Study of steroid-protein binding by means of competitive adsorption: Application to cortisol binding in plasma

CLINICA CHIMICA ACTA

STUDY

361

OF STEROID-PROTEIN

ADSORPTION:

W. HEYNS,

APPLICATION

H. VAN

BAELEN

AND

BINDING

P. DE

Rega Instituut, Laboratorium ZIOOY Exfierimentele Katholieke Universiteit te Leuven (Belgium) (Received

BY

TO CORTISOL

MEANS BINDING

OF COMPETITIVE IN PLASMA

MOOR Geneeskunde,

July 19th. 1967)

SUMMARY

The distribution of a steroid between a solution of steroid-binding proteins and a steroid-adsorbing solid added to this solution, depends not only on the properties of the adsorbent but also on the properties of the binding proteins and thus may be used for the study of steroid-protein interactions. Different types of distribution can be distinguished and were applied to the study of cortisol binding in plasma. (I) Endogenous steroids were removed from plasma by Incubation with a rather large amount of adsorbent. (2) The cortisol-binding capacity of plasma transcortin was measured after incubation with a balanced amount of adsorbent and cortisol. (3) A relative index of cortisol-protein binding proportional to the ratio of bound to unbound cortisol, was determined by incubation of charcoal-treated plasma with a trace of [r,@H]cortisol and appropriate amounts of adsorbent. (4) From this relative index of cortisol-protein binding in plasma an estimate of the cortisol-binding affinity of plasma transcortin was derived.

Steroid-protein binding is of great importance for the transport of the steroid hormones in plasma and for their action at the cellular level. The binding properties of protein solutions for a steroid may be characterised by different parameters: the amount of protein sites available for binding with the steroid is generally expressed as the capacity (concentration of binding sites); the afinity or strength of the interaction is expressed by the association constant of these binding sites for the steroid’. The dimensionless product of the concentration of the binding sites with their association constant has been called the index of net binding1~2. Numerous methods have been devised to study steroid-protein interaction9; have equilibrium dialysi9, ultrafiltration5, gel filtrations and ultracentrifugation’ been used most extensively. In the present paper the binding proteins have been studied using solid adsorbents, which compete proteins for the steroid. C&z. Chim.

characteristics of with the binding

Acta,

18 (1967)

361-370

HEYNS &! al.

362 THEORETICAL CONSIDERATIONS

When a solid adsorbent is added to a solution of a steroid, this steroid is removed from the solution until equilibrium occurs between the adsorbed* quantity of steroid and the concentration of steroid remaining in solutions. This equilibrium is defined by the adsorption constant of the adsorbent (Appendix I). When, however, the adsorbent is added to a steroid solution, containing also steroid-binding proteins, the adsorbent and these proteins interact and compete for the steroid through the unbound steroid concentration. Finally, an equilibrium occurs between the steroid adsorbed by the solid, the unbound steroid concentration and the protein-bound steroid (Appendix 2). According to the quantity of the added adsorbent and its properties, this equilibrium may be completely in favour of the adsorbent: the steroid is almost entirely removed from the binding solution. This situation may be applied for the elimination of endogenous steroids from the protein solution. The experimental conditions can also be chosen so that only free or slightly bound steroids are adsorbed by the solid. In this case the binding capacity of strongly binding proteins can be determined or steroid levels can be measured by competitive protein-bindinga. Finally, the adsorbent and the binding proteins may be of about equal strength. Since the adsorbed amount of steroids, known from the decrease of the steroid concentration that occurs after addition of the adsorbent, is related quantitatively to the unbound steroid concentration, it can be used to estimate this unbound steroid concentration. For the exact calculation of the unbound steroid concentration however, the adsorption constant of the adsorbent under the conditionsoftheexperiment must be known (Appendix 2). This indirect calculation of the unbound steroid concentration is only entirely valid under limited conditions. The binding of steroid to protein and to the adsorbent must be in equilibrium at the time of the measurement. The adsorbent must be inert with regard to the binding protein and to the steroid and small individual variations of the composition of the protein solution (e.g. plasma) should not modify the adsorption constant of the adsorbent or must be excluded by standardisation of the measuring conditions. MATERIAL AND METHODS

Fuller’s Earth (BDH) and Norit A (Nutritional Biochemicals Corporation) were used as such. Tritium-labelled cortisol (IO mC/mg) was purchased at CEN (Mol, Belgium) and purified before use by paper chromatography. A Packard Instruments Model 3003 Liquid Scintillation Counter was used for the measurement of radioactivity. Protein concentrations were measured by the biuret reactionlo; plasma corticoids were determined fluorimetricallyll; the measurement of the binding capacity of plasma transcortin was done by a gel filtration techniques. Adsorption of steroids from solutions by solid adsorbents was performed as follows: a given volume of a solution of labelled steroids in water, in buffer solution or in protein solution was shaken in a thermostatised incubator with a known amount * Adsorption is used in this paper for the interaction interaction of steroid and protein. C&z. Chim. Acta,

18 (1967)

361-370

of steroid and adsorbent,

binding for the

COMPETITIVE

ADSORPTION

OF

363

STEROIDS

of adsorbent. After a given period the adsorbent was centrifuged down and the concentration of radioactivity in the supernatant was measured and compared to the concentration before addition of the adsorbent. Ultrafiltration was done under vacuum at 25’. The ends of a piece of Visking dialysis tubing (8/32; length 12 cm), washed with distilled water, were slipped over two glass canulas, perforating a rubber stopper and fixed by drying these ends under gentle heating and by rubber 0 rings. Then the stopper was placed upon a vacuum flask and the U-shaped Visking tube inside the flask was preliminary stretched and dried by vacuum. An accurately weighed counting vial was fixed on the lower end of the U by means of a bended hairpin. After reapplication of the vacuum 4 ml of diluted plasma were brought into the tubing and ultrafiltration was allowed to occur for 30 min. The counting vials, containing between 0.35 to 0.45 g of ultrafiltrate were weighed again and the radioactivity was measured after addition of water to 0.5 g and of scintillation solution. The radioactivity was also determined in the plasma before and after the ultrafiltration, the latter value being IO to IS”/, higher and the mean of these values was considered as the bound plus unbound radioactivity. PRACTICAL

APPLICATIONS

AND

RESULTS

Co&sol

adsorption properties of sume solid adsorbents Of the numerous substances, checked for adsorption of steroids Fuller’s Earth and powdered charcoal (Norit A) were retained for further studies. The equilibration was followed by the disappearance of [r,z-3H]cortisol from water after addition of the adsorbent, until a stable low concentration was attained. This occurred within minutes for Fuller’s Earth but took several hours for Norit A, that had extremely strong adsorption properties (adsorption constant for [I,z-~H]cortisol in phosphate buffer (pH 7.30; I 0.20) at 22”: 4.10~ ml/g). The adsorption constant of Fuller’s Earth was not stable in aqueous solutions but diminished

very slowly. A similar decrease

of the adsorption

constant

was found

Fig. I. Adsorption constant (in ml/g) of Fuller’s Earth measured at 25’ after I h of incubation in function of buffer composition and pH. Open squares: phosphate buffer I 0.2; black dots: Tris buffer I 0.2. Fig. 2. Comparison of the cortisol-binding capacity (CBC) of plasma transcortin, individual plasma samples by incubation with charcoal and by gel filtration. Clin. China. Acta,

estimated

18 (1967)

in

361-370

nEYNs et al.

364

when the adsorbent was preincubated with water before adding the steroid. For this reason the incubation time was standardised at I hour when this adsorbent was used. In Fig. I the influence of pH and buffer composition is given on the adsorption constant of Fuller’s Earth. It appears that Fuller’s Earth is very pH-dependent. Furthermore Tris-buffer diminishes the adsorption constant of this adsorbent. Fuller’s Earth was not completely inert with regard to the composition of the solution. It produced a slight decrease of the pH of the solution, buffered between pH 7 to 8 and also adsorbed protein (about 7 mg per IOO mg of adsorbent), so that in dilute solutions almost all protein was removed from the solution. The [r+G’H]cortisol was not affected by contact with the adsorbent; indeed after 5 h of incubation of a larger quantity of tritiated cortisol with Fuller’s Earth no degradation was found by paper chromatography and radio-scanning. In conclusion, the studied adsorbents do not fulfil the theoretical requirements of stable equilibrium and of inertness, desirable for the study of steroid-protein interactions by competitive adsorption. Nevertheless, as demonstrated further, satisfactory results can be obtained practically, when the experimental conditions are carefully chosen and sufficiently standardised. Elimination of endogenous steroids by adsorption on powdered charcoal Samples of heparinised plasma were gently shaken for 30 min at room temperature with 50 mg per ml of Norit A. Then the adsorbent was removed by centrifugation (IO min at 4000 rev./min), filtration over Schleicher and Schull paper No. 595 and a second centrifugation. In this way the charcoal was removed from most plasma samples, although in some instances (lipemic and postprandial plasma) centrifugation at high speed (10000 rev./min) was necessary. After charcoal treatment the endogenous corticoid levels, measured fluorimetrically, were reduced to very low values (2.6 & 1.0 (S.D.) ,ug per IOO ml; n: 30) in normal samples and in samples taken during ACTH infusion. Of added trace amounts of j r,2-3H]cortisol only 1.4% (range o-3.2:/,; n: 28) remained in the solution. Endogenous dehydroepiandrosterone (androst-5-ene-3/Lol-I7-one) sulfate, as measured by gas chromatography12 and small loads of tritiated testosterone or dehydroepiandrosterone sulfate also were removed from plasma. This treatment with charcoal did not produce a change of the protein content of plasma. The pH of the samples increased with 0.27 & 0.08 (S.D.) units (n: zg). The cortisol-binding capacity of plasma transcortin measured in 24 normal samples showed an increase of 1.4 & 1.7 (SD.) ,ug per IOO ml, i.e. from 22.9 * 3.8 (SD.) to 24.3 i 3.6 (S.D.) pg per IOO ml. In 4 individual plasma samples various chemical parameters were measured by standard automatic procedures (Technicon SMA 12) : the sodium, potassium, chloride, total protein, calcium and alcaline phosphatase content were not affected by this procedure (differences
China.

A&z,

18 (1967)

361-370

COMPETITIVE

ADSORPTION

was considered in normal

OF STEROIDS

to approximate

and in pregnancy

365

the cortisol-binding plasma

capacity

a very good agreement

of plasma (CBC). Indeed was found between

results of this procedure and the CBC measured by gel filtration (Fig. z). The equilibration time and the amount of cortisol were not critical.

the

Identical

results were obtained with equilibration periods of 30, 45 and 60 min, or with loads of 4, 5 and 6 pug of cortisol. When [r,z-3H]cortisol was used, the concentration remaining in the plasma after equilibration with Norit A could also be calculated from measurements of the radioactivity. These values were very similar to the fluorimetrical data, the coefficient as duplicates being

of variation 5.1%

(n:

IZ),

between the results of both techniques considered and this without correction for the endogenous

corticoids. Estimation of an index of steroid-protein At very low steroid concentration after removal approximates (Appendix

3b).

binding in plasma (i.e., with a trace

load of labelled

steroid

of endogenous steroids) the ratio of protein-bound to unbound steroid the total index of binding of the plasma proteins for this steroid When a steroid-adsorbing

solid is added to such a system

the ratio

of not adsorbed to adsorbed steroid (S,/(Si-S,)) is related to the index of protein binding of the solution through a factor, determined by the adsorption constant (Appendix 2). Although for the conversion of the ratio of not adsorbed to adsorbed steroid into the index of steroid-protein binding of the solution the adsorption constant must be exactly known, the latter ratio can be used as arelative index of steroidprotein binding on the only condition that the adsorption index of the solid is reproducible from sample to sample. Since factors as pH, ionic strength, buffer composition and temperature influence the properties of the adsorbent (and, of course, those of the binding proteins), they must be kept constant for this determination. Measurement of a relative index of cortisol-protein binding in plasma. Two ml of a solution of bovine albumin (7 g/roe ml in phosphate buffer pH 7.30; I a trace amount of labelled cortisol were added to 0.5 ml of charcoal-treated

and plasma

0.20)

and 2 ml of this mixture were incubated with 80 mg of Fuller’s Earth for I h at 25” in a shaking incubator. Then the adsorbent was centrifuged down. From measurements of the radioactivity before and after the incubation with adsorbant the ratio of not adsorbed to adsorbed cortisol (S,/(Si-S,)) was calculated and used as a relative index of cortisollprotein binding. The temperature of 25’ was chosen for practical reasons since centrifugation was possible only at room temperature. The use of charcoal-treated plasma eliminated the interference of endogenous steroids and other components with the binding of cortisol to proteins and with the adsorption of cortisol to Fuller’s Earth. The five fold dilution with buffer permitted a sufficient standardisation of the conditions of the measurement (pH, buffer composition) while adsorption to the adsorbent of specific binding proteins was minimalised by the addition of weakly binding bovine albumin. With the amount of Fuller’s Earth used, about half of the cortisol was adsorbed in normal plasma, so that the precision of the calculation was optimal. The within-sample reproducibility of this relative index of cortisol-protein binding was good (coefficient of variation for duplicates 3.6%, n: 30) and the variation between samples of the pH was sufficiently small (7.33 * 0.02). The relative index of cortisol-protein binding measured in individual plasma samples was in good correClin. Chim.

Actn,

18 (1967) 361-370

HEYNS et al.

366

lation with the CBC values of these samples (Fig. 3). This means that both the adsorption constant of the adsorbent and the affinity of plasma transcortin are fairly reproducible or that their variations are compensatory, what is unlikely. A good correlation was found also between the cortisol-binding capacities individual

plasma

samples

and their

somewhat

lower

relative

index

of

of binding

measured by another procedure (Fig. 4), wherein plasma was diluted with I volume of phosphate buffer (pH 7.30; I o.zo; 70 g/l albumin) and incubated for I h at 25”, with IOO mg/ml of Fuller’s Earth (i.e. the same proportion of Fuller’s plasma protein). The pH of a few plasma samples however, was insufficiently by this method,

resulting

in erroneous

Earth and stabilized

results.

C”“, 10

20

30

40 pg

per

loon?”

Fig. 3. Comparison of the relative index of binding (RBI) as determined by competitive adsorption on Fuller’s Earth (first procedure) and the cortisol-binding capacity (CBC) measured by gel filtration of individual plasma samples. Fig. 4. Comparison of the relative index of binding (RBI) as determined by competitive adsorption on Fuller’s Earth (second procedure) and the cortisol-binding capacity (CBC) measured by gel filtration of individual plasma samples.

Conversion of the relative index of cortisol-protein binding into its absolute value. Although in many experiments the relative index of binding is a useful parameter, a conversion to its absolute value should be preferred. Therefore the adsorption constant of the solid must be determined. An extrapolation of the data obtained in buffer solution of the same pH, ionic strength to plasma may be erroneous since other factors (i.e. proteins) also intervene. In solutions containing binding proteins the adsorption constant can only be calculated indirectly by comparison of the relative index of binding with a direct measurement of the index of binding, for instance by ultrafiltration. This was done for the 7 g per IOO ml solution of bovine albumin (using less adsorbent, because of the weak binding) and for 8 individual plasma samples, diluted with 4 volumes of the albumin solution as in the first procedure of competitive adsorption. In buffer solution a somewhat higher adsorption constant (450 ml/g) is found than in the albumin solution (250 ml/g) or in the diluted plasma samples (246 & 6 (SD.) ml/g). When the latter adsorption constant is used, the kM/V value (Appendix 3) for 40 mg/ml of Fuller’s Earth becomes 9.8. The index of cortisolprotein binding in undiluted plasma is obtained by a further multiplication by 5. C&n. Chim. Acta, 18 (1967) 361-370

COMPETITIVE

ADSORPTION

Relation

OF STEROIDS

of the index

index of cortisol-protein

of cortisol-protein

binding

totalises

367 binding

in plasma

the index of cortisol

to transcortin. binding

The

to different

proteins (Appendix 3), but in plasma at 25O the binding of trace amounts of cortisol is mainly due to transcortin. Hence the affinity (association constant) of plasma transcortin may be estimated by dividing the index of cortisol-protein binding in plasma by the cortisol-binding capacity, measured separately, although this calculation includes a small error for binding to other proteins. (Fig. 3) an affinity of plasma trancortin of 7.2.10’ tained

In 25 different plasma samples & 0.9.10~ (S.D.) l/mole was ob-

(at 25’).

DISCUSSION

In the study of steroid-protein interactions little or no use has been made of competition between binding proteins and added adsorbents, although this principle was already mentioned by Klotz 13. Adsorbents were employed in the determination of steroid concentrations to extract steroids from biological solutions not containing protein+, or to remove the unbound steroid in methods based on competition for binding proteins of endogenous and labelled steroids 9J6. Techniques which show some similarity with competitive adsorption are : the estimation of cortisol binding to plasma proteins by Farese and PlagerlE, who measured the uptake of [14C]cortisol by red blood cells and the estimation of testosterone binding in plasma by Pearlman and Crepy17, who used Sephadex for equilibrium dialysis and found some adsorption of testosterone to the gel matrix. The use of red blood cellQ8, ion exchange resinsls or SephadexZO for the study of thyroxin binding, are also in some way related to the principle of competitive adsorption. The use of charcoal for the elimination of non-protein material from protein solutions is well knownZ1. By incubation with Norit A cortisol, testosterone and dehydroepiandrosterone sulfate were almost completely removed from plasma without signs of alteration of the protein or the transcortin content, This simple method may be very useful, since it permits the study of the interaction with proteins of known concentrations of an added steroid, without interference of its endogenous levels or of steroids with competitive properties. When plasma samples were incubated with balanced amounts of cortisol and charcoal the concentrations of corticoids measured in the supernatant fluid after centrifugation were very similar to the cortisol-binding capacities of these samples measured by a gel filtration technique. After this equilibration a situation seems to be present where nearly complete saturation of transcortin occurs, although the unbound and albumin-bound cortisol are still sufficiently low. The unbound cortisol is stabilized against variations of the concentration of endogenous steroids or of transcortin by the interaction of the large load of cortisol and the adsorbent. This easy method is very suitable for the determination of cortisol-binding capacities in multiple samples. For the determination of the ratio of bound to unbound steroid in protein solutions, ultrafiltration and competitive adsorption each have their own specific advantage and drawbacks. Ultrafiltration gives direct information on the ratio of bound to unbound steroid and almost physiological conditions can be chosen. On the other hand, ultrafiltration is technically more difficult and the interference of Clin. Chim. Acta,

18

(1967) 361-370

HEYi%

368 radioimpurities

may be very important,

et at.

when only a small fra.ction of the steroid is

unbound, i.e., at low temperature in undiluted plasmaz2. The interference of radioimpurities is much smaller in competitive adsorption since the conditions are chosen so that about half of the labelled steroid is removed by the adsorbent. Competitive adsorption however gives only indirect information on the ratio of bound to unbound steroid, since the adsorption constant of the adsorbent intervenes in the calculation. For Fuller’s Earth this adsorption constant is very dependent of the experimental conditions, so that non-physiological dilutions and temperatures have to be applied. Since the adsorption constant is also dependent on the nature of the steroid, competitive adsorption is less suitable for studies of competition of different steroids, although it may be used as a rapid screening test. The techmque of competitive adsorption provides already an easy, standardised and reprociucible method for the comparison of steroid binding in multiple samples; it may be considerably improved by the introduction of adsorbents, corresponding better to the theoretical requirements. In the present paper the practical evaluation of methods based on adsorption was limited to the binding of cortisol in plasma. It is evident that these or similar methods have a more general use in the study of binding phenomena. ACKNOWLEDGEMENTS

The valuable technical assistance of Miss Lies D’hoore, Miss Mady Jorissen and Miss Anne Marie Van de Gaer is gratefully acknowledged. This work was financed in part by a grant No. 853 of the Nationaal Onderzoek. APPENDIX

Fonds voor Wetenschappelijk

Geneeskundig

: CALCULATIONS

Symbols Si

S, B U V M k X K, C,

initial steroid concentration steroid concentration after equilibration with the adsorbent concentration of steroid bound to protein unbound steroid concentration volume (in ml) amount of adsorbent (in g) adsorption constant of the adsorbent (in ml/g) quantity of steroid adsorbed by the adsorbent association constant of a protein binding site n concentration of a protein binding site n

(I)

Steroid adsorption from solutions containing no binding protein9 When only very low concentrations of steroids are used, the classical adsorption isotherm (Freundlich) X = k.M.Cl/”

Clin. Chim. Acta, 18 (1967) 361-370

COMPETITIVE

ADSORPTION

may be replaced

369

OF STEROIDS

by a simple linear equation

X = k.M.C. Since also

X = V.(Si-Se)

and

c = S, Si--Se

k.M

1’

S, (2)

Steroid adsorjdion from solutions Since the quantity of steroid

unbound

containing adsorbed

= V*(Si-Se)

or

U = (Si-Se).

GM

but also

S, = B + U B+U

k.M

S,

v

U B k.M -=v.Si-s,-I U

or

by the

steroid concentration X = k*M*U

and

binding proteins at equilibrium is determined

sr-s, Se

For adsorbents as Sephadex with an internal volume (Wr .M) that excludes the proteins but not the steroids, this equation becomes: B

S,.(k+Wr).M

U

Sr.\‘-SS,(V’-Wr.M)

I

(3)

Binding of steroids ha protein solutions (I, 2) (a) Solutions with I protein binding site ~a From the equilibrium B = U.K,.(C,-B)

the following equation

may be derived

B _ K,.C,.C I+K,.U At

very

low steroid

-

B

U

concentrations

U. K, < < I

= K,.C, Clip. Chim. .4cta, 18 (1967) 361-370

HEYKS

370 this product may be called the index of binding of the solution. (0) Solutions with diferent uot internctitag hidil2g sites From the equation for a single binding site it follows

or at very low steroid

CZi,/. CA&m. Acta,

concentrations

18 (1907) 301-370

et al.