Steroid-protein interactions

ANALYTICAL

BIOCHEMISTRY

t?&

48-58

(1969)

Steroid-Protein XXII.

Extrapolation

Technique

Corticosteroid-Binding WALTER

Interactions

HOFFMANN’

for

Globulin AND

Determination by Gel

ULRICH

Biochemistry Department, Reynolds

University of Louisville Building, Loukville, Kentucky

Received February

of

Filtration

WESTPHAL* School of Medicine. 40??08

17, 1969

The quantitative determination of the corticosteroid-binding globulin (CBG) level in serum by a rapid and convenient procedure remains an important objective in biochemical and clinical studies. Among the numerous principles applied for the measurement of steroid-protein interaction (l-3), equilibrium dialysis (4), gel filtration (5#, 6)) and ultrafiltration (7) are most extensively used for CBG analysis. The advantages and limitations of the equilibrium dialysis and gel filtration methods for the analysis of the various binding parameters have been discussed in detail (8). A relatively simple and rapid method for the assessment of steroid bound with high affinity to serum proteins is filtration over a Sephadex gel. The protein-bound steroid is excluded by the dextran gel, e.g., Sephadex G-50 or G-25, and is eluted in association with the protein fraction. The main disadvantage of the gel filtration is the continuous dissociation of the steroid-protein complex as the serum is filtered over the length of the column (6, 9). The portion of bound steroid in the eluate therefore is a minimum quantity; the extent of loss by dissociation during filtration is unknown. Possible adsorption of the steroid to the dextran matrix (10) is another factor that may affect the result. The gel filtration method used in its simple form does not determine the unbound steroid present in the original serum sample; modifications have been proposed to find the unbound portion in a gel equilibration procedure (10). The present study was initiated in an effort to correct for losses such as those caused by dissociation during the gel filtration. ’ Present address: Frauenklinik ’ To whom requests for reprints

der Universitlit, Heidelberg, should be addressed. 48

West

Germany

DETERMINATION

MATERIALS

OF

CORTISOL

AND

BINDING

49

METHODS

Steroids. Cortisol (llP,17,21-trihydroxy-pregn-4-ene-3,20-dione) was a commercial preparation recrystallized to the melting point reported in the literature. Cortisol-4-1”C (specific activity 46 mc/mmole) was obtained from New England Nuclear Corp., Boston, Mass., and was checked at least every two months for radiochemical purity by thin-layer and paper chromatography in several solvent systems (chloroform/methanol (9/l), benzene/ethanol (4/l), and Bush C, i.e., toluene/ethyl acetate/ methanol/water (9/l/5/5), all v/v). Only material with a radiochemical purity of 96% or higher w,asused. The radioactive steroid solutions were stored in a 9/l (v/v) benzene/methanol mixture at 4”. Radioactivity was determined in a Packard liquid scintillation spectrometer model 314 EX. The scintillation solvent was that described by DeMoor and Steeno (ll)-it consists of 100 gm naphthalene, 10 gm PPO (2,4-diphenyloxazole) , and 0.25 gm dimethyl POPOP (1,4-bis-2(4-methyl-5-phenyloxazolyl) benzene) dissolved in 1 liter reagent-grade p-dioxane. A mean counting efficiency of 55% was obtained. The chromatograms were analyzed in a Packard radiochromatogram scanner model 7200. Human Serecm. Four pooled samples were used in the present study; two were prepared from outdated ACD (acid-citrate-dextrose) blood pooled from normal individuals (N, and N,) ; serum P II was obtained from a pregnant woman in the second trimester, and serum P III from a w,oman in the third trimester of pregnancy. Serum from whole ACD blood was prepared by recalcification of the plasma obtained by centrifugation at 4”; calcium chloride was added to a final concentration of 11.5 mg CaCl, per 100 ml. After clotting at room temperature and centrifugation the serum was tested for absence of fibrinogen. All sera and intermediate serum preparations were stored at -85”. Removal of Endoqenous Steroids (Stripping). Sephadex G-25 fine, beaded, was washed with buffer and packed into a jacketed column (2.2 cm diameter) to a bed height of 45 cm. The temperature of the column was equilibrated by a Haake Ultra-Thermostat, set at 45” + 0.01”. Five milliliters of the serum was applied to the column in small portions and washed in with a minimal quantity of buffer. After emergence of the void volume (about 60 ml) the protein was collected in approximately 20 ml. The combined eluates from several runs were dialyzed for 48 hours at 4” against two changes of 0.2% NaCl and lyophilized. The dry protein was dissolved in buffer and adjusted to a concentration of 6.5 gm % by use of the biuret technique (12). It has been found that removal of cortirosteroids and progesterone is practically complete (>99%) and that

50

HOFFMANN

AND

WESTPHAL

the binding affinity of CBG in serum is not affected by this procedure (13). For all experiments, the water used was double glass-distilled and deionized; the buffer was 0.05 M phosphate of pH 7.4. Stability of Cortisol-4-Y?. In view of the ready self-decomposition of radiolabeled cortisol (14)) preliminary experiments were carried out to examine if cortisol breaks. down under the conditions chosen. Cortisol-414C alone in buffer, or with human serum in buffer, was incubated in an Erlenmeyer flask at 4”, 37”, and 50” for 1, 3, 24, and 48 hours. Furthermore, cortisol-4-14C in a buffer solution in the presence of a serumcontaining dialysis bag w,as subjected to the same conditions. Penicillin (500 units/ml) was added to the incubations lasting longer than 3 hours. After the various time intervals the solutions were extracted three times with equal volumes of chloroform or dichloromethane and the combined extracts were evaporated under vacuum or by a stream of cotton-filtered nitrogen. An aliquot was taken for the evaluation of the total recovery of radioactivity, which was found to be at least 95%. The remaining material was subject,ed to thin-layer and paper chromatography in at least two of the three systems mentioned above. Cortisol-4-14C w,as found to remain radiochemically pure in the incubations lasting up to 24 hours at the three temperatures. After incubation for 48 hours at 37” and 50°, the chromatograms showed, in addition to cortisol, two peaks more polar and two peaks less polar than cortisol. One of these had the Rf values of 11-/3-hydroxyandrost-4-ene-3,17-dione (14). The chromatograms. from the extracts of the cortisol-protein-buffer solutions indicated less decomposition than those from the extracts from cortisol solutions in free buffer. The decomposition products amounted to a total of 8 to 12% after 48 hours at the higher temperatures. Decomposition of cortisol-4-W, therefore, did not occur under the conditions chosen for the present experiments. The protein concentrations were determined by a biuret technique (12) using an analyzed crystalline bovine albumin preparation for calibration (Protein Standard Solution, Armour Pharmaceutical Co., Kankakee, Ill.). The presence of proteins in the eluates from the chromatographic columns was assessedby absorbance measurements at 280 rnp in a Zeiss spectrophotometer model PMQ II. Cortisol in serum N, was determined by a calorimetric procedure (15) and was found to be 8.3 pg %. Sephadex Gel Filtration. Sephadex G-25 fine, beaded, Sephadex G-50 coarse, unbeaded, and three jacketed columns type K25/45 were obtained from Pharmacia Fine Chemicals, Inc., New Market, N. J. For accurate volume adjustment, 3.5, 7.0, and 10.5 gm of Sephadex G-50 were suspended in buffer and packed into the three columns, which were equilibrated by a temperature bath fmodel 2095-2, Forma Scientific, Inc..

DETERMINATION

OF

CORTISOL

BINDING

51

Marietta, Ohio) set at 4’ Z!I 0.02”. The heights of the beds were measured, This procedure was repeated five times and the average values marked on the columns. A large amount of Sephadex was then thoroughly washed w’ith buffer and decanted several times to remove the fines. The three columns were packed to the marks so that bed heights corresponded to 3.5, 7.0 and 10.5 gm of Sephadex, respectively. The quality and homogeneity of the beds were checked by Blue Dextran 2000 (Pharmacia) . After each run the columns were washed with at least 300. mI of buffer. A mixture of unlabeled cortisol and cortisol-4-14C (approximately 3 X lo” to 6 X lo5 cpm) in amounts corresponding to concentrations of 48-52 and 98-102 kg per 100 ml for the experiments with stripped normal and pregnancy serum, respectively, was evaporated to dryness in a 25 ml Erlenmeyer flask under a stream of cotton-filtered nitrogen. To the remaining film of dry cortisol, 4 ml of the stripped test serum was added and the flask incubated at 37” for 3 hours in a water bath with gentle shaking. After an additional 60 minutes at 4”, 0.5 ml of this equilibrated serum was placed in the sample applicator of the column and was allowed to drain. This was followed by two washes of approximately 0.1 ml of buffer before elution was started. The flow rate was adjusted to 1 ml/mm; however, increasing the flow rate to 2, 3, 4 and 5 ml/min did not affect the results. After volumes of 10, 25, and 40 ml, respectively, had emerged from the three columns the subsequent eluates were collected in 30-drop fractions (approximately 1 ml) directly into the counting vials containing 10 ml of the counting solution. Quadruplicate l-ml samples, of the 1: IO diluted original equilibrated serum were also counted and used as the basis for the calculations. The quantity, pg cortisol bound per 100 ml serum, was obtained by the formula Cpl~~bound

x

F

waotal

is the radioactivity of the protein filtrate, cpmt,t,l the where cpmbound counts for 0.5 ml serum before gel filtration, and F its cortisol concentration in pg/lOO ml. By assuming one cortisol binding site for human CBG and a molecular weight of 52,000 (16, 17), the value of bound cortisol can easily be converted into weight concentration of CBG in human serum. Multiplying the pg of bound cortisol per 100 ml by 1.44 yields mg CBG per liter of serum. DeMoor et aZ. (5) and Doe et al. (6) have reported that the gel filtration procedure applied to serum does not measure albumin-bound cortisol; they found that cortisol after equilibration with human serum albumin did not appear in the protein fraction when the equilibrated mixture was

52

H~FFMANN

ANDWESTPHAL

filtered over Sephadex G-50. It is assumed that the cortisol-albumin complex dissociates completely during gel filtration, since its dissociation constant is considerably higher than that of the cortisol-CBG complex. Therefore, when whole human s.erum is equilibrated with cortisol and subjected to gel filtration, the radioactive cortisol eluted in association with the protein is considered bound to CBG. Equilibrium Dialysis. In general the procedure previously reported (13) was followed. Samples of 4 ml of the 1: 10 diluted stripped serum were placed in dialysis bags (Visking 24 mm flat diameter) and dialyzed against 8 ml of the equilibrated steroid solution containing a total of 1, 3, 10, 30, 60, and 100 ng of radioactive cortisol per milliliter. This corresponds to concentrations of 2.76 X 10eg M (1 ng) to 2.76 ‘A 1c7M (100 ng) cortisol in the outside solution. After 48 hours of equilibration on a rotating disc at 4”, the volumes of the inside solutions were measured and the radioactivity of each bag determined in triplicate l-ml samples. At the end of the experiment, quadruplicate l-ml s,amples of the starting solution and of the outside solutions were counted after their volumes were measured. The concentration of bound steroid, [S,,] , was calculated as the difference between the total concentration inside the bag and the concentration of unbound steroid, [S], in the outside solution. The combining affinity, C, was calculated according to (18) :

where [P] is the total protein concentration in the bags in gmjliter. has been as.certained experimentally that the C values obtained with IO-fold diluted sera are identical with those of the undiluted sera The C value, therefore, can be applied to a calculation of the bound unbound portion of cortisol in the undiluted serum. When x equals per cent unbound steroid and 100 -x the percentage bound, then

It the (8). and the

c = 100 - Z

x . [PI and .z: is obtained by using the protein concentration (gmJliter) of the diluted serum. The concentration of bound and unbound steroid is tained in @g/100 ml by multiplying (100 --5) and x, respectively, by total. cortisol concentration in pg/lOO ml, divided by 100. Additional tails have been reported (8). RESULTS

AND

unobthe de-

DISCUSSION

Different amounts of Sephadex G-50 were tried in order to find the smallest quantity that still gave a distinct separation of bound and un-

DETERMINATION

OF

CORTISOL

BINDING

53

bound steroid. An amount of 3.5 gm Sephadex per column fulfilled this requirement under the conditions chosen. This amount was doubled and tripled for the two other filtrations so that column No. 1 contained 3.5 gm, column No. 2, 7.0 gm, and column No. 3, 10.5 gm, of Sephadex G-50. The separation of bound and unbound cortisol after filtration over the three columns is shown in Figure 1, where the peaks represent the cortisol bound to protein, and the shaded bars’ indicate t’he presence of the protein measured by absorbance at 280 mp. The peaks of free cortisol are not shown in this graph; however, it is evident that the interval between protein-bound and unbound cortisol can be clearly distinguished. When cortisol alone was filtered through the columns no radioactivity appeared in the fractions in which the protein is eluted under our experimental conditions. The total recovery of radioactivity was’ found to be at least 96%. The method of gel filtration does not provide an equilibrium between protein-bound and unbound cortisol but separates the two species in a continuously changing system. The component parts of the CBG-cortisol complex are held together by noncovalent bonds. Therefore, continuous clissociation occurs as the complex moves down the column. For this reason the cortisol concentration in the interval between the bound and unbound steroid never reaches zero. Under the given conditions the total

PROTEIN

FIG. 1. Separation of protein-bound and unbound cortisol4’“C after equilibration at 370 for 3 hours with stripped human serum (NJ by three different amounts of Sephadex G-50: (0) 3.5 gm Sephadex G-50 (Column 1). (0) 7.0 gm Sephadex G-50 (Column 2)) (A 1 10.5 gm Sephadex G-50 (Column 3). Shaded bars indicate presence of protein in the three eluates.

54

HOFFMANN

0

AND

3.5 GRAMS

SEPHADEX

WESTPHAL

7.0 G-50

10.5

FIG. 2. Determination of CBG capacity by extrapolation of bound values obtained after filtration over three different amounts of Sephadex G-50. PIT1and N, represent values of stripped sera from normal individuals, P II and P III those from women in the second and third trimester of pregnancy, respectively. For the results obtained with serum N1 the standard deviations have been calculated for the three columns; specified in Figure 1: Column Column Column

1: 26.9 f 0.45 pg/lOO ml (26 experiments) 2: 24.0 + 0.61 pg/lOO ml (8 experiments) 3: 21.3 * 0.57 rg/lOO ml (8 experiments)

of dissociated cortisol will be proportional to the amount of Sephadex used for the filtration. Plotting the bound cortisol as determined in the eluates from the three columns versus the amount of Sephadex in the columns and extrapolating to a zero amount of Sephadex is therefore expected to yield a binding value that is corrected for loss by dissociation and possible reversible adsorption. This is shown in Figure 2. The binding capacity values obtained for the sera N,, N,, P II, and P III are 29.5, 21.9, 40.8, and 47.2 pg per 100 ml, respectively. The straight lines, obtained in various experiments for the different sera demonstrate validity of the assumption that t.he extent of dissociation and amount of Sephadrs are proport’ional. The four sera used in the present experiments (two normal sera and two sera obtained at different stages,of pregnancy) were selected in order to cover a wide range of CBG concentration as it is normally encountered in clinical mat.erial. Since it was desirable to compare the results with those obtained by

quantity

DE~~~RMINATIoN

Binding

Serum

55

BINDING

TABLE 1 Parameters for CortisoM-1% in Four Obtained by Equilibrium Dialysis at 4”

Cortisol-4~W Outside soln. before equilibration

OF CORTISOL

concentration Inside

Sera

in fig/100

ml

soln.

equilibration

after

Bound Total

Unbound

Bound

Unbound”

Nl

0.1 0.3 1.0 3.0 6.0 10.0

1.86 5.55 18.5 37.4 61.3 92.2

0.004 0.012 0.052 1.37 4.26 9.74

1 86 5 54 18 4 36 0 57.1 82.4

502 473 356 26.3 13.4 8.5

N2

0.1 0.3 1.0 3.0 6.0 10.0

1.96 5.51 17.1 32.9 56.0 87.7

0.004 0.013 0.115 1.93 5.70 12.2

1.96 5 50 17.0 31.0 50.2 75.6

490 433 148 16.1 8.8 6.2

P II

0.2 0.6 2.0 6.0 12.0 20.0

3.50 10.8 33.1 65.4 108 171

0.006 0.023 0.239 3.84 10.7 22.5

3.49 10.8 32.9 61.6 97.6 148

600 472 138 16.0 9 1 66

P III

0.3 0.9 3.0 9.0 18.0 30.0

5.48 16.5 47.4 88.4 157 244

0.007 0.021 0.545 6 62 20.9 42.2

-5.47 16.5 46.9 81.7 136 201

781 78.5 86 12.0 65 4.8

a The seeming discrepancies between some quotients and the corresponding values for bound and unbound cortisol given in the two preceding columns are caused by rounding off the concentration figures, especially those for low concentrations of unbound cortisol, to the decimals given.

other, established procedures, the same sera (N,, N,, P II and P III1 were also subjected to the equilibrium dialysis technique described under “Materials and Methods.” The calculations and t.he graphic evaluations of the binding capacity were performed by two methods (13, 19). Table 1 shows the values for the total, bound, and unbound cortisol obtained in the equilibrium dialysis experiment. The graphic representation of t,he results according to reference (13) is given in Figure 3. It is apparent that at very low concentrations, of added eortisol the unbound

56

HOI’FMANN

AND

WIWTPHAL

FIQ. 3. Determination of CBG capacity in the four stripped human sera described in Figure 2 by equilibrium dialysis according to Westphal (13). The unbound (unbd) c0rtis01values for the high-aflinity binding system (CBG) are too low to be distinguished from the abscissa. The three points on each line represent low-&nity binding to albumin. Intersection of the lines with the abscka provides a meaeurement of the CBG binding capacity. The dotted lines are extensions to the values obtained by the extrapolation shown in Figure 2.

cortisol fraction increases very little due to the high binding affinity of CBG. When all available binding sites of the CBG become sat,urated, thr unbound portion of cortisol rises at a much greater rate with increasing concentration of total cortisol, indicating binding to albumin. The intersection of the two curves provides a measure for the concentration of CBG. The dotted lines in Figure 3 extend the curves to the points on the abscissa which represent the binding capacity values obtained from Figure 2. These extensions appear as acceptable extrapolations of the curves N,, Nz, and P II. In the case of P III, the extrapolation would intersect with the abscissa at about 56 ~~g,/loO ml instead of 47.2 pg/lOO ml resulting from Figure 2. This discrepancy illustrates the uncertainty of binding values obtained by equilibrium dialysis with a limited number of concentrations, M will be pointed out below. The binding data obtained by equilibrium dialysis for the four sera studied (Table 1) have also been evaluated according to Rosenthal (19). This method represents a graphic procedure for a binding system consisting of one ligand and one or more binding macromolecules. Determination of the high-affinity binding capacity of the four sera by Rosenthal’s technique yielded values in agreement with the results shown in Figure 3. The two graphic methods thus gave essentially the same values

DEXERMINATION

OF CORTISOL

BINDING

57

as the present extrapolation technique. However, it should be emphasized tliat the analysis of the equilibrium dialysis data by the two graphic methods (13, 19) is not free of uncertainty. This may be apparent from an inspection of Figure 3. Evidently, many more experimental points would be necessary in order to obtain a clearly defined intersection between the lines for the CBG-binding and albumin-binding systems. For a complete, thermodynamically valid determination of binding parameters, i.e., association constants and concentration of binding sites, a procedure is required that measures the bound and unbound portion of the ligand at equilibrium; pertinent methods, such as equilibrium dialysis, cannot be easily replaced. However, equilibrium dialysis is time consuming and exposes the solutions for many hours to potentially harmful conditions. In contrast, gel filtration is rapid. Combining the gel filtration technique with an extrapolation procedure eliminates the main disadvantage caused by dissociation and possible reversible adsorption. The method described in the present paper thus provides a reliable and convenient determination of the concentration of binding sites. It should prove useful in all cases in which knowledge of the unbound portion of the ligandmacromolecule system is not essential. SUMMARY

A method has been developed for the quantification of the corticosteroid-binding globulin (CBG) which eliminates the disadvantages of the gel filtration technique (dissociation, possible reversible adsorption). Binding capacity values are obtained with three different amounts of Sephadex G-50, and are extrapolated to a gel volume of zero. CBG concentrations have been measured for two normal and two pregnancy sera. The results have been found in agreement with those obtained by two independent procedures based on equilibrium dialysis. ACKNOWLEDGMENTS The authors are indebted to Dr. D. M. Haynes and Dr. W. M. Wolfe, Department of Obstetrics and Gynecology, University of Louisville School of Medicine, for their cooperation in providing the pregnancy sera. They also wish to acknowledge the expert assistance of Miss N. Rust and Mr. G. B. Harding. Outdated blood was obtained through Grant NC-2955 of the National Red Cross Blood Program from the Louisville Regional Blood Center. This work was supported by grants from the National Institute of Arthritis and Metabolic Diseases (AM-04040 and AM-08369) and by a Research Career Award from the Division of General Medical Sciences (GM-K6-14,138) of the United States Public Health Service. REXERENCES U., in “Mechanism of Action of Steroid Hormones” (C. A. Villee and L. L. Engel, eds.), p. 33. Pergamon, New York-Oxford, 1961.

1. WEBTPWL,

58

HOFFMANN

2. &NDBERG,

A. A., ROSENTHAL,

AND

H., SCHNEIDER,

WETESTPHAL S. I,., AND SLAUNWHITE,

W. R., JR., in

‘Steroid Dynamics” (G. Pincus, T. Nakao, and J. F. Tait, eds.), p. 1. Acndemic Press, London-New York, 1966. 3. DIXON, P. F., BOOTH, M., AND BUTLER, J., in “Hormones in Blood” (C. H. Gray and A. L. Bacharach, eds.), 2nd ed., Vol. 2, p. 305. Academic Press, LondonNew York, 1967. 4. DAUGHADAY, W. H., J. C&n. Invest. 35, 1428 (1956). 5. DEMOOR, P., HEIRWEGH, K., HEREMANS, J. F., AND DECLERCK-RASKIN, M., J. clin. Invest. 41, 816 (1962). 6. DOE, R. P., l?ERN.4NDEZ, R., AND SEAL, U. S., J. C&n. Endocrinol. 24, 1029 (1964). 7. SANDBERG, A. A., SLAUNWHITE, W. R., JR., AND ANTONIADES, H. N., Recent Progr. Hormone Res. 13, 209 (1957). 8. WESTPHAL, U., in “Methods in Enzymology,” Vol. 15 (R. B. Clayton, pd.). Aca-

demic Press, London-New York, 1969. 9. QUINCEY, R. V., AND GRAY, C. H., J. Endocrinol. 26,509 (1963). 10. PEARLMAN, W. H., AND CREPT, O., J. Biol. Chem. 242, 182 (1967). 11. DEMOOR, P., AND STEENO, O., J. Endocrinol. 26, 301 (1963). 12. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. ( 1949). 13. WESTPHAL, 14. WESTPHAL, 15. PETERSON, 16. SEAL, U.

177,

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Biochem. Biophys. 118, 556 (1967). U., CHADER, J. G., AND HARDING, G. B., Steroid 10, 155 (1967). R. E., KARRER, A., AND GUERRA, S. L., Anal. Chem. 29, 144 (1957). S., AND DOE, R. P., in “Steroid Dynamics” (G. Pincus, T. Nakao,

and

U., Arch.

T. F. Tait, eds.), p. 63. Academic Press, London-New 17. MIJLDOON, 18. DAUGHADAY, 19. ROSENTHAL,

T.

G., AND WESTPHAL, W. H., J. Cl&. Invest. H. E., Anal. B&hem.

U., J. Biol. Chem. 37, 511 (1958). 20,525 (1967).

242,

York, 1966. 5636

(1967).