moles of acetic acid per mole of protein, whereas RBP reduced and alkylated in the absence of urea contained an average of only 0.5 mole of acet.ic acid per mole of protein.

Reductive alkylation of RBP produced significant changes in the ultraviolet absorption spectrum of the protein (Fig. 7). RBP alkylated in the absence of urea showed a relative reduction of 330 rnp absorbance, as compared to 280 rnp absorbance, of 25 to 3090. This suggests the loss of about 25 to 30% of the

r* I I I I * I I. e I I I 9, 400 380 360 340 320 300 280 260

WAVELENGTH ( rnfl)

FIG. 7. Ultraviolet absorption spectra of untreated and of alkylated RBP. Curve A represents untreated RBP; Curves B and C represent RBP reduced and alkylated in the absence or presence of urea, respectivelv. The protein concentrations were similar (about 0.3 to 0.4 mg per ml) but not identical in the three solutions whose spectra are shown.

protein-bound retinol from the RBP preparation. RBP alkylated in the presence of urea apparently lost all of its bound retinol, as evidenced by the complete disappearance of the 330 rnp absorption peak. In addition, the spectrum of this protein preparation showed accentuation of a shoulder peak near 292 rnp. Disc gel electrophoresis of mixtures of untreated RBP and of RBP alkylated in the presence or absence of urea showed that the ‘*C of the RBP alkylated in the absence of urea was recovered in t,he gel slices containing the H-l and H-d bands of untreated RBP. In contrast, the 14C of the RBP alkylated in the presence of urea was distributed over a wide portion of the gel, suggesting that this RBP preparation was considerably modified as a result of the alkylation.

The ability of the alkylated RBP preparations to form a complex with untreated PA was examined, with mixtures of PA and alkylated RBP in molar ratios of about 1.1: 1. From the distribution of radioactivity in the complexed and free RBP peaks, it appeared that the affinity of RBP, alkylated without urea, for PA was only slightly reduced, since approximately 90% of this RBP preparation was recovered as the PA-REP complex. In contrast, RBP alkylated in the presence of urea showed a markedly reduced affinity for PA, with only 20% of this BP preparation recovered as a complex with PA.

Iodination of Prealbumin-Three equal-sized portions of PA (about 5 mg each) were iodinated with three levels of ICI, each level containing the same amount of 1311. Table IV shows the number of microgram atoms of iodine incorporated per pmole of PA for each preparation. The ultraviolet absorption spectra of the three iodinated PA preparations and of untreated PA showed progressive changes in the spectrum of the protein with increasing extent of iodination (see Fig. 8).

Mixtures of iodinated PA and of untreated PA were analyzed by disc gel electrophoresis, and the mobility of the iodinated PA


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1910 Interactions Involved in Retinal Transport Vol. 245, No. 5

preparations was determined by the distribution of radioactivity on the sliced gels. The iodinated PA preparations were also tested for their ability to form a complex with RBP, with mixtures of iodinat,ed PA and untreated RBP in molar ratios of about 1.1: 1. The results of these experiments are presented in Table IV. Iodination of PA with approximately 4.6 atoms of iodine per molecule did not significantly alter its binding affinity for RBP or its electrophoretic mobility on polyacrylamide gel. However, increased amounts of iodination to t,he extent of 12.5 and 24.8 atoms of iodine per molecule of protein resulted in a progressive and striking decline in the binding affinity of PA for RBP and also in a change in the electrophoretic mobility of the PA.

Each of the 1311-labeled PA preparations was tested for its binding capacity for thyroxine, with 1251-labeled thyroxine to determine thyroxine concentrations. The results of these studies are shown in Fig. 9. The PA-I-l preparation bound thyroxine with an affinity only slightly reduced from that of untreated PA. With increasing iodination, however, the thyroxine binding affinity decreased progressively; the affinities of the PA-I-2 and PA-I-3 preparations for thyroxine were ap-

TABLE IV

Studies with iodinated prealbumin

Preparation 1 ~$~$i,“lf 1 Z$i& 1 Distribution of ‘311 after disc gel electrophoresis

PA-I-l

PA-I-Z

PA-I-3

f$ am dine/

pnzole PA

4.6

12.4

24.8

97

33

5

proximately one-tenth and one-fiftieth, respectively, that of untreated PA.

Iodination of Retinal-binding Protein-RBP was iodinated with three different levels of ICl as shown in Table V. The efficiency of incorporation of iodine into RBP in all three preparations was quite constant, at about 52%. As with PA, the ultraviolet absorption spectra of the three iodinated RBP preparations showed progressive changes in the spectrum of the protein with increasing amounts of iodination (see Fig. 10). For RBP con taining approximately 2 atoms of iodine per molecule (RBP-I-l),

the spectrum was similar to that of untreated RBP, except that the retinol peak was shifted from 330 rnM to about 325 rnp.

Iodination up to the level of 4.3 atoms of iodine per molecule (RBP-I-2) did not result in a significant reduct,ion in the relative intensity of the 325 to 330 rnp absorption peak (compared with 280 rnp absorption), indicating that the binding of retinol to RBP was not significantly disrupted by this level of iodination.

The iodinated RBP preparations were tested for their ability

1.6

n 1.4

O-0 UNTREATED

A---A PA-I-1 U---17 PA-I-2

T-T PA-I-3

>95y0 of I311 migrated with untreated PA band

Half of 1311 migrated with PA band; rest migrated more slowly

>95% of 1311 associated with a wide, slowly migrating band

0.6-

a Expressed as the percentage of RBP recovered as a PA-RBP complex.

FIU. 9. Effect of iodination on the interaction of PA with thyroxine. The estimated values for the apparent association constants (k, values) were as follows: untreated PA, 1.5 X 107; PA- I-l, 1.3 X 107; PA-I-2, 0.15 X 107; PA-I-3, 0.03 X 107. See Table IV for the characterization of the PA-I-l, 2, and 3 preparations.

TABLE V

Studies with iodinated retinal-binding protein

360 340 320 300 280 260 WAVELENGTH (mu 1

FIG. 8. Ultraviolet absorption spectra of untreated and of iodinated PA. See Table IV for the characterization of the PA- I-l, 2, and 3 preparations.

Preparation

RBP-I-l RBP-I-2 RBP-I-3

Extent of iodination

pg atom $&?x/~??&

2.0 4.3 8.3

Bindii;gpaA~ity

80 63 20

Q Expressed as the percentage of RBP recovered as a PA-RBP complex, as determined by the distribution of I311 between the PA-RBP complex and free RBP peaks.


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WAVELENGTH (lll,U 1

FIG. 10. Ultraviolet absorption spectra of iodinated RBP. See Table V for charact,erization of the BBP-I-l, 2. and 3 preparations. The protein concentrations were almost id&t&al (approximately 0.37 my per ml) in the three solutions whose spectra are shown.

to form a complex with untreated PA, with mixtures of PA and RBP in molar ratios of about I .l: 1. The results of these studies are presented in Table V. The binding capacity of iodinated RBP for PA was inversely related to the extent of iodination.

Since retillol contains four conjugated double bonds, it was possible that the retinol itself underwent iodination under the conditions used. To examine this possibility, a portion of the RBP-I-1 preparation was partially extracted with heptane, and approximat,ely 25% of the retinol was recovered in the heptane phase. No radioactivity was found in the retinol-containing heptane phase, indicating that the double bonds of retinol were not iodinated in the RBP preparation.

DISCUSSION

RBP is isolated from human plasma in the form of a proteins protein complex, together with plasma PA. The complex i- dissociated by electrophoresis and can be formed again by mixing together soIutions of the isoIated proteins. Each of the two proteins also interacts with a particular small molecule: PA with L-thyroxine and RBP with retinol. The aim of the experiments reported here was to examine in more detail some of the chemical and physical aspects of each of the three interactions in this system, namely that of the two proteins with each other, and of each protein with its ligand. In these studies the effects of changes in temperature, pH, and urea concentration, and the effects of reduction and alkylation of disulfide bonds and of iodination, were assessed.

The analytical ultracentrifuge studies reported here clearly establish the formation of a 1: 1 molar complex between RBP and PA. When the two proteins were mixed together in nearly equimolar amounts they migrated together and appeared as a single homogeneous component in both sedimentation velocity and equilibrium analyses. The estimated molecular weight of the complex (70,200) was almost exactly the sum of the molecular weights of the individual proteins (PA, 49,400, and RBP,

21,300). These results suggest that no major structural changes occur in e&her protein as a result of the formation of the complex. The molecular weight estimate for RBP confirms our previous estimate of approximately 21,000 (I). The studies also indicate that RBP and PA only interact with each other in 1: 1 molar proportions. Thus, when RBl’-1’9 mixtures containing either protein in amounts in excess of a 1: 1 molar ratio were analyzed, the results showed the presence, in these mixtures, of the 1: 1 molar RBP-PA complex together with the free uncomplexed protein present in excess of this molar ratio.

The stability of the PA-RI{P complex was similar throughout the temperature range tested, namely 4-42.5”. This suggests that,, in viva, elevation of body temperature to as high as 42” would probably not cause significant dissociation of the complex. The complex appeared to be quite stable in the pH range of 5.8 to 7.5, with considerable dissociation occurring mainly between pH 8.6 and 10.3. These results suggest that ionic interactions or hydrogen bonding or both may play a major role in the association of the two proteins.

From the values in Table III, t’he association constant (k,) for formation of a complex between PA and RBP at III-I 8.2 was roughly estimated to be at least 2 x lo5 liters per mole. This value was calculated by assuming that the equilibrium present in the sample when it was loaded onto the Sephadex column was not changed during gel filtration. However, since a 40.fold increase in volume occurred during gel filtration, the true value of k, at pH 8.2 is probably closer to 106. The value of k, at pH 7.5 could not be approximated in the same way, since the estimates of the concentrations of free RBP and PA were much less accurate at this pH because of the very small proportion of RBP present in the free form. It is, however, clear that k, at pH 7.5 is considerably larger than that at pH 8.2. Moreover, from studies with the undissociated “native” complex (the D preparation) compared with mixtures of purified PA and RBP, it is clear that the association between the “native” proteins is stronger than that between previously separated PA and RBP (the latter consisting of a mixture of H-d, H-I, and 11 forms). With the data at hand, and given these considerations, we estimate that the value of k, for the protein-protein interaction at physiological pH in z&o is probably of the order of lo*; this estimate may, however, be incorrect by an order of magnitude 01 more. It should, in fact, be stressed that our estimates of the association constants for the RBP-PA interaction at different pH values represent, at best, only rough approximations.

The formation of the RBP-PA complex has a number of physiological and chemical consequences. As discussed previously (I), the protein-protein interaction clearly serves to protect RTSP by preventing the glomerular filtration of the relatively small RBP molecule. It also appears that the interaction of retinol with RBP is stabilized by t,he formation of the RBP-PA complex (II). As reported here, the formation of the RBP-PA complex alters the chromatographic behavior of RBP on DEAE-Sephadex. Thus, the RBP-PA complex had an affinity for DEAE-Sephadex which was greater than that of RBP alone, and which was similar to that of PA alone.

Purified RBP is micro-heterogeneous on disc gel electrophoresis and consists of three bands, two fluorescent and one nonfluores- cent, all with CQ mobilit’y. The observations presented here suggest that during purification and analysis the naturally occurring form of RBP (H-2) is gradually converted to a more rapidly migrating form of the holoprotein (N-l), perhaps by loss of a labile amide group, and in turn to the retinol-free apoprotein


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(A). The three RBP bands are immunologically indistinguish- able and, in studies to be reported in detail elsewhere, were found to react identically with an antiserum prepared against purified RBP. We previously reported (1) that purified RBP consists of two bands, one containing and one without retinol. It now appears that during the earlier purification procedure, which was longer and involved considerably more handling of the RBP, essentially all of the H-2 form was converted to the H-i form, thus resulting in a preparation consisting of two bands, H-l and A.

Urea, at a concentration of 6 M, caused complete dissociation of the PA-RBP complex. In addition, the binding affinity of PA for thyroxine was reduced approximately 15.fold in the presence of 6 M urea. In contrast, 6 M urea did not disrupt the retinol-RBP complex and hence did not appear to interfere with the association of retinol with RBP. The effects of urea on the association of PA with both RBP and thyroxine were completely reversible. The possible mechanisms responsible for the effects of urea (e.g. disruption of the structure of water, interaction with nonpolar amino acid side chains, and formation of hydrogen bonds with peptide groups) have been discussed by Gordon and Warren (12).

Chemical modifications of PA and RBP were carried out in order to determine the accessibility of some of their functional groups to modifying agents and the effects of such modifications on the various interactions in the system. Reductive alkylation of disulfide bonds under standard conditions was shown to be incomplete for both proteins. Results with other proteins (13-15) have shown that the quantitative reduction of disulfide groups can sometimes be obtained only in the presence of de- naturing agents. In the present studies, only one of the two disulfide bonds in PA was reduced in the absence or in the presence of 6 M urea; the effect of prolonged reduction time in the presence of urea was not tested. Reduction and alkylation of one disulfide bond in PA did not significantly affect the binding of either RBP or thyroxine to PA. It thus appears that this accessible disulfide bond in PA does not play an important role in determining and maintaining the conformation of PA, at least with regard to those aspects of PA structure involved in its interaction with RBP and with thyroxine. It should be noted that previous studies have shown that the interaction of PA with thyroxine is independent of the PA-RBP interaction (2).

RBP contains 5 to 6 half-cystine residues per molecule (1) and no accessible monothiols (Table I). It is therefore likely that RBP contains either three disulfide bonds or two disulfide bonds and one inaccessible monothiol. In any event, in contrast to PA, all disulfide bonds in the RBP molecule were highly resistant to reduction; prolonged exposure to a reducing agent in the presence of urea was necessary in order to effect reduction. In the absence of urea, 0.5 mole of acetic acid was incorporated per mole of RBP after reduction. This value suggests that approximately one-fourth of the RBP molecules underwent reduction of one disulfide bond. Approximately one-fourth of the retinol content was lost from this partially reduced preparation, as compared to untreated RBP. These observations, taken together, suggest that reduction of the first disulfide bond in RBP significantly alters the structure of RBP with regard to the retinol-binding side, resulting in a decreased affinity for retinol and a loss of retinol from the holoprotein. In the presence of 6 M urea almost complete reduction and alkylation of two disulfide bonds of RBP were achieved. This modification com-

pletely disrupted the retinol-RBP complex and resulted in complete loss of the retinol previously bound to the protein. In addition, this degree of reduction of the disulfide bonds of RBP greatly reduced the affinity of RBP for untreated PA, although some interaction did occur between reduced RBP and PA.

Iodination of either PA or RBP produced progressive changes in the ultraviolet absorption spectrum of the protein. Iodina- tion of PA at a level of 4.6 atoms of iodine per molecule of PA did not alter the affinity of PA for RBP and only slightly reduced its affinity for thyroxine. In contrast, iodination of RBP with only 2 atoms of iodine per molecule of RBP significantly reduced the affinity of RBP for PA; further iodination of RBP produced progressive reductions in the affinity of RBP for PA. However, iodination of RBP up to the level of 4.3 atoms of iodine per molecule of RBP did not appear to interfere with the interaction of retinol with RBP, since there was no loss of retinol from the RBP preparation iodinated to this extent.

These studies demonstrate that it is possible to interfere selectively with the interaction of RBP with either PA or retinol. Addition of 6 M urea, for example, completely disrupts the RBP-PA complex, whereas the retinol-RBP complex remains fully intact. Similarly, iodination of RBP up to the level of 4.3 atoms of iodine per molecule of RBP selectively interferes with the interaction of RBP with PA, without disrupting the retinol-RBP complex. In contrast, when the disulfide bonds of RBP are reduced and alkylated the binding of retinol by RBP is completely disrupted, whereas the altered RBP retains a slight (although greatly reduced) affinity for PA. It is thus clear that different factors are involved in the lipid-protein (retinol-RBP) and the protein-protein (RBP-PA) interactions of RBP. Fur- ther studies of these interactions, and of that of PA with thyroxine, are necessary in order to define more fully the factors controlling each of these interactions in vitro, and their relation- ships to the biological functions of the retinol transport system in viva.

Ac&owZe&me&s-We are grateful to Drs. W. Poillon and P. Feigelson for analytical ultracentrifuge analyses and to Miss E. Miller for expert assistance.

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Amiram Raz, Tatsuji Shiratori and DeWitt S. GoodmanRetinol Transport in Plasma

Studies on the Protein-Protein and Protein-Ligand Interactions Involved in

1970, 245:1903-1912.J. Biol. Chem.

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