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The chemistry of human transcortin
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
182, 437-442
The Chemistry The Effects
of Human Transcortin
of pH, Urea, Salt, and Temperature Progesterone’
DANIEL Departments
(1977)
of Biochemistry3
W. CHAN2
AND
on the Binding
W. R. SLAUNWHITE,
December
and
JR.
and Pediatrics, Schools of Medicine and Dentistry, York at Buffalo, Buffalo, New York 14207 Received
of Cortisol
State
University
of New
21, 1976
The interaction of cortisol and progesterone with pure transcortin was investigated. The temperature dependence of cortisol and progesterone binding is a result of the predominantly negative enthalpy of binding which suggests a very good fit between ligand and protein such that the bonds formed are of the van der Waals type. The optimal pH of cortisol (8.0) and progesterone (8.5) binding suggests involvement of cysteine, histidine, and/or tyrosine residues in the binding process. Transcortin is irreversibly denatured at pH 4.0. The effect of sodium chloride on the binding of both steroids is small. At lower sodium chloride concentrations (less than 0.15 M), binding decreases somewhat with decreasing salt concentration. Urea produces a progressive decrease in the association constants of both steroids which is completely reversible up to 2.0 M and 30% reversible at 3.0 M. Scatchard analysis of cortisol binding in the presence of a constant amount of progesterone and vice versa confirms earlier data obtained on plasma that cortisol and progesterone do not bind at two independent sites. It is not possible, however, to decide whether they bind at the same site or at two interdependent (interacting) sites.
At the time this work was done, very little data on pure transcortin had been reported aside from molecular constants and amino acid carbohydrate composition (l-4). As a prelude to a systematic investigation of the structure of transcortin and its binding site(s), it was important to investigate the properties of transcortin in response to the usual perturbations employed by protein chemists. We found pure transcortin to be a reasonably stable glycoprotein.
It is remarkable that transcortin, the plasma binding protein for cortisol, has a high equilibrium constant of association (of the order of lo8 M-‘) for both cortisol and progesterone, two such ‘dissimilar steroid hormones (l-3). Cortisol with three hydroxy groups is hydrophilic in nature, while progesterone without hydroxy groups is hydrophobic. Early investigations based solely on experiments with plasma suggested that cortisol and progesterone bind at the same site on transcortin since they displace each other (1). 1 This
work
was supported
by USPHS
Grant
MATERIALS
GM-
AND
METHODS
Transcortin was purified from human plasma (American Red Cross, Buffalo, New York) by a modification (5) of an affinity chromatographic method (6). Binding parameters were calculated by Scatchard analysis. Transcortin solutions were prepared for equilibrium dialysis by dissolving 0.5 mg of pure transcortin in 5 ml of gelatin-phosphosaline buffer lo.075 M NaCl, 0.025 M sodium phosphate, pH 7.0 (or as specified), containing 0.1% gelatin and 0.02% sodium azide]. Gelatin buffer (O.l%), which was used
21045.
2 This work is taken from a dissertation submitted by D.W.C. to the State University of New York at Buffalo Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 3 Address reprint requests to the Department of Biochemistry, State University of New York at Buffalo. P. 0. Box U, Station B, Buffalo, New York 14207. 437 Copyright All rights
0 1977 by Academic of reproduction
Press, Inc. in any form reserved.
ISSN
0003-9861
438
CHAN AND SLAUNWHITE
to stabilize transcortin, bound neither steroid. Most of the cortisol was removed by adding 5 ml of XAD-2 resin (polystrene, Rohm and Haas) and incubating for 30 min at 20°C. The cortisol-poor transcortin solution was diluted to 100 ml with the same buffer. Endogenous cortisol was measured by the method of Keane et al. (7). Equilibrium dialysis was performed on a Labquake rotator (Lab Industries) (16 rotations/min) for 40 hr. Pure transcortin solution (1.0 ml) placed inside dialysis tubing (Fisher) was dialyzed against 3.0 ml of gelatin-phosphosaline buffer containing 50 nCi of [3Hlcortisol (specific activity 53 Ci/mmol) or [3Hlprogesterone (specific activity 57.4 Ci/mmol) (New England Nuclear) and various amounts of nonradioactive cortisol or progesterone (Steraloids). The tritiated steroids were used without purification since they were found to be greater than 98% pure by thin layer chromatography. Radioactivity was counted on a liquid scintillation spectrometer (Packard Tri-carb) with an efficiency of 35% and an accuracy of 1%. The pH of buffer solutions was measured before and after dialysis on an Accumet pH meter (Fisher). RESULTS
Temperature. Temperature has a marked effect on the binding of cortisol and progesterone. Scatchard analysis showed that the association constants for both steroids increased lo- to E-fold in going from 37 to 4°C while the number of binding sites remained constant (Fig. 1). The free energy and the enthalpic and entropic changes in the cortisol- (progesterone-) transcortin reaction were calculated from the relationships: AG” = AH” - TAP and R In K = - AH’/T + AS”. The results (Table I) show that the free energy change of cortisol and progesterone binding to transcortin is derived predominantly from enthalpy changes. PH. The binding of cortisol and progesterone to transcortin is sensitive to pH. Scatchard analysis showed that the binding of cortisol to transcortin is maximal at pH 8.0, while the binding of progesterone to transcortin is best at pH 8.5 (Fig. 2). No binding was observed at pH 4.0 for either cortisol or proges&rone. Reversibility of the pH effect was examined by adjusting the pH of transcortin solutions back to 7.0 and then placing them in new dialysis sacks, replacing the outside solution with the same concentration of steroid but at pH 7.0, and repeating equilibrium di-
*Or \
\. I\ \ \
k
1.6
il2
04
0.6
FIG. 1. Temperature dependency of the binding of transcortin. Transcortin solutions (5 pg/ml), pH 7.0, were dialyzed for 40 hr. Cortisol: 0, 4°C; A, 20°C; 0, 37°C; progesterone: n , 4°C; A, 20°C; 0, 37°C.
alysis. Full recovery was observed for all pH values except pH 4.0 and 11.5 (Table II). Salt. The effect of NaCl on the binding of cortisol and progesterone to transcortin is small. The greatest effect is at the lower ionic strengths, i.e., less than 0.15 M NaCl (Fig. 3). The binding affinities of both steroids were depressed by decreasing NaCl concentration. Urea. Urea decreased the binding afflnity of cortisol and progesterone to transcortin (Fig. 41, but n remained essentially constant up to 2.0 M. In 3.0 M urea, a slight decrease of n was observed. Reversibility was tested by removing urea and repeating equilibrium dialysis. The effects of urea were reversible except at 3.0 M where partial recovery was observed. Competition. Cortisol and progesterone bind strongly to transcortin, a protein which has a single binding site for each steroid. Do they bind at the same site or at different sites? Two experiments were designed to examine these possibilities. The first experiment involved the displacement of [3Hlcortisol with increasing
PHYSIOCHEMICAL
STUDIES TABLE
THERMODYNAMIC
PARAMETERS
Steriod
439
OF TRANSCORTIN I
FOR TRANSCORTIN-CORTI~OLIPMGESTERONE
K,”
(lOa M-‘1
COMPLEXES
Average AG” (kcabmol)
Average AH” (kcabmol)
Averaa; ASS” deg. mol)
Cortisol
4 20 37
2.1, 2.7 0.82, 1.02 0.1, 0.3
-10.7 -10.7 -10.4
-13
-8
Progesterone
4 20 37
3.0, 3.8 1.0, 1.4 0.13, 0.33
-10.9 -10.9 -10.5
-14
-11
a One-half of the K,, values were obtained from Fig. 1. TABLE REVERSIBILITY
OF THE pH EFFECT
II
ON TRANSCORTIN-CORTI~OL/PROGESTERONE
Cortisol
pH (original) Before” K (108%1, 0 0.29 2.49 4.26 2.2 0.91
4.0 5.0 7.0 8.0 9.0 11.5
Afterb n 0 0.94 0.98 1.0 0.99 0.80
COMPLEXES
Progesterone
(la-‘) 0.47 2.4 2.5 2.4 2.5 0.98
After*
Before’ n 0.06 1.0 0.98 0.98 0.99 0.80
(lik’) 0 0.97 3.58 5.21 4.55 1.97
n 0 0.98 1.0 0.99 0.98 0.80
a These values were obtained from Fig. 2. b After adjusting the pH of transcortin solutions to 7.0 and repeating equilibrium were calcuiated by Scatchard analysis.
TO-
W-
30 li
/
/ 10 :A
/
,_/-1_1_1+-I-1 50 60
7D
90
BD
rao
110
121)
PH FIG. 2. The association constants of transcortin for cortisol (0) and progesterone (0) from pH 5.0 to 11.5. Transcortin solutions (5 pg/ml) were dialyzed at 4°C for 40 hr. The pH values of both inside and outside solutions were measured after as well as before dialysis. Scatchard analysis was used to calculate association constants and the number of binding sites. I represents two independent determinations.
(k-9 0.48 3.5 3.3 3.4 3.6 1.2
n 0.1 0.98 0.99 0.99 1.0 0.79
dialysis. The K,, and n
amounts of nonradioactive progesterone, and vice versa. Figure 5a shows that cortisol and progesterone can displace each other. The second experiment involved the binding of [3Hlcortisol in the presence of increasing amounts of nonradioactive cortisol plus a constant amount of nonradioactive progesterone, and vice versa. Figure 5b shows that the association constants of either cortisol or progesterone were depressed to about the same extent in the presence of a constant amount of the other steroid. The number of binding sites, however, remained the same. DISCUSSION
The binding of cortisol to transcortin is highly pendent (pH 7.0). The stants of cortisol at 4°C M-‘) and 37°C (0.2 * 0.1
and progesterone temperature deassociation con(2.4 + 0.3 x lo* x lOa M-‘) agree
440
CHAN AND SLAUNWHITE NaCl
‘1
NaCl(Ml FIG. 3. The binding affinities of transcortin at various salt concentrations. Transcortin, 5 pg/ml, in sodium chloride solutions, pH 7.0, containing 0.1% gelatin and 0.02% sodium azide were dialyzed at 4°C for 40 hr. Scatchard analysis was used to calculate the association constants. I represents two inde\ pendent determinations. Urea 4r
$s 3‘0 ‘; 22 e io-
PC 0
PC 0.5
PC IO
PC
20
h I PC
transcortin appears to react with both steroids with such a good fit that multiple bonds, probably of the van der Waals type, are formed (12). The goodness of fit may be facilitated by an induced conformational change in transcortin during the binding process. The binding of cortisol and progesterone to transcortin is sensitive to pH (Fig. 2). The maximal K,,, for cortisol at pH 8.0 agrees with those reported in plasma studies. De Moor reported the maximal percentage bound at pH 8.0 (13) and Westphal reported the maximal value of the bound/ unbound ratio at pH 7.9 (3). During the preparation of this paper, Gaillard et al. reported the maximal percentage of cortisol bound to pure transcortin at pH 8.0 (14). The difference in maximal K,,, for cortisol (pH 8.0) and progesterone (pH 8.5) is small but probably significant. The pH profiles suggest the possible involvement of cysteine, histidine, and/or tyrosine in the binding process. Recently, Khan and Rosner (15) reported that cysteine may be Competition of Gxtisolond
Progesterone
3.0
Urea Concentratian(M) FIG. 4. The binding affinities of transcortin at various urea concentrations. Transcortin solutions (5 pg/ml), pH 7.0, was dialyzed at 4°C for 40 hr in the presence of urea. Urea was crystallized from distilled water before use. Reversibility experiments were done by removing urea (transcortin solutions were dialyzed against 100 vol of phosphosaline), adding fresh outside solutions, and repeating equilibrium dialysis in the absence of urea. Scatchard analysis was used to calculate the association constant. C, Cortisol; P, progesterone; 0, K,, in the presence of urea; q , K,,, after the removal of urea; I represents two independent determinations.
closely with most of the reported values (811). The association constant (K,,,) of progesterone at 4°C (3.4 + 0.4 x lo8 M-‘) is about the same as’ reported while the I&,,, at 37°C (0.23 + 0.1 x lo* ~-9 is lower than the reported value (0.8 x lo* M-‘1 (2). The free energies of cortisol and progesterone binding to transcortin are derived predominantly from negative enthalpy changes (Table I). These suggested that
F
5
FIG. 5. Competition of cortisol and progesterone. The preparation of transcortin solutions (pH 7.0) is described under Materials and Methods. (Al Scatchard analysis (4°C) in the presence of (1) 13H]cortisol and nonradioactive progesterone, and (21 13Hlprogesterone and nonradioactive co&sol. (B) C, Cortisol Scatchard plot; P, progesterone Scatchard plot; C + P,, cortisol Scatchard plot in the presence of a constant amount of progesterone (46 nmol, the molar ratio of progesterone to transcortin being 0.51); P + C, progesterone Scatchard plot in the presence of a constant amount of cortisol (49 nmol, the molar ratio of cortisol to transcortin being 0.54).
PHYSIOCHEMICAL
STUDIES
OF
TRANSCORTIN
441
They both one of the amino acids involved in the group is quite hydrophobic. bind to transcortin with nearly equal affmbinding of cortisol to transcortin. Since the effect of sodium chloride on the ity at 37°C; at 4”C, progesterone binds twice as strongly as cortisol. All evidence binding affinity is small, the involvement of protein-protein interaction or of the points to a single binding site for each hormone. Do they bind at the same site or charged groups on the surface of transcortin is probably insignificant. This is in different sites? Early evidence from plasma studies indicated that cortisol and accord with experience and prediction. Human transcortin has not been observed progesterone can displace each other from to aggregate spontaneously (9,10,16); Ros- plasma transcortin although progesterone is less effective than cortisol due to its ner has, however, reported the polymeristronger binding to serum albumin (18). zation of human transcortin (4). The polymer can be deaggregated in dodecyl sul- This suggests that they are binding at the fate gel electrophoresis with 1% mercaptosame site. Plasma, however, has many ethanol. Recently, Gaillard et al. observed proteins and small molecules which might affect the binding of either steroid. Therethe polymerization of human transcortin both spontaneously and after heating at fore, the binding of both steroids to pure 60°C for 15 min (11). Polymerization was transcortin was examined. Figure 5a partially reversible during SDS gel elec- shows that progesterone can displace trophoresis and completely reversible in [3H]cortisol from transcortin, and vice the presence of dithiothreitol. It is un- versa; progesterone and cortisol have eslikely that electrostatic bonds would be sentially equal capability of displacing involved in the binding of cortisol or pro- each other. In addition, Scatchard plots of gesterone since they are neutral molecortisol in the presence of a constant cules. On the other hand, urea depressed amount of progesterone and vice versa inthe binding affinity of both cortisol and dicate that they do not bind at two indeprogesterone. This suggests the possible pendent sites. It is not possible, however, involvement of hydrogen bonds as well as to decide whether they bind at the same hydrophobic interactions in the binding site or at two interdependent (interacting) sites. The small difference in pH required process. One of the characteristics of transcortin to produce maximal binding is consistent is its irreversible denaturation in plasma with the latter. below pH 5.0 (13, 17). The present study REFERENCES confirms that the inhibition of cortisol and 1. SANDBERG, A. A., ROSENTHAL, H. E., SCHNEIprogesterone binding to transcortin at pH DER, S. L., AND SLAUNWHITE, W. R., JR. (1966) 4.0 is irreversible (Table II). Recently, in Steroid Dynamics (Pincus, G., Nakao, T., Gaillard et al. (14) showed a 93% decrease and Tait, J. F., eds.), pp. 1-61, Academic of cortisol-binding activity at pH 4.0. On Press, New York. the other hand, the effect of urea is revers2. SEAL: U. S., AND DOE, R. P. (1966) in Steroid ible, except at 3.0 M, in which case partial Dynamics (Pincus, G., Nakao, T., and Tait, J. recovery was observed. From our experiF., eds.), pp. 63-90, Academic Press, New ence, transcortin solution (100 pg/ml) in York. phosphosaline at pH 7.0 is stable for at 3. WESTPHAL, U. (1971) in Steroid-Protein Interacleast 2 weeks at 4 or 20°C. tions. Monographs on Endocrinology, Vol. 4, pp. 198-199, 213-214, Springer-Verlag, BerThe effects of temperature, ionic lin. strength, urea, and pH on the binding of 4. RQSNER, W. (1972) J. Steroid Biochem. 3, 531cortisol and progesterone to transcortin 542. are quite similar except for a small differ5. CHAN, D. W., SHARMA, M., AND SLAUNWHITE, ence in the pH maximum. Although cortiW. R. Submitted for publication. sol and progesterone are C-21 steroids, 6. RQ~NER, W., AND BRADLOW, L. (1971) J. Clin. they are very different in nature. Cortisol Endocrinol. 33, 193-198. with three hydroxy groups is rather hydro7. KEANE, P., STUART, J., MENDEZ, J., BARBALXIRO, philic, while progesterone with no hydroxy J., AND WALKER, W. (1975) Clin. Chem. 21,
442
CHAN
AND
1474-1478. 8. SEAL, U. S., AND DOE, R. (1962) J. Biol. Chem. 237, 3136-3144. 9. MULDOON, T., AND WESTPHAL, U. (1967)J. Biol. Chem. 242, 5636-5643. 10. SCHNEIDER, S., AND SLAUNWHITE, W. R., Jr. (1971) Biochemistry 10, 2086-2093. 11. GAILLARD, F., HAN, K., AND DAUTRAVAUX, M. (1975) Biochimie 57, 559-568. 12. KEANE, P., WALKER, W., GAULDIE, J., AND ABRAHAM, G. (1976) Clin. Chem. 22,70-73. 13. DE MOOR, P., STEENO, O., AND DECK, R. (1963) Actu Endocrinol. 44. 107-118.
SLAUNWHITE 14. GAILLARD, F., AUBERT, J. P., DAUTREVAUX, M., AND LOUCHEUX-LEFEBVRE, M. H. (1976) FEBS Lett. 64, 278-284. 15. KHAN, M., AND ROSNER, W. (1976) Abstract 371 of the 58th Annual Meeting of the Endocrine Society. 16. SEAL, U., AND DOE, R. (1962) J. Biol. Chem. 237, 3136-3140. 17. DAUGHADAY, W., AND MARIX, I. (1961) Metab. Clin. Exp. 10, 936-940. 18. ROSENTHAL, H., SLAUNWHITE, W. R., JR., AND SANDBERG, A. (1969) J. Clin. Endocrinol. 29, 352-367.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
182, 437-442
The Chemistry The Effects
of Human Transcortin
of pH, Urea, Salt, and Temperature Progesterone’
DANIEL Departments
(1977)
of Biochemistry3
W. CHAN2
AND
on the Binding
W. R. SLAUNWHITE,
December
and
JR.
and Pediatrics, Schools of Medicine and Dentistry, York at Buffalo, Buffalo, New York 14207 Received
of Cortisol
State
University
of New
21, 1976
The interaction of cortisol and progesterone with pure transcortin was investigated. The temperature dependence of cortisol and progesterone binding is a result of the predominantly negative enthalpy of binding which suggests a very good fit between ligand and protein such that the bonds formed are of the van der Waals type. The optimal pH of cortisol (8.0) and progesterone (8.5) binding suggests involvement of cysteine, histidine, and/or tyrosine residues in the binding process. Transcortin is irreversibly denatured at pH 4.0. The effect of sodium chloride on the binding of both steroids is small. At lower sodium chloride concentrations (less than 0.15 M), binding decreases somewhat with decreasing salt concentration. Urea produces a progressive decrease in the association constants of both steroids which is completely reversible up to 2.0 M and 30% reversible at 3.0 M. Scatchard analysis of cortisol binding in the presence of a constant amount of progesterone and vice versa confirms earlier data obtained on plasma that cortisol and progesterone do not bind at two independent sites. It is not possible, however, to decide whether they bind at the same site or at two interdependent (interacting) sites.
At the time this work was done, very little data on pure transcortin had been reported aside from molecular constants and amino acid carbohydrate composition (l-4). As a prelude to a systematic investigation of the structure of transcortin and its binding site(s), it was important to investigate the properties of transcortin in response to the usual perturbations employed by protein chemists. We found pure transcortin to be a reasonably stable glycoprotein.
It is remarkable that transcortin, the plasma binding protein for cortisol, has a high equilibrium constant of association (of the order of lo8 M-‘) for both cortisol and progesterone, two such ‘dissimilar steroid hormones (l-3). Cortisol with three hydroxy groups is hydrophilic in nature, while progesterone without hydroxy groups is hydrophobic. Early investigations based solely on experiments with plasma suggested that cortisol and progesterone bind at the same site on transcortin since they displace each other (1). 1 This
work
was supported
by USPHS
Grant
MATERIALS
GM-
AND
METHODS
Transcortin was purified from human plasma (American Red Cross, Buffalo, New York) by a modification (5) of an affinity chromatographic method (6). Binding parameters were calculated by Scatchard analysis. Transcortin solutions were prepared for equilibrium dialysis by dissolving 0.5 mg of pure transcortin in 5 ml of gelatin-phosphosaline buffer lo.075 M NaCl, 0.025 M sodium phosphate, pH 7.0 (or as specified), containing 0.1% gelatin and 0.02% sodium azide]. Gelatin buffer (O.l%), which was used
21045.
2 This work is taken from a dissertation submitted by D.W.C. to the State University of New York at Buffalo Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 3 Address reprint requests to the Department of Biochemistry, State University of New York at Buffalo. P. 0. Box U, Station B, Buffalo, New York 14207. 437 Copyright All rights
0 1977 by Academic of reproduction
Press, Inc. in any form reserved.
ISSN
0003-9861
438
CHAN AND SLAUNWHITE
to stabilize transcortin, bound neither steroid. Most of the cortisol was removed by adding 5 ml of XAD-2 resin (polystrene, Rohm and Haas) and incubating for 30 min at 20°C. The cortisol-poor transcortin solution was diluted to 100 ml with the same buffer. Endogenous cortisol was measured by the method of Keane et al. (7). Equilibrium dialysis was performed on a Labquake rotator (Lab Industries) (16 rotations/min) for 40 hr. Pure transcortin solution (1.0 ml) placed inside dialysis tubing (Fisher) was dialyzed against 3.0 ml of gelatin-phosphosaline buffer containing 50 nCi of [3Hlcortisol (specific activity 53 Ci/mmol) or [3Hlprogesterone (specific activity 57.4 Ci/mmol) (New England Nuclear) and various amounts of nonradioactive cortisol or progesterone (Steraloids). The tritiated steroids were used without purification since they were found to be greater than 98% pure by thin layer chromatography. Radioactivity was counted on a liquid scintillation spectrometer (Packard Tri-carb) with an efficiency of 35% and an accuracy of 1%. The pH of buffer solutions was measured before and after dialysis on an Accumet pH meter (Fisher). RESULTS
Temperature. Temperature has a marked effect on the binding of cortisol and progesterone. Scatchard analysis showed that the association constants for both steroids increased lo- to E-fold in going from 37 to 4°C while the number of binding sites remained constant (Fig. 1). The free energy and the enthalpic and entropic changes in the cortisol- (progesterone-) transcortin reaction were calculated from the relationships: AG” = AH” - TAP and R In K = - AH’/T + AS”. The results (Table I) show that the free energy change of cortisol and progesterone binding to transcortin is derived predominantly from enthalpy changes. PH. The binding of cortisol and progesterone to transcortin is sensitive to pH. Scatchard analysis showed that the binding of cortisol to transcortin is maximal at pH 8.0, while the binding of progesterone to transcortin is best at pH 8.5 (Fig. 2). No binding was observed at pH 4.0 for either cortisol or proges&rone. Reversibility of the pH effect was examined by adjusting the pH of transcortin solutions back to 7.0 and then placing them in new dialysis sacks, replacing the outside solution with the same concentration of steroid but at pH 7.0, and repeating equilibrium di-
*Or \
\. I\ \ \
k
1.6
il2
04
0.6
FIG. 1. Temperature dependency of the binding of transcortin. Transcortin solutions (5 pg/ml), pH 7.0, were dialyzed for 40 hr. Cortisol: 0, 4°C; A, 20°C; 0, 37°C; progesterone: n , 4°C; A, 20°C; 0, 37°C.
alysis. Full recovery was observed for all pH values except pH 4.0 and 11.5 (Table II). Salt. The effect of NaCl on the binding of cortisol and progesterone to transcortin is small. The greatest effect is at the lower ionic strengths, i.e., less than 0.15 M NaCl (Fig. 3). The binding affinities of both steroids were depressed by decreasing NaCl concentration. Urea. Urea decreased the binding afflnity of cortisol and progesterone to transcortin (Fig. 41, but n remained essentially constant up to 2.0 M. In 3.0 M urea, a slight decrease of n was observed. Reversibility was tested by removing urea and repeating equilibrium dialysis. The effects of urea were reversible except at 3.0 M where partial recovery was observed. Competition. Cortisol and progesterone bind strongly to transcortin, a protein which has a single binding site for each steroid. Do they bind at the same site or at different sites? Two experiments were designed to examine these possibilities. The first experiment involved the displacement of [3Hlcortisol with increasing
PHYSIOCHEMICAL
STUDIES TABLE
THERMODYNAMIC
PARAMETERS
Steriod
439
OF TRANSCORTIN I
FOR TRANSCORTIN-CORTI~OLIPMGESTERONE
K,”
(lOa M-‘1
COMPLEXES
Average AG” (kcabmol)
Average AH” (kcabmol)
Averaa; ASS” deg. mol)
Cortisol
4 20 37
2.1, 2.7 0.82, 1.02 0.1, 0.3
-10.7 -10.7 -10.4
-13
-8
Progesterone
4 20 37
3.0, 3.8 1.0, 1.4 0.13, 0.33
-10.9 -10.9 -10.5
-14
-11
a One-half of the K,, values were obtained from Fig. 1. TABLE REVERSIBILITY
OF THE pH EFFECT
II
ON TRANSCORTIN-CORTI~OL/PROGESTERONE
Cortisol
pH (original) Before” K (108%1, 0 0.29 2.49 4.26 2.2 0.91
4.0 5.0 7.0 8.0 9.0 11.5
Afterb n 0 0.94 0.98 1.0 0.99 0.80
COMPLEXES
Progesterone
(la-‘) 0.47 2.4 2.5 2.4 2.5 0.98
After*
Before’ n 0.06 1.0 0.98 0.98 0.99 0.80
(lik’) 0 0.97 3.58 5.21 4.55 1.97
n 0 0.98 1.0 0.99 0.98 0.80
a These values were obtained from Fig. 2. b After adjusting the pH of transcortin solutions to 7.0 and repeating equilibrium were calcuiated by Scatchard analysis.
TO-
W-
30 li
/
/ 10 :A
/
,_/-1_1_1+-I-1 50 60
7D
90
BD
rao
110
121)
PH FIG. 2. The association constants of transcortin for cortisol (0) and progesterone (0) from pH 5.0 to 11.5. Transcortin solutions (5 pg/ml) were dialyzed at 4°C for 40 hr. The pH values of both inside and outside solutions were measured after as well as before dialysis. Scatchard analysis was used to calculate association constants and the number of binding sites. I represents two independent determinations.
(k-9 0.48 3.5 3.3 3.4 3.6 1.2
n 0.1 0.98 0.99 0.99 1.0 0.79
dialysis. The K,, and n
amounts of nonradioactive progesterone, and vice versa. Figure 5a shows that cortisol and progesterone can displace each other. The second experiment involved the binding of [3Hlcortisol in the presence of increasing amounts of nonradioactive cortisol plus a constant amount of nonradioactive progesterone, and vice versa. Figure 5b shows that the association constants of either cortisol or progesterone were depressed to about the same extent in the presence of a constant amount of the other steroid. The number of binding sites, however, remained the same. DISCUSSION
The binding of cortisol to transcortin is highly pendent (pH 7.0). The stants of cortisol at 4°C M-‘) and 37°C (0.2 * 0.1
and progesterone temperature deassociation con(2.4 + 0.3 x lo* x lOa M-‘) agree
440
CHAN AND SLAUNWHITE NaCl
‘1
NaCl(Ml FIG. 3. The binding affinities of transcortin at various salt concentrations. Transcortin, 5 pg/ml, in sodium chloride solutions, pH 7.0, containing 0.1% gelatin and 0.02% sodium azide were dialyzed at 4°C for 40 hr. Scatchard analysis was used to calculate the association constants. I represents two inde\ pendent determinations. Urea 4r
$s 3‘0 ‘; 22 e io-
PC 0
PC 0.5
PC IO
PC
20
h I PC
transcortin appears to react with both steroids with such a good fit that multiple bonds, probably of the van der Waals type, are formed (12). The goodness of fit may be facilitated by an induced conformational change in transcortin during the binding process. The binding of cortisol and progesterone to transcortin is sensitive to pH (Fig. 2). The maximal K,,, for cortisol at pH 8.0 agrees with those reported in plasma studies. De Moor reported the maximal percentage bound at pH 8.0 (13) and Westphal reported the maximal value of the bound/ unbound ratio at pH 7.9 (3). During the preparation of this paper, Gaillard et al. reported the maximal percentage of cortisol bound to pure transcortin at pH 8.0 (14). The difference in maximal K,,, for cortisol (pH 8.0) and progesterone (pH 8.5) is small but probably significant. The pH profiles suggest the possible involvement of cysteine, histidine, and/or tyrosine in the binding process. Recently, Khan and Rosner (15) reported that cysteine may be Competition of Gxtisolond
Progesterone
3.0
Urea Concentratian(M) FIG. 4. The binding affinities of transcortin at various urea concentrations. Transcortin solutions (5 pg/ml), pH 7.0, was dialyzed at 4°C for 40 hr in the presence of urea. Urea was crystallized from distilled water before use. Reversibility experiments were done by removing urea (transcortin solutions were dialyzed against 100 vol of phosphosaline), adding fresh outside solutions, and repeating equilibrium dialysis in the absence of urea. Scatchard analysis was used to calculate the association constant. C, Cortisol; P, progesterone; 0, K,, in the presence of urea; q , K,,, after the removal of urea; I represents two independent determinations.
closely with most of the reported values (811). The association constant (K,,,) of progesterone at 4°C (3.4 + 0.4 x lo8 M-‘) is about the same as’ reported while the I&,,, at 37°C (0.23 + 0.1 x lo* ~-9 is lower than the reported value (0.8 x lo* M-‘1 (2). The free energies of cortisol and progesterone binding to transcortin are derived predominantly from negative enthalpy changes (Table I). These suggested that
F
5
FIG. 5. Competition of cortisol and progesterone. The preparation of transcortin solutions (pH 7.0) is described under Materials and Methods. (Al Scatchard analysis (4°C) in the presence of (1) 13H]cortisol and nonradioactive progesterone, and (21 13Hlprogesterone and nonradioactive co&sol. (B) C, Cortisol Scatchard plot; P, progesterone Scatchard plot; C + P,, cortisol Scatchard plot in the presence of a constant amount of progesterone (46 nmol, the molar ratio of progesterone to transcortin being 0.51); P + C, progesterone Scatchard plot in the presence of a constant amount of cortisol (49 nmol, the molar ratio of cortisol to transcortin being 0.54).
PHYSIOCHEMICAL
STUDIES
OF
TRANSCORTIN
441
They both one of the amino acids involved in the group is quite hydrophobic. bind to transcortin with nearly equal affmbinding of cortisol to transcortin. Since the effect of sodium chloride on the ity at 37°C; at 4”C, progesterone binds twice as strongly as cortisol. All evidence binding affinity is small, the involvement of protein-protein interaction or of the points to a single binding site for each hormone. Do they bind at the same site or charged groups on the surface of transcortin is probably insignificant. This is in different sites? Early evidence from plasma studies indicated that cortisol and accord with experience and prediction. Human transcortin has not been observed progesterone can displace each other from to aggregate spontaneously (9,10,16); Ros- plasma transcortin although progesterone is less effective than cortisol due to its ner has, however, reported the polymeristronger binding to serum albumin (18). zation of human transcortin (4). The polymer can be deaggregated in dodecyl sul- This suggests that they are binding at the fate gel electrophoresis with 1% mercaptosame site. Plasma, however, has many ethanol. Recently, Gaillard et al. observed proteins and small molecules which might affect the binding of either steroid. Therethe polymerization of human transcortin both spontaneously and after heating at fore, the binding of both steroids to pure 60°C for 15 min (11). Polymerization was transcortin was examined. Figure 5a partially reversible during SDS gel elec- shows that progesterone can displace trophoresis and completely reversible in [3H]cortisol from transcortin, and vice the presence of dithiothreitol. It is un- versa; progesterone and cortisol have eslikely that electrostatic bonds would be sentially equal capability of displacing involved in the binding of cortisol or pro- each other. In addition, Scatchard plots of gesterone since they are neutral molecortisol in the presence of a constant cules. On the other hand, urea depressed amount of progesterone and vice versa inthe binding affinity of both cortisol and dicate that they do not bind at two indeprogesterone. This suggests the possible pendent sites. It is not possible, however, involvement of hydrogen bonds as well as to decide whether they bind at the same hydrophobic interactions in the binding site or at two interdependent (interacting) sites. The small difference in pH required process. One of the characteristics of transcortin to produce maximal binding is consistent is its irreversible denaturation in plasma with the latter. below pH 5.0 (13, 17). The present study REFERENCES confirms that the inhibition of cortisol and 1. SANDBERG, A. A., ROSENTHAL, H. E., SCHNEIprogesterone binding to transcortin at pH DER, S. L., AND SLAUNWHITE, W. R., JR. (1966) 4.0 is irreversible (Table II). Recently, in Steroid Dynamics (Pincus, G., Nakao, T., Gaillard et al. (14) showed a 93% decrease and Tait, J. F., eds.), pp. 1-61, Academic of cortisol-binding activity at pH 4.0. On Press, New York. the other hand, the effect of urea is revers2. SEAL: U. S., AND DOE, R. P. (1966) in Steroid ible, except at 3.0 M, in which case partial Dynamics (Pincus, G., Nakao, T., and Tait, J. recovery was observed. From our experiF., eds.), pp. 63-90, Academic Press, New ence, transcortin solution (100 pg/ml) in York. phosphosaline at pH 7.0 is stable for at 3. WESTPHAL, U. (1971) in Steroid-Protein Interacleast 2 weeks at 4 or 20°C. tions. Monographs on Endocrinology, Vol. 4, pp. 198-199, 213-214, Springer-Verlag, BerThe effects of temperature, ionic lin. strength, urea, and pH on the binding of 4. RQSNER, W. (1972) J. Steroid Biochem. 3, 531cortisol and progesterone to transcortin 542. are quite similar except for a small differ5. CHAN, D. W., SHARMA, M., AND SLAUNWHITE, ence in the pH maximum. Although cortiW. R. Submitted for publication. sol and progesterone are C-21 steroids, 6. RQ~NER, W., AND BRADLOW, L. (1971) J. Clin. they are very different in nature. Cortisol Endocrinol. 33, 193-198. with three hydroxy groups is rather hydro7. KEANE, P., STUART, J., MENDEZ, J., BARBALXIRO, philic, while progesterone with no hydroxy J., AND WALKER, W. (1975) Clin. Chem. 21,
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AND
1474-1478. 8. SEAL, U. S., AND DOE, R. (1962) J. Biol. Chem. 237, 3136-3144. 9. MULDOON, T., AND WESTPHAL, U. (1967)J. Biol. Chem. 242, 5636-5643. 10. SCHNEIDER, S., AND SLAUNWHITE, W. R., Jr. (1971) Biochemistry 10, 2086-2093. 11. GAILLARD, F., HAN, K., AND DAUTRAVAUX, M. (1975) Biochimie 57, 559-568. 12. KEANE, P., WALKER, W., GAULDIE, J., AND ABRAHAM, G. (1976) Clin. Chem. 22,70-73. 13. DE MOOR, P., STEENO, O., AND DECK, R. (1963) Actu Endocrinol. 44. 107-118.
SLAUNWHITE 14. GAILLARD, F., AUBERT, J. P., DAUTREVAUX, M., AND LOUCHEUX-LEFEBVRE, M. H. (1976) FEBS Lett. 64, 278-284. 15. KHAN, M., AND ROSNER, W. (1976) Abstract 371 of the 58th Annual Meeting of the Endocrine Society. 16. SEAL, U., AND DOE, R. (1962) J. Biol. Chem. 237, 3136-3140. 17. DAUGHADAY, W., AND MARIX, I. (1961) Metab. Clin. Exp. 10, 936-940. 18. ROSENTHAL, H., SLAUNWHITE, W. R., JR., AND SANDBERG, A. (1969) J. Clin. Endocrinol. 29, 352-367.