Vol. 253, No. 22, Issue of November 25, pp. 8221-8228, 1978 Printed m U.S A

New Biospecific Adsorbents for the Purification of Estradiol Receptor*

(Received for publication, January 4, 1978)

Robert Bucourt, Michel Vignau, and Vesperto Torelli

From Roussel UclaL 93230 Romainville, France

H618ne Richard-Foy,$ Claudine Geynet, Claude Secco-Millet, GBrard Redeuilh, and Etienne-Emile Baulieu

From the Znstitut National de la Sant6 et de la Recherche Mkdicale, Unit& de Recherches sur le Mktabolisme Molkdaire et la Physio-Pathologie des Stkro’ides, Dtpartment de Chimie Biologique, Facultk de Mkdecine de Bicitre, 94270 BicZtre, Frances

The synthesis of biospecific adsorbents for the puri- fication of the cytosol estrogen receptor from calf uterus is described. The characteristic of several estra- diol derivatives, spacer chains, and insoluble matrix were systematically studied. Estradiol derivatives sub- stituted at positions C2, 3, 4, 7cu, 17a, and 17/I were tested for their affinities for the receptor; positions 7a: and 17a: were the most suitable. Acidic compounds had lower affinities than their methylester analogues. Long chain derivatives bound the receptor less firmly than corresponding shorter chains. However, when these ligands were attached to an insoluble matrix, the long spacer chain derivatives (214 atoms) were more effi- cient than the shorter ones. There was a satisfactory parallelism between affinities of free ligands and recep- tor binding to the respective biospecific adsorbents. On the basis of their stability in the presence of cytosol (no release of ligand), due to the absence of ester bonds, long chains were selected as spacers. Both acrylamide and agarose biospecific adsorbents displayed some ionic exchange capacity and consequently nonspecifi- cally bound proteins; the influence of this nonspecific binding on the purification of the receptor was studied.

On the basis of their stability, of their binding speci- ficity, and of their selectivity for the receptor, the es- tradiol-‘la derivative adsorbents were selected for the purification of the receptor.

Affinity chromatography, based on the principle of specific protein-ligand recognition, is an efficient method for the pu- rification of proteins and has been successfully used for nu- merous enzymes (1). In contrast, for “binding proteins,” such as steroid hormone receptors, several problems are encoun- tered (2-5). This is due to a number of specific problems related to the properties of these macromolecules and of their ligands. 1) The equilibrium dissociation constants (K,)) of enzymes for substrates or inhibitors are of the order of 1 to 100 PM, whereas they are of the order of 0.1 nM for the receptor interacting with hormones. These differences in affinities are

* This work was partially supported by the DBlbgation G&&ale B la Recherche Scientifique et Technique, the Centre National de la Recherche Scientifique, and the Ford Foundation. The costs of pub- lication of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Preceding papers were published under the name of Truong (see Refs. 7 and 14).

8 Postal address, Lab Hormones, 94270 BicBtre, France.

mainly due to the slow dissociation rate of hormone-receptor complexes at 4°C as well as 25°C (6, 7). Consequently, the displacement of the receptor from the adsorbent (biospecific elution) may be difficult. 2) The activity of enzymatic proteins is easily measured even in the presence of competitive inhib- itors. In contrast it is difficult to determine receptor binding activity in the presence of nonradioactive competitive ligands, again because of the slow rate of dissociation of the complexes. Raising the temperature increases the dissociation rate of the receptor-ligand complexes but may also lead to receptor in- activation. 3) For most enzymes and other binding proteins, water-soluble ligands are used for the elution from the bio- specific adsorbent but steroids are not very soluble in aqueous media (maximum solubility of estradiol, 30 PM). This charac- teristic limits the concentration of ligand which can be used to elute the receptor from biospecific adsorbents. 4) The degree of purification required to obtain homogenous enzymes is usually lO’“-fold, whereas it is 10” for steroid receptors. This last point makes it particularly important to develop an affin- ity chromatography step for the receptor purification. Affinity chromatography is the only method providing the possibility to obtain in a single step a purification >10”-fold, high recov- ery, and concentration of the protein.

Three constituents of a biospecific adsorbent are involved in determining its properties: the bound ligand, the spacer arm, and the polymeric matrix. 1) For binding the estrogen receptor, it is necessary to use a ligand which has a signifi- cantly higher affinity for the receptor than for other binding proteins also present in the extracts (e.g. plasma proteins). Several steroid derivatives interact with albumin with an apparent KI, = 10 P.M.’ I f the receptor is to be selectively retained, it is necessary that its Ku for the ligand coupled to the adsorbent be ~0.1 PM. The structural requirements of the steroid derivatives which can bind to the receptor are very strict. 2) The length of the spacer chain should allow accessi- bility of the ligand to the receptor binding site with minimal interfering effects (e.g. folding of the chain). The spacer chain should also be sufficiently resistant to hydrolysis in aqueous biological media (5). Otherwise there will be formation of (free) ligand-receptor complexes in the effluent fraction in- stead of binding to the adsorbent since the receptor generally has a higher affinity for free than for matrix-bound ligand. Consequently, a precise estimation of the amount of retained receptor will be difficult (8, 9) and interpretation of the properties of the biospecific adsorbent are likely to be erro- neous. 3) Finally the solid matrix should be stable, inert, and not provoke denaturation of the receptor during the chroma- tography.

’ F. Faure and E. E. Baulieu, unpublished results.

8221

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

a222 Biospecific Adsorbents for Estradiol Receptor Purification

The aim of the studies reported here was the preparation of biospecific adsorbents suitable for the purification of the es- tradiol receptor from calf uterus cytosol. We systematically investigated the binding characteristics of estradiol spacer derivatives and of estradiol spacer matrix materials.

MATERIALS AND METHODS

See miniprint supplement.’

RESULTS

Interaction of the Receptor with the Estrogen-Spacer Derivatives

In order to select the most efficient ligand for receptor purification, several estradiol derivatives were synthetized (see miniprint supplement). The binding of these estradiol derivatives to the receptor was measured by competition against [“Hlestradiol. The radioactive complex was measured by the charcoal technique (10) and K, was calculated from Dixon plots (see miniprint supplement).

Attachment of Spacer to Ring A (Aromatic) of Estradiol-The affinity of the 3-hemisuccinate derivative could not be measured since total hydrolysis, regenerating estradiol, occurred spontaneously during incubation. The 2- and the 4-diazo-phenyl derivatives have a K, approximately lo” times higher than estradiol, thus excluding them for selec- tive retention of the receptor (Fig. la) (9).

Estradiol-17,8 Acylates-Estradiol-17P hemisuccinate has some affinity for the receptor (Fig. lb). However, when a longer chain is attached in position 17/3 by an ester link, the affinity for the receptor is very low (Fig. lc). Moreover, the easy cleavage of the ester bond by cytosol preparations, men- tioned in particular for deoxycorticosterone-hemisuccinate ad- sorbents is not very encouraging (5). However, some investi- gators have obtained satisfactory results with this series of 17p compounds, even if some release of the ligand is men- tioned (4).

Estradiol-17a-Spacer Derivatives-17a-Ethynyl estradiol (Fig. 2a) has much affinity for the receptor and a relatively reduced affinity for plasma proteins as compared to estradiol (11) and, therefore, is an interesting starting compound from a biospecific viewpoint. When a short side chain is grafted on at position 17a, the affinity is decreased (Fig. 2~) and even more dramatically when there is a free carboxyl group (Fig. 2b). However, affinity can be partially restored by esterifica- tion (Fig. 2d). As in the 17p series, lengthening of the chain decreases the affinity (Fig. 2e) and again this result is not very favorable even when there is no problem with the stability of these compounds.

Estradiol7aSpacer Derivatives-It is conceivable that the graft of spacer away from the two hydroxyl groups could lead to high affinity derivatives suitable for affinity chromatogra- phy since both groups are involved in the binding of estradiol. The estradiol-7a-methyl derivative (Fig. 3a) has a high affinity for the receptor (12) and a very low affinity for the plasma protein SBP (13). It was again observed that the introduction of a carboxyl group on a short chain (Fig. 3b) leads to very low (nondetectable) affinity of the derivative for the receptor. However, the corresponding alcohol (Fig. 3c) is very efficient. Moreover, a longer neutral derivative (Fig. 3d) has very high affinity. Finally, if the carboxyl function is maintained 10

’ Portions of this paper (including Figs. 7 to 10) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, Md. 20014. Request Document No. 78M-14, cite author(s), and include a check or money order for $2.70 per set of photocopies.

a: Kiz 10~ M

KiE 0.1 p M

0-CO-(CH,);COO-

LIP

KelOpM

C.

HO 1:

FIG. 1. Estradiol spacer derivatives: Grafting of the spacer onto the ring A, or by esterification of 17,@-OH. *, two alternative positions for substitution of the chain.

OH K,zl nM

OH K,E10 pM

OH K,E 0.2 p M

Kiz 0.1 p M

Fro. 2. Estradiol spacer derivatives: 17~ series.

carbon atoms away from the steroid nucleus (Fig. 3e), a good result is still obtained.

Binding of the Receptor to Biospecific Adsorhents

It would seem to be good practice to select an estradiol- spacer derivative of highest affinity in order to make as great as possible the difference between affinities for the receptor and other binding proteins. However it cannot be predicted whether the situation will be the same after linkage of deriv- atives to the matrix. For this reason, most of the derivatives were coupled to solid matrix and then assayed for receptor binding.

Polystyrene Derivative-Polystyrene is hydrophobic and, consequently, difficult to wet and to wash. The swelling in water is very poor. Apparently, the receptor was not bound by the adsorbent but partially denaturated in the presence of the polymer.

Polyacrylamide Derivatives-Polyacrylamide is of great interest because the adsorbent can be stored in a dry state at room temperature for long periods of time, without danger of contamination by bacteria or splitting of the derivative. The 3-hemisuccinate of estradiol linked to the acrylamide (Fig.

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Biospecific Adsorbents for Estradiol Receptor Purification 8223

a

HO K,E 0.1-I n M

no competitor

K,Y 0.1 p M

K,zlO nM

OH

f!

HO K,z 0.1 p M

FIG. 3. Estradiol spacer derivatives: 7a series

4d), even though it was relatively stable in the dry state, could not be used for chromatography since the ligand was released when it was suspended in the buffer and very rapidly when exposed to the cytosol. Table I shows that the same decrease in the binding activity is observed after exposure of the adsorbent to the cytosol and to the dichloromethane extract. This suggests that the adsorbent releases a compound, pro- voking the same effect as the exposure to the adsorbent. The receptor was not bound to the adsorbent but remained present in the cytosol after exposure to the adsorbent, complexed with the nonradioactive ligand. Exchange measurements allowed recovery of all the binding activity. Results of Table I had shown that incubation with the buffer is sufficient for provok- ing the release of this ligand, but when the cytosol is present, an enzymatic cleavage cannot be exluded (5). It can be ex- cluded that these phenomena are only due to an incomplete wash of the adsorbent after synthesis. The washing procedure for this adsorbent was the same as that used for the other adsorbents, which are not significantly contaminated by free steroid (see Table II). The comparison of the binding prop- erties of the 7cu and 17cu-estradiol derivatives presented in Table II, (A&o&e&s a, b, c, e, and fof Fig. 4) indicates that the best binding is obtained with the estradiol-7a-acrylamide adsorbent (4a-1) and with the estradiol-17n-acrylamide ad- sorbent (4f). Exchange measurements and measurement of the possible contaminant by receptor radioimmunoassay con- firm these results.

These data lead to several comments: 1. The length of the spacer is important. When the ligand

is linked to the acrylamide through a short chain (adsorbents 4b and 4e), the binding of the receptor is very low.

2. For a similar length of chain (adsorbents 4a and 4c), the difference in the cross-linking of the solid matrix is important. Adsorbent 4c does not bind the receptor. The main difference between this adsorbent and adsorbent 4a is the cross-linking. The swelling of 4c in the water is approximately 5 times less than that of 4a. For the adsorbent 4c, it is important to notice that a significant amount of contaminant was present in the effluent fraction of the column. It is surprising to detect it by

TABLE I

Binding of receptor to e&radio1 d-hemisuccinate acrylamide adsorbent

See Fig. 4d. % of control cytosol

Direct measure- Exchange mea- merit surement

Cytosol + dichloromethane ex- 99.0” tract of buffer

Cytosol exposed to the adsorb- 3.0” ent 14.0h looh

Cytosol + dichloromethane ex- 2.5” tract of buffer incubated with the adsorbent

a In this experiment, adsorbent was incubated batchwise with calf uterus cytosol (binding activity, 10,600 cpm/ml) or buffer (for details see miniprint supplement). After exposure to the adsorbent, cytosol and buffer were separated by centrifugation. The buffer, exposed or not to the adsorbent, was then extracted with dichloromethane, and the extracts were dried and incubated concomitantly with untreated cytosol and 1 nM [“Hlestradiol. Cytosol exposed to adsorbent was incubated in parallel with the same amount of [“Hlestradiol. The binding activities (in counts per min per ml) were measured by the charcoal technique.

h In a different experiment, 10 ml of calf uterine cytosol (binding activity 104,000 cpm/ml) were loaded on a column containing 1 ml of adsorbent. The receptor binding activities were measured in the cytosol and in the pool of the effluent fractions by direct and exchange hydroxylapatite techniques (for details see miniprint supplement).

TABLE II

Binding of e&radio1 receptor to biospecific adsorbents The structures of the adsorbents are presented in Figs. 4 and 5 and

the letters refer to these figures. Numbers refer to different prepara- tion batches of the same adsorbent. One-milliliter columns were packed with the absorbents. Acrylamide absorbents were washed with 200 ml of a water/dioxane mixture (v/v), 2 liters of water and equilibrated with 500 ml of Tris buffer. Trypsin-treated cytosol was loaded onto the columns under a flow rate of 18 to 20 ml/h for the acrylamide adsorbents and 25 to 30 ml/h for the agarose adsorbents. During the loading, the effluent fractions were collected and pooled. The binding activities were measured by the charcoal technique. The amount of receptor bound to the columns was calculated by difference between the amounts loaded onto the columns and present in the effluent fractions. Direct measurements (see miniprint supplement) are expressed in per cent of the loaded receptor bound to the adsorb- ent. Exchange measurements (see miniprint supplement) are ex- pressed in per cent of the value obtained by the direct measurement. The amounts of “contaminant” free ligand were measured by receptor radiocompetition assay (see miniprint supplement), and the results expressed in estradiol equivalents were: 0.5, 0, 2.3, 0.1, and 0.5 nM for adsorbents 4a-I, 4b, 4c, 4e, and 4f, respectively. The amounts of steroid coupled to the different adsorbents were: 4a-1, 0.15 mg/ml of gel; 4a-2, 0.02 mg/ml of gel; 4b, 0.4 mg/ml of gel; 4c, 0.12 mg/ml of gel; 4e, not measured; 4f, 0.008 mg/ml of gel; 5-1, 0.03 mg/ml of gel (theoretical value) and 0.1 mg/ml of gel (measured value); 5-2, 1.37 mg/ml of gel (theoretical value) and 0.3 to 0.6 mg/ml of gel (measured value). (For details and discussion of these values, see miniprint supplement.)

Loaded receptor ~pmol)

Adsorbents

76 228 380 1097 380 1097

Bound receptor (8)

Direct meawrement Exchange measure- ment

4a-1 55 47 40 98 4a-2 14 7 4b 20 17 15 94 4c 28 31 4e 32 29 26 100

4f 54 48 43 95 5-1 78 74 70 5-2 100 90 90 69 91

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8224 Biospecific Adsorbents for Estradiol Receptor Purification

t

OH

a CO

\ OH OH

b i

C / \

CO-NH-(CH,~NH-CO-(CH2~.. - #

d

3 aF OH

e CO-NH-(CHpkNH-CO-c&. OH

/ OH

f t

\

@ CO-NH-(CH+NH-CO-(CH~OO-(cH,~EC--- OH

FIG. 4. Acrylamide adsorbents.

the receptor radioassay, because the derivative used for the synthesis had no affinity for the receptor. It is probable that this contamination is not due to an incomplete washing of the adsorbent, but to a leakage occurring by a split at the acryl- amide level.

3. The best results were obtained with adsorbent 4a-1 and 4f, indicating a good correlation between the affinities of the estradiol-spacer derivatives for the receptor and the binding to the corresponding biospecific adsorbent containing similar amounts of immobilized steroid.

4. The 17a-butynol derivative can probably be used for receptor purification, but the ester linkage in the chain is not very stable. When the gel is stored for a long time in Tris buffer, release of the ligand is observed. Extensive washing of the gel before use is absolutely necessary.

Agarose Derivatives-On the basis of the results obtained with the acrylamide derivatives, only one adsorbent type was studied (Fig. 5 and Table II). Fixation of the receptor was obtained for the two adsorbents containing different amounts of coupled steroid. In both cases the binding was higher than for adsorbents 4a-1 and 4f. This is not surprising for adsorbent 5-2 which contains high amounts of immobilized steroid. The difference in the capacities between adsorbents 4a-1, 4f, and 5-l can be explained either by an underestimation of the amount of coupled derivative for adsorbent 5-l (see miniprint supplement) or by a difference in the cross-linking of the adsorbents.

El&ion of the Receptor Bound to Biospecific Adsorbents

The ultimate test of the success of affinity chromatography is the ability to elute and recover the receptor from the biospecific adsorbent with reasonable yield and purity.

Results presented in Table III refer to three adsorbents able to bind the estradiol receptor (see Table II), through a biospecific mechanism and not in a nonspecific manner. In all cases it was observed that, in the absence of an excess of free estradiol, the increase of the temperature leads to a very low recovery of the bound receptor (wash, 32°C in the table). On

OH

/ 1 CO-NH-(CHZ);NH-(CH2);NH-CO-&H&;, -

8 OH

FIG. 5. Agarose adsorbent.

the contrary in the presence of 30 pM estradiol, the receptor is eluted and the degree of purification is high in all cases. The elution recovery is higher for adsorbent 4a-1 than for the other two. Elution recoveries explain the difference in the degree of purification obtained.

These differences in the elution recoveries could be inter- preted in different ways:

1. The affinity of the receptor adsorbent 4a-1 would be lower than for the other two and consequently for similar amounts of coupled steroid, and, under similar conditions, the elution of the receptor would be easier. In fact this does not seem to be the case since the affinity for the receptor is higher for the 7a than for the 17a-estradiol derivatives and the estradiol-spacer derivative is the same for adsorbents 4a- 1 and 5-l.

2. The amount of coupled steroid would be lower for ad- sorbent 4a-1. This hypothesis is not in agreement with the measured values, even if it is difficult to measure precisely the amount of coupled steroid (see miniprint supplement).

3. The receptor binding capacity would be different for the three adsorbents. This last possibility is in agreement with data presented in Table II indicating binding 4a-1 < 4f < 5-l. For higher recoveries of the receptor from the last two ad- sorbents, it would be necessary to decrease the concentration of coupled steroid (the amount of eluting steroid cannot be increased over the used concentration, because of the limit of solubility of estradiol in aqueous medium) (14).

Adsorbent 4a-1 is the most efficient for estradiol receptor purification. It allows a 1700-fold purification of estradiol receptor from cytosol with a recovery of 30%.

Ionic Exchange Properties of the Biospecific Adsorbents

Results presented in Table III indicated there is also pro- teins bound to biospecific adsorbents, probably by an ionic exchange mechanism, whereas the binding of the receptor is essentially due to a biospecific phenomenon. The amount of receptor bound to the column by ionic interaction is low, as indicated by the low amount of receptor present in the wash at 32°C (Table III), or eluted by increasing the ionic strength (14) or decreasing pH (Fig. 6). The presence of large amounts of free estradiol is necessary for elution of the receptor. When large volumes of cytosol are loaded onto the column, the contribution of ionic interactions is minimized, 50 ml of cytosol saturating the ionic groups of a l-ml column.

The influence of the titer of acrylamide derivatives (ami- noethylacrylamide and estradiol-7a-undecanoate-aminoeth- ylacrylamide) and of the ionic strength on the binding of proteins and receptor was investigated. Results presented in Table IV show that the binding of the receptor to the bio- specific adsorbent was not significantly modified by the ionic strength and that it was higher than the binding to acrylamide and aminoethylacrylamide. Aminoethylacrylamides of differ- ent titers were used. An important binding of nonreceptor proteins to the aminoethylacrylamide was observed. The de- crease of the titer and the increase of the ionic strength minimized considerably this phenomenon. The binding of the receptor at high ionic strength indicated that it is highly probable that most receptor is bound by a biospecific mech-

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Biospecific Adsorbents for Estradiol Receptor Purification

TABLE III Comparison of the successive elution ofproteins and receptor bound to biospecific adsorbents without or with estradiol

Adsorbents (for nomenclature see Table II and Figs. 4 and 5) were packed in l-ml columns. After loading with trypsin-treated cytosol the columns were washed at 32OC by Tris buffer until Arx,, ,,,,, fell to zero. The fractions containing proteins were pooled and assayed for receptor and proteins. Batchwise incubation of the adsorbents in the presence of 30 PM [ ‘Hlestradiol (specific radioactivity, 4 to 10 Ci/

__-~ Adsorbent Fraction Actimty Protein

4a-1

4f

5-1

Cytosol Wash, 32°C Bound to ad-

sorbent Eluate

cytoso1 Wash, 32°C Bound to ad-

sorbent Eluate

cytoso1 Wash, 32°C Bound to ad-

sorbent Eluate

Cpmx lO”/fraction

t t 9

40. ‘k.+ %.n.

30.

pmol/ml 4.40 0.18

7.8

4.40 0.46

4.12

4.5 0.05

3.8

w/ml 8.25 0.067

0.0088

8.25 0.037

0.007

11 1.3

0.015

FIG. 6. Elution of the receptor by a pH gradient: Fifteen milliliters of a partially purified “4s-trypsin” form of the receptor (cytosol precipitated by ammonium sulfate 30% of saturation; purification 5- fold; proteins, 3.2 mg/ml; specific activity, 37,600 cpm/mg of protein) were loaded at 4°C onto a l-ml column containing the estradiol-7a- acrylamide adsorbent (Fig. 4a-1). After washing with 20-ml Tris buffer, receptor was eluted at 4°C by applying a pH gradient (buffer consisting of citrate (0.05 M) + potassium phosphate (0.1 M) solutions, containing bovine serum albumin (1 mg/ml) which served to protect the receptor). Then 2.5-ml fractions were collected. Each fraction, after measurement of pH, was diluted by addition of 20 ml of Tris (0.1 M) buffer, pH 7.5, in order to reach neutrality. Receptor binding sites were measured by the hydroxylapatite exchange technique. Receptor was eluted at a pH close to 6, at which this molecular form of the receptor is stable. A reversible inactivation is observed for pH 4.5 (15), a pH at which no receptor binding activity was detected. The eluted receptor represents 2%~ of the receptor bound to the adsorbent and is probably receptor bound by an ionic exchange mechanism.

anism. However, this adsorbent also exhibited some ionic exchange properties, as indicated by column studies. These results indicate that the control of the ionic charge of biospe- cific adsorbent is important. All the acrylamide adsorbents used for the receptor purification have titers <O.l N. Similar observations were made with the agarose adsorbents. How- ever, it is more difficult to control their titer than for acryl- amide adsorbents.

8225

mmol) for l/r h at 29°C was then performed in order to elute the receptor biospecifically bound. After filtration of these fractions (“Kluate”) through Sephadex G-50 columns in order to eliminate the excess free steroid, specific binding was measured by the HAP tech- nique (14). Binding activities given in the table for the eluate fractions were corrected for the dilution introduced by the gel filtration.

Specific activity Purification Total activity -Reco”ery

pmol/mg pm01 ‘i

0.52 1 730 100 2.68 2.34

215 29 899 1730 70.4 32.5

0.52 1 1263 100 12.43 10.6

319 25 588 1130 36.9 11.5

0.41 1 1315 100 0.038 0.4

868 66 253 617 106 12

TABLE IV

Ionic exchange properties of acrylamide and modified acrylamide gels

The gels were incubated batchwise with rat cytosol, and the recep- tor binding activities were measured by the charcoal technique in the cytosol after exposure to the gels (for details see “Materials and Methods”). The control binding activities were 6,200 and 2,600 cpm/ ml and the protein concentrations were 0.55 and 0.58 mg/ml for the cytosol in the Tris and Tris/KCl (0.5 M) buffers, respectively. The structure of the biosoecific adsorbent is oresented in Fig. 4”.

Tris buffer Tris/KCl (0.5 M) buffer

Binding ac- Protein % Binding ac- tivity % of of control tivity % of Protein %

control control of control

Acrylamide 97 Aminoethylacrylamide, 11

0.75 N

100 100 100 18 50 86

Aminoethylacrylamide, 0.27 N

19.5 48 64 84

Aminoethylacrylam- ide-li:r, 0.27 N

6 57 6.5 84

-_____

DISCUSSION

Purification of steroid hormone receptors, and in particular the cytosol estradiol receptor, raises certain specific problems due to the extremely low concentration of the receptor in the extracts and the presence of substantial quantities of other proteins binding the same hormone. Thus, utilization of bio- specific adsorbents capable of selectively retaining the recep- tor is necessary. This selectivity can be achieved by attaching the estradiol molecule in such a way that the affinity of the receptor for the derivative, compared to that of estradiol, is impaired as little as possible. The affinity of the receptor for the immobilized derivative will be a function of the fixation position of the spacer chain on the steroid molecule, as well as the character of the spacer chain itself. For the biospecific adsorbents described in the literature, the choice of the deri- vation position for the spacer chain has been more a function of the facility of synthesis of the derivatives than of the previsible selectivity for receptor retention. However, in order to carry out the purification of steroid hormone receptors to homogeneity, an affinity chromatography step more efficient than those previously described is necessary (l-5).

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8226 Biospecific Adsorbents for Estradiol Receptor Purification

The aim of the work presented here was to study the efficiency of different new biospecific adsorbents obtained by immobilization of estradiol for the purification of estradiol receptor from uterine cytosol. We have systemically measured the affinity of the estrogen receptor for estradiol derivatives bearing different spacer chains substituted at different posi- tions on the steroid molecule. As a result, it has been possible to select several derivatives for which we observed a parallel- ism between binding of their neutralized forms and the cor- responding adsorbents obtained by coupling them to an insol- uble matrix.

The 7~y and 17a derivatives exhibited highest affinity for estradiol receptor and showed no affinity for plasma transport proteins (11, 13).

However, the requirement of a long spacer chain, previously described for other biospecific adsorbents (4), is not a general rule for all steroid binding proteins. For instance, for plasma steroid binding proteins a short chain is convenient (16) and for Ar,+,,3-oxo-steroid isomerase a short one is even preferable (17).

In both 17a and 7n derivatives series, significant receptor binding to biospecific adsorbents was observed if the spacer chain contained more than 14 atoms, except if the long chain was obtained by the condensation of the spacer derivative on a long diamine coupled to the acrylamide. The coupling to acrylamide of such a diamine increases considerably the cross- linking of the adsorbent and the binding of the receptor becomes negligible.

For the purification of the estrogen receptor, the 7o deriv- atives were routinely preferred to the 17a derivatives. The former provides complete accessibility to the A and D ring of estradiol, which are implicated in the recognition and binding of estradiol by the receptor (12). Moreover, the suitable 17a derivative adsorbent described here has an ester bond in the chain, resulting in a stability lower than that of the corre- sponding 7a adsorbent.

Two types of adsorbents were synthesized, using either acrylamide or agarose. The acrylamide adsorbents can be stored in a dry state and, under these conditions, the ligand is not released, while the adsorbents do not survive in storage once swollen in aqueous medium. In general, the agarose adsorbents have better mechanical properties than the acryl- amide adsorbents.

It should be noted that all the biospecific adsorbents studied bind a significant amount of proteins nonspecifically. This binding is essentially due to ionic interactions. The titration is more easily monitored for acrylamide adsorbents than for agarose. The ionic exchange capacity of the adsorbents is not a major inconvenience for the purification of the receptor since appropriate washing can remove the contaminating pro- teins prior to the biospecific elution by the ligand (14). Bio- specific adsorbents obtained by coupling estradiol 7n or 17a derivatives to acrylamide or to agarose routinely allowed a single sten 600 to 2.000-fold nurification of the “4Strvnsin”

” I 1 “l

form of the estradiol receptor from calf uterine cytosol, with a yield of 10 to 30%.

From results presented in this paper, some requirements for the most suitable biospecific adsorbent can be proposed:

1) attachment of the spacer chain at the 7n position of estradiol (low impairment of the affinity compared to estra- diol) allowing a high selectivity for receptor retention; 2) long spacer chain, stable in the presence of the sample, during storage, washing, etc. . . ; 3) for the insoluble matrix, agarose is to be preferred to acrylamide (better mechanical proper- ties).

Systematic studies of the parameters influencing the puri- fication of estradiol receptor by affinity chromatography and the use of this method for achieving purification of estradiol receptor to homogeneity will be reported elsewhere” (18).

Acknowledgment-We thank L. Helie for technical assistance.

REFERENCES

1. Cuatrecasas, P., and Anfinsen, C. B. (1971) Anna. Rev. Biochem. 40, 259-278

2. Jensen. E. V.. DeSombre, E. R., and Jungblut, P. W. (1967) in Proceedings of the 2nd International Congress of Hormonal Steroids. Milan. 1966 (Martini, L., Franscini, F., and Motta, M., eds) pp. 492-500, Excerpta Medica Foundation, Amsterdam

3. Vonderhaar, B., and Mueller, G. C. (1969) Biochim. Biophys. Acta 176,626-631

q Truong, H., and Baulieu, E.-E. (1971) Biochim. Biophys. Acta i.

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

18.

237;167-172 Ludens, J. H., De Vries, J. R., and Fanestil, D. D. (1972) J. Biol.

Chem. 247, 7533-7538 Best-Belpomme, M., Richard-Foy, H., Secco-Millet, C., and Bau-

lieu, E.-E. (1976) Biochimie (Paris) 58, 863-869 Milgrom, E., and Baulieu, E.-E. (1969) Biochim. Biophys. Acta

194, 602-605 Vermeulen. A., and Verdonck, L. (1970) Acta Endocrinol. 147,

(Suppl) 239-256 Geynet, C., Millet, C., Truong, H., and Baulieu, E.-E. (1972)

Gynecol. Invest. 3, 2-29 Renoir, M. (1973) Diplome d’Etudes Superieures, Universite Paris

VI Truong, H., and Baulieu, E.-E. (1974) FEBS Lett. 46, 321-325 Soulignac, O., Truong, H., and Baulieu, E.-E. (1974) C. R. Acad.

Sci. Paris 278, 2955-2958 Le Gaillard, F., Racadot, A., Racadot-Lero, N., and Doutrevaux,

M. (1974) Biochimie 56,99-108 Vincent, F. (1976) These de Doctorat d’Etat es Sciences, Univer-

site Paris XI Richard-Foy, H. (1977) These de Doctorat d’Etat es Sciences,

Universite Paris VII

4. Sica, V., Parikh, I., Nola, E., Puca, G. A., and Cuatrecasas, P. (1973) J. Biol. Chem. 248,6543-6558

5. Kuhn, R. W., Schrader, W. T., Smith, R. G., and O’Malley, B. W. (1975) J. Biol. Chem. 250,4220-4228

6. Best-Belpomme, M., Fries, J., and Erdos, T. (1970) Eur. J. Bio- them. 17, 425-436

Additional references are found on p. 8228.

‘G. Redeuilh, R. Richard-Foy, E. E. Baulieu, R. Bucourt, M. Vignau, and H. Richard-Foy, manuscript in preparation.

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Biospecific Adsorbents for Estradiol Receptor Purification a227

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8228 Biospecific Adsorbents for Estradiol Receptor Purification

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

R Bucourt, M Vignau and V TorelliNew biospecific adsorbents for the purification of estradiol receptor.

1978, 253:8221-8228.J. Biol. Chem. 

  http://www.jbc.org/content/253/22/8221Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/253/22/8221.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on August 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from