ARCHIVES
OF BIOCHEMISTRY
AND
168, 473-482 (1975)
BIOPHYSICS
Steroid-Protein Fluorescence
Quenching
of Progesterone-Binding
Glycoprotein STEPHEN Department
D. STROUPE,
of Biochemistr\:
Interactions’
Upon Binding SU-LI
Ufliuersit.v
CHENG,
of Louisuille.
Globulin
of Steroids AND
ULRICH
School of Medicine,
Received September
and al-Acid
WESTPHAL
Louisuille,
Kentuckv
40201
9, 1974
The influence of progesterone and four other steroids on the intrinsic fluorescence of progesterone-binding globulin was investigated. The corresponding effect of progesterone on a,-acid glycoprotein was also studied. The intrinsic fluorescence of the progesteronebinding globulin and of a,-acid glycoprotein was quenched by about 60 and 17%, respectively, upon forming stoichiometric complexes with progesterone. Graphical analysis of fluorescence quenching titrations with progesterone gave affinity constants at 23°C of 2 x lo9 M- 1 for progesterone-binding globulin and 1 x lo6 M ’ for a,-acid glycoprotein. With progesterone-binding globulin, affinity constants of 1 x lo9 Mu ’ were determined for desoxycorticosterone. 1 x lOa M ’ for testosterone, and 2 x lo6 M ~1 for cortisol. The fluorescence quenching of PBG by 5-pregnen-3@ol-20-one, 5a-pregnanedione, and 5ppregnanedione, steroids lacking the A’-3-keto grouping, was too small to be evaluated; however, binding of the pregnanediones to progesterone-binding globulins was demonstrated when the progesterone-progesterone-binding globulin complex was “unquenched” as a result of competitive displacement of progesterone by addition of the pregnanediones. The quenching phenomenon is assumed to be mainly due to radiationless transfer from protein to the near uv (n - **) absorption band of steroids containing the A’-3-keto chromophore.
Progesterone-binding globulin (PBG) 2 is the specific progesterone-binding macromolecule of pregnant guinea pig plasma. The circulating level of PBG rises to relatively high levels during pregnancy (1, 2); it can be separated from the corticosteroidbinding globulin by chromatographic procedures (3). Binding affinity is high for progestogens and androgens, but low for corticoids (4, 5). PBG is a glycoprotein of very high carbohydrate content (6, 7). Purification of PBG and investigation of its physical and chemical properties have ’ This article is Paper XXX1 in the series, “Steroid-protein Interactions.” Paper XXX is MacLaughlin, D.T., and Westphal, U. (1974) Biochim. Biophys. Acta 365, 3755388. 2 Abbreviations used: AAG, a,-acid glycoprotein or orosomucoid; PBG, progesterone-binding globulin; K,,, association constant. 473 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved
been reported from several laboratories (8-10). The characteristics of this glycoprotein make it an attractive model to study the structural relationships between ligand and binding site in a steroid-binding protein of high affinity and distinct specificity. For rapid quantitation of active PBG, and for a study of the influence of steroid structure on the binding affinity to the glycoprotein, the equilibrium dialysis method has disadvantages. It is time consuming and requires radiolabeled steroids when applied at practicable concentrations. In search of a method that was free of these drawbacks, the effect of steroid binding on PBG fluorescence was explored (11). It was found that the intrinsic fluorescence of PBG was strongly quenched upon binding to progesterone. This afforded the basis
474
STROUPE,
CHENG
for a convenient assay of steroid-PBG interaction. Exploitation of data on the absorption spectrum of progesterone and the fluorescence spectrum of PBG led to information on the protein-steroid interaction at the molecular level. The present report describes the basic phenomena and the procedure of fluorescence quenching titration for the determination of binding site concentration and affinity constants of steroid-protein complexes. MATERIALS
AND
METHODS
PBG was prepared from pooled pregnant guinea pig serum as previously described (12). AAG was obtained from the American Red Cross and further purified (13). The steroids were obtained from commercial sources and recrystallized; the melting points were checked. The progesterone used in the uvabsorption studies was a highly purified (14) preparation. All other chemicals were reagent grade and water was glass redistilled and deionized. Fluorescence measurements were made in an Aminco-Bowman spectrofluorometer equipped with a xenon lamp and a liquid-cooled cell compartment. The temperature of the cell contents was maintained at 23 i 1°C with a Haake circulating water bath. Both activation and emission monochromators were calibrated and the settings verified with a mercury discharge lamp as described in the instrument instruction manual. Activation and emission spectra were corrected only for blank fluorescence. Fluorescence quenching titrations. All protein solutions were prepared at pH 7.4 in either 50 mM phosphate buffer or 10 mM Tris chloride containing 0.1 M NaCl. The titrations were performed by adding with a microliter syringe small aliquots of relatively concentrated steroid solutions (about lo-“-lo-“M in 50% ethanol) to 2.0 or 3.0 ml of the protein solution in the cell. Ethanolic steroid solutions were prepared by weight and the concentrations verified (where applicable) by measuring the ultraviolet absorbance using previously reported extinction coefficients (15). Thorough mixing after each addition was necessary to obtain reproducible results. Excitation was usually at 280 nm and emission was read at 340 nm, i.e., at the respective maxima. The absorbance of the protein solutions was below 0.02 to fulfill conditions for fluorescence changes to be proportional to concentration changes (16). More concentrated solutions were either diluted or they were excited at a wavelength other than that of maximum absorption. The fluorescence corrected for dilution was plotted as a function of added steroid, the data yielding a characteristic curve (see, for example, Fig. 3). The linear portion at excess steroid was extrapolated to zero steroid concentration to correct for nonspecific
AND WESTPHAL quenching due to ethanol in the steroid solvent and inner filter effects. The initial linear portion was extended to intersect the extrapolated baseline thus yielding the equivalence point. At this point, the concentration of ethanol was approximately 0.25%. Since PBG is polydisperse (lo), it is not possible to relate the concentration of binding sites to protein weight. The PBG concentration was therefore operationally defined as the concentration of binding sites. Equilibrium dialysis data evaluated by the Scatchard procedure (10) are in agreement with the assumption that all steroid binding sites in PBG are identical and independent of each other. Furthermore, as seen in Figs. 3 and 5, the change of fluorescence over the range of complete binding is linear demonstrating that the binding sites are fluorometrically the same. Therefore all binding sites are considered independent and identical. The affinity constant was determined from the expression:K. = a/ (1 - a)*P where a is the degree of association as given by the ratio of quenching at the equivalence point over total quenching, and P is the binding site concentation at the equivalence point. The degree of quenching at the equivalence point was obtained by drawing a smooth curve through all data points between the two linear portions (see above), thereby basing the value of a not on a single point, but rather on an averaged value. The data representing the quenching curve between the linear portions can be presented as a Scatchard plot. Since the affinities obtained by both methods of calculation are the same within experimental error, Scatchard analysis was not routinely used. The calculation procedures described by Steiner et al. (17) and Halfman and Nishida (18) were also utilized to calculate affinity constants. However, both methods yielded approximately the same results as those from the above expression; therefore, the calculations were made according to the simpler formula. Ultrauiolet absorption spectra were obtained with a Cary 15 spectrophotometer at room temperature. The absorption spectra of steroids in the presence of PBG (difference spectra) were measured after setting the baseline with the same solution of PBG in both the reference and sample cells. The steroid was then added to the sample cell and an equal volume of solvent to the reference cell. Analysis of the overlap of PBG fluorescence and progesterone absorption spectra was made utilizing Forster’s theory of radiationless energy transfer (19, 20). The corrected equation given by Latt et al. (21) was applied: R B = 0
90001n10K2Q
128r5n’N
J
’
where R, is the distance for equal probability of radiationless energy transfer or fluorescence. K2 is an orientation factor varying from 0 to 4 (22); a value of 4
PROTEIN
FLUORESCENCE
was applied. Q is the donor quantum yield; it was determined relative to tryptophan (23). The value of 0.13 for the quantum yield of tryptophan given in (24) was utilized. The symbol n, the refractive index of the solvent surrounding the fluorophore and chromophore has been assumed to be 1.5 (25); N is Avogadro’s number. J is the overlap integral defined by: J = I c(x) F(h) A’ dX, where F(x) is the donor fluorescence
normalized
(RLm6 l+(RJR)” ’ efficiency
Excitation
Emission
itic. : 2 ;4f ‘G %20. 200
e(x) is the acceptor molar extinction coefficient. From R, one can calculate R, the separation between donor and acceptor, by:
where E is the overall
81
475
BINDING
to 1:
IF(x) dz+ = 1;
E=
AND STEROID
of energy transfer
250
300
350
400
450
FIG. 2. Fluorescence excitation and emission spectra of AAG (solid lines) and AAG-progesterone complex (broken lines). Emission of the excitation spectra was measured at 340 nm; excitation of the emission spectra was at 282 nm. Concentration of both AAG and the complex was 1.3 x 10m5 M, determined by titration of binding sites.
(20). RESULTS
Figures 1 and 2 give the fluorescence excitation and emission spectra of PBG and AAG and of their progesterone complexes. PBG has an excitation maximum at 280 nm and an emission maximum at 340 nm; the excitation of AAG is maximal at 282 nm and its emission at 340 nm. The excitation and emission properties of both proteins are characteristic of polypeptides containing tryptophan. When either protein forms a progesterone complex, the excitation and emission wavelengths are not shifted; however, in both cases the fluorescence is quenched. The PBG-progesterone complex has a fluorescence only 40% that of the free protein; that is, the 2.5
1
200
Excitation
250
Emission
300
350
400
450
FIG. 1. Fluorescence excitation and emission spectra of PBG (solid lines) and PBG-progesterone complex (broken lines). Emission of the excitation spectra was measured at 340 nm; excitation of the emission spectra was at 280 nm. Concentration of both PBG and the complex was 3.3 x lo-’ M, determined by titration of binding sites.
fluorescence of PBG is quenched by about 60% upon binding progesterone. On the other hand, AAG fluorescence is quenched by only about 17% upon forming a stoichiometric complex with progesterone. Figure 3 is a fluorescence quenching titration curve of PBG with progesterone. The initial linear fluorescence decrease is readily extrapolated to the baseline defining the equivalence point at a PBG concentration of 2.70 x 1O-8 M. The fraction dissociated at equivalence yields an affinity constant of 2 x 10s M- I. The inset in Fig. 3 shows the same data in the form of a Scatchard plot giving a K, of 2 x lo9 M- ’ and a concentration of 2.77 x 1Om8M PBG. Note that the data in the Scatchard plot span only about 25% of the total binding range, reflecting the fact that under the conditions used binding is “fluorometritally complete” over the initial portion of the titration curve. In Fig. 4, fluorescence quenching titration of AAG with progesterone is shown. The slope of the baseline is greater than that obtained in Fig. 3. This steeper slope was always observed with AAG and may result from greater exposure of fluorophores to solvent and added steroid than is the case with PBG. Extrapolation of the initial fluorescence decrease to the baseline affords an active AAG concentration of 1.3 x 10m5 M. The degree of dissociation at equivalence corresponds to an affinity con-
STROUPE,
r 0
CHENG
AND WESTPHAL
1.0 1.5 Progesterone/PEG
0.5
2.0
, 2.5
FIG. 3. Fluorescence quenching titration of PBG with progesterone. At the equivalence point, 12.6 ~1 of 4.32 x lo-’ M progesterone had been added to 2.0 ml of PBG solution, yielding a binding site concentration of 2.70 x 1Oe8 M. The solid curve yields a K, of 2 x 10QM- I. The inset gives the data from the nonlinear region as a Scatchard plot. The filled circles correspond to the actual data points whereas the open circles are taken from the solid curve. The Scatchard plot gives a concentration of 2.77 x 10m8 M and a K, of 2.1 x lo9 M-‘.
I 0
1.0
2.0
3.0
Progerfcrone/AAG
FIG. 4. Fluorescence quenching titration of AAG with progesterone. At equivalence, AAG concentration was 1.3 x lo-” M. A K, of 1 x lo6 M-’ was calculated from the solid curve.
stant, K,, of 1 x lo6 M- I. This affinity constant, determined at 23”C, is in agreement with a value of 1.5 x lo6 M-r obtained at 4°C by equilibrium dialysis (26) when corrected by the previously published temperature dependence of AAG-progesterone interaction (13). The AAG binding site concentration of 1.3 x 10m5M and the total protein concentration of 1.2 x IOe5 M (assuming M, = 41,000) give a value of n = 1.1 binding site per molecule AAG, which agrees with the established value determined by equilibrium dialysis (27). Although the signal change is not large,
titration curves with AAG are reproducible and are routinely used in this laboratory. Eleven titrations over a period of several months3 gave an average n value of 0.88 i 0.13 (SD). In tests of the reproducibility of the method, a series ‘of 15 titrations of progesterone vs PBG, performed over a period of several months, afforded an average K, of 2.0 f 0.6 x log M-l (fiducial limits calculated (28) for 99% confidence level = 1.6 to 2.5 x log M-‘). In evaluating the precision of concentration determinations by fluorescence quenching titrations, quadruplicate determinations on the same solution gave a binding site concentration of 2.84 * 0.18 x 1O-8 M. This standard deviation corresponds to an error of 6.4%. In another test of concentration determinations, a different solution was titrated six times over a period of three weeks; between tests the sample was stored frozen. The average binding site concentration was found to be 8.41 h 0.13 x 10e7 M, i.e., the standard deviation was 1.5%. The interaction of other steroids with PBG was studied by the fluorometric method. Figure 5A-C shows the fluorescence quenching titrations of PBG by desoxycorticosterone, cortisol, and testosterone. The results are reported as a function 3 Unpublished
results by Mr. Timothy
Kute.
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AND STEROID
477
BIiYDIIKG
50
40
30
20 0
0.5
Testosterone/PBG
1.5 1.0 Stemid/PBG
20
25
FIG. 5. Fluorescence quenching titrations of PBG with: A, desoxycorticosterone, K, = 1 x lo9 M- ‘, concentration at equivalence = 1.57 x lo-’ M; B, cortisol, K, = 2 x lo6 M-‘, concentration at equivalence = 8.37 x 10m7 M; C, testosterone, K, = 1 x 10’ Mm’, concentration at equivalence _ 8.50 x 10m7 M; D, pregnenolone and progesterone, concentration at equivalence for both = 8.42 x lo-’ M.
of the steroid to protein ratio; the concentration of active PBG was determined separately by titration with progesterone. Duplicate determinations with desoxycorticosterone gave an average K, of 1 x lo9 M-i, four determinations with testosterone an average K, of 1 x 10s M-l. A single determination with cortisol corresponded to a K, of 2 x lo6 M- ‘. Note that different protein concentrations were utilized for the titrations in Fig. 5. All values are in agreement with previously published studies using a competitive equilibrium dialysis method (4). Figure 5D gives the titration of two identical PBG samples with progesterone pregnenolone (5-pregnen-3@01-20and one). Even though pregnenolone has been reported to bind to PBG with an affinity constant of 3 x lo7 M-i (4) (an order of magnitude greater than cortisol) fluorescence of PBG is quenched only 6% by pregnenolone compared to 26% by cortisol and 62% by progesterone. To explore the reasons for lack of quenching by pregnenolone, uv absorption spectra of progesterone and pregnenolone, both complexed with PBG were obtained (Fig. 6). The spectra are reported as molar absorptivities based on moles of complex. The progesterone-PBG spectrum in Fig. 6
-4
i i 1 ,I meJ ‘j 220
240
260 “In
280
300
FIG. 6. Difference spectra of PBG complexes with progesterone (solid line) and pregnenolone (broken line). The baseline was recorded with the same PBG solution in reference and sample cuvettes. Steroid solution was then added to the sample cuvette with an equal volume of solvent added to the reference cuvette and the difference spectrum recorded. Concentration of both complexes was 1.7 x 10 6 M.
has a broad maximum near 245 nm and a sharp peak at 233 nm. The longer wavelength peak is assigned to the major transition of progesterone whose maximum is at 249 nm in water, but is shifted to shorter wavelengths in nonpolar solvents and when bound to proteins (29). The peak at 233 nm
478
STROUPE.
CHENG
AND WESTPHAL
is not due to the progesterone chromopoint results in the expected fluorescence phore; rather it must arise as a difference decrease; however, when 5a-pregnanedione spectrum between the liganded and unlior 5fi-pregnanedione are added to the ganded protein. This follows from the preg- quenched complex, the fluorescence innenolone curve which does not show ab- creases again and approaches the initial sorption at 245 nm because the A4-3-keto fluorescence at higher concentrations of group is absent; however, there is a sharp competing ligand. Progesterone is being maximum at 232 nm. Since pregnenolone displaced; the curves show that 5cY-pregitself does not absorb at 232 nm the signal nanedione is bound more firmly than 5pmust again be due to a difference in pregnanedione, in agreement with pubabsorption between liganded and unlilished results (4). The inset in Fig. 7 gives ganded PBG. Both progesterone (a A4-3- the titration of unliganded PBG with 5pketosteroid) and pregnenolone (lacking pregnanedione; no quenching is observed. this chromophore) appear to induce a simiFurther insight into the mechanism of lar conformational change in PBG resultthe fluorescence quenching by A4-3-ketoing in the difference signal at 232 or 233 steroids is offered in Fig. 8. The near-uv nm. Therefore the absence of quenching absorption spectra of progesterone in hepseen in Fig. 5D cannot result from lack of tane, methanol, and water with 10% ethainteraction between PBG and pregnenonol, representing a wide range in polarity, lone. are given. Changes in solvent polarity are To demonstrate that both A4-3-ketosterwell known to alter the wavelength of lacking the (Y,& maximum absorption of A4-3-ketosteroids oids and steroids unsaturated keto grouping bind at the (30); however, such changes also result in same site on PBG, displacement experisignificant differences in the near-uv specments were performed using 5/3-preg- tra of A4-3-ketosteroids. Of particular innane-3,20-dione (5@-pregnanedione) and terest is the spectrum in heptane which 5-a-pregnane-3,20-dione (5a-pregnanediexhibits vibrational fine structure. As seen one); in both steroids the A4-3-keto chro- for progesterone in Fig. 8, there are well mophore is absent. In Fig. 7, .titration of defined maxima at 337 nm (t =39), 323 nm PBG with progesterone to the equivalence (t = 40), and 296-297 nm (E= 44). Shoulders are evident at 365-370 nm, 350 nm, 307 nm, and 288 nm. The spectrum in methanol is smooth with a single broad maximum at 292-293 nm (t = 106). In water containing 10% ethanol there is no discernible maximum in the near-uv region; however, a shoulder is observed around 290-300 nm with t -200 (not shown). These molecular extinction coefficients are of the same magnitude as those observed in the near ultraviolet for other A’-3-ketosteroids (31). Also presented in Fig. 8 is the normalized fluorescence of PBG illustrating the overFIG. 7. Fluorescence quenching titration of PBG lap of PBG fluorescence and progesterone with progesterone (open circles and triangles) followed by addition of Sa-pregnanedione (filled circles) absorption. The quantum yield of PBG or 5 P-pregnanedione (filled triangles). At the end of fluorescence was 72% that of tryptophan; the progesterone titrations, both samples were 4.95 x utilization of the value of 0.13 (24) results 10 7 M in progesterone binding sites and 5.35 x 10 ’ M on 0.094 as the quantum yield of PBG. The in progesterone. When the competing steroids were product t(x) F(X) x4 used to calculate the added, the PBG samples were “unquenched” by 50% critical distance R, in Fijrster’s radiationat 1.0 x 10e6 M 5a-pregnanedione and at 3.0 x 10m6M less energy transfer theory is also shown for 5/Spregnanedione. The inset shows the fluorescence the absorption spectrum in 10% ethanol. titration of an identical PBG sample with 5/3-pregTable I gives the values of the overlap nanedione.
PROTEIN
FLUORESCENCE
160
60
0 260
260
300
320
340 "ml
360
360
400
42:
FIG. 8. Ultraviolet absorption spectra of progesterone in water containing 10% ethanol (-. -. ). methanol (---I and heptane (. .) illustrating the overlap with PBG fluorescence (-0-l. PBG fluorescence was normalized as explained in the text. The overlap product c(h) F(h) h’ t-Cl-1 is given for the absorption spectrum in 10% ethanol. TABLE
I
OVERLAP INTEGRALS, J, AND CRITICAL DISTANCES, R,, FOR PBG-PROGESTERONE COMPLEX Solvent
J(cm6/mmoll
90% Water + 10% ethanol Methanol Heptane
6.49 x lO-‘¶ 6.64 x lo-‘” 5.90 x 10-I”
R, (Aja 5.9 5.9 5.8
“The value for n is assumed to be 1.5; K* is taken to be 4.
integral, J, for all three solvents and the critical distance, R,, for 50% energy transfer. Note that in spite of wide differences in the absorption spectra, all solvents give nearly the same value of J which results in similar values for R, no matter what solvent best approximates the near-uv spectrum of progesterone bound to PBG. DISCUSSION
Fluorescence quenching has been utilized in the study of many protein-ligand systems; a recent review (22) covers some of them. Concerning protein-steroid interactions, binding of 19-nortestosterone to A5-3-ketosteroid isomerase quenched the protein intrinsic fluorescence by 75% (32). The quenching was ascribed to a proteinto-steroid energy transfer, and it was sug-
AND STEROID
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479
gested that tyrosine residues are involved in the steroid binding site (33). The interaction between bovine serum albumin and various androstane derivatives has also been investigated fluorometrically by determining “formation constants” and stoichiometries for various steroids (34). No details have been reported on the energy transfer in fluorescence studies involving steroids. The decrease of intrinsic fluorescence exhibited by PBG upon interaction with A4-3-ketosteroids is of interest for two reasons. Fluorescence quenching titrations can be utilized to quantitate the steroid binding protein in solution and to measure the affinity of the interactions. Secondly, the quenching phenomenon can be evaluated to afford information on the nature of the steroid-protein interaction at the molecular level. The study of the influence of progesterone on PBG fluorescence was undertaken in an attempt to develop a convenient method of measuring the concentration of active PBG and its affinity constant in one experiment without resorting to equilibrium dialysis. Therefore emphasis was placed on obtaining stoichiometric binding over a large part of the titration to provide a reliable measure of PBG binding sites upon which to base the calculation of the affinity constants. In testing the precision of concentration determinations by this method, standard deviations of 6.4% at 2.84 x 1Om8M and 1.5% at 8.41 x 10e7 M were obtained. In equilibrium dialysis and Scatchard analysis, five replications of a 1:200 dilution of the same pool of pregnant guinea pig serum gave a binding site concentration of 4.2 i 0.2 x 10e8 M with a standard deviation of 7%. Therefore, both methods are considered comparable in precision. However, fluorescence quenching titrations generally gave concentrations lo-25% lower than equilibrium dialysis. In a direct comparison of four different PBG solutions the concentrations determined by fluorescence quenching titration were on the average 23% lower than those obtained by equilibrium dialysis. This deviation is outside the experimental error found for the two methods. Since no such discrep-
480
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CHENG
ancy was observed for AAG, the difference may be specific for PBG. Concerning the affinity constants, 15 titrations gave a K, value of 2.0 f 0.6 x log M-l at 23°C for PBG-progesterone interaction. A comparable series of equilibrium dialysis studies on both diluted pregnant guinea pig serum and purified PBG gave a K, of 2.1 f 0.4 x log M-’ at 4°C in 16 separate experiments. However, the affinity constants by the two methods were obtained at different temperatures; three determinations by equilibrium dialysis at 23°C gave an average K, value of 1.0 x log M-’ (fiducial limits (28) at 99% confidence level 0.7-1.3 x log M-l). Thus fluorescence titrations are seen to overestimate the equilibrium dialysis affinity constant by more than experimental error. Comparison of the two methods for accuracy is difficult because of the relatively wide range of values actually obtained with the fluorescence quenching method. For the 15 titrations mentioned above the range was 1.1 to 3.0 x log M-l. Similar variations (up to twofold) were observed when the method was applied to measurements on a larger series of steroids.’ Therefore, to obtain acceptable precision, affinity constants must be based on at least quadruplicate determinations. If such an average is taken, the fluorometric method gives the correct order of magnitude for the affinity constants for the various steroids and correct relationships of the affinities to each other. The method of fluorescence quenching titration has the advantage of generally using less binding protein (typically 0.1 nmol PBG per experiment) and being much more rapid than equilibrium dialysis. It does not require radiolabeled steroids. However, the protein must be in purified form and the steroid must have the appropriate ultraviolet absorption. Analysis of fluorescence quenching can also be used in assaying for PBG activity during purification procedures without introducing radiosteroids: the fluorescence of the relevant fractions read with and without a large excess of progesterone affords a mea’ Unpublished
results by Mr. A. T. Blanford.
AND WESTPHAL
sure of the progesterone-induced quenching which is proportional to PBG purity. For the second aspect mentioned above, the quenching phenomenon can offer information concerning the molecular basis of the PBG-progesterone interaction. Fiirster (20) has listed four mechanisms by which energy can be transferred from an excited state: (1) nontrivial transfer [resonant transfer], (2) reabsorption, (3) complexing, (4) encounter. The quenching is not due to reabsorption because the optical densities are kept low, and the percent quench is invariant at 60% over a hundred-fold concentration range from 10e6 to lo-* M PBG. Complexing, with an attendant alteration in the absorption spectrum is unlikely to be the major source of quenching. A small difference spectrum (not shown in Fig. 6) is induced between 280 and 300 nm upon steroid binding, but the change is less than 5% of the total absorption, too small to account for the large decrease in fluorescence. Encounter quenching will arise if there is a conformational difference between PBG and the PBG-steroid complex and if the PBG fluorophores in the complex are more exposed to solvent. This can be rejected on two accounts. First, pregnenolone, 5a-pregnanedione, and 5P-pregnanedione all form complexes with PBG, but without significant quenching. Second, in a comparison between a titration in buffer with and without 20% sucrose, the quenching at equivalence was 60% in both cases; less quenching would be expected in the more viscous medium if quenching resulted from increased encounter transfer in the complex. The remaining transfer mechanism is resonant transfer which classically is proven by enhanced acceptor fluorescence upon donor excitation. If a A4-3-ketosteroid is the acceptor, this method of proof is unavailable because molecules with an n + S* transition as their lowest energy transition are nonfluorescent (35). The absorption spectrum of progesterone overlapping the fluorescence emission spectrum of PBG makes resonant transfer possible, but does not prove it. In accordance, we did not observe quenching by steroids lacking the overlapping absorption. Furthermore,
PROTEIN
FLUORESCENCE
when a quenched complex was backtitrated with a nonquenching steroid, the complex fluorescence increased to that of unliganded PBG. These two results prove that merely forming a complex between PBG and a steroid is not sufficient to quench fluorescence; rather the steroid must possess an overlapping absorption band to accept energy from the PBG excited electronic state. Resonant transfer is the only mechanism of energy transfer consistent with these observations. Although important information is lacking for a quantitative interpretation of the overlap integrals (Table I), the following comments are made in an attempt to understand the mechanism of the interaction. Using the simplest case for energy transfer, we assumed only one of several tryptophan residues to be fluorescent. The calculations utilize K* = 4, the most favorable case for transfer with resultant distances being maximum values. With an R, value of 5.9 A and the 60% quench observed with progesterone, a maximum separation of 5.5 A is calculated. This small separation is predicated by the low extinction coefficient of the steroid in the fluorescence overlap region. Since testosterone and desoxycorticosterone quench PBG fluorescence to about the same extent as progesterone, it is most likely that these steroids are bound at the same site and in the same mode as progesterone. In contrast, cortisol quenches the fluorescence of PBG by only 26% at saturation. The overlap integral of cortisol (and indeed of other A*-3-ketosteroids) ought to be approximately the same as that of progesterone as long as the similarity of the steroid chromophore system in ring A is maintained. It can be calculated, therefore, that cortisol is located up to 6.9 A from the critical tryptophan in the complex. A detailed physical interpretation of separation distances smaller than the molecules themselves is meaningless. However, in the case of cortisol, since the value for J should be the same as for progesterone, the diminished quenching must result either from an orientation less favorable to transfer (change in K’), or an increase in separation (change in R,), or both. Whatever change there is in
AND STEROID
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481
the PBG-cortisol complex, it results in a stability three orders of magnitude lower than that of the PBG-progesterone complex. The proposed mechanism of fluorescence quenching by energy transfer from protein fluorophores to the nAr* absorption band of a conjugated carbonyl group in the steroid is general. The mechanism implies that any protein-steroid interaction will quench the intrinsic fluorescence of the protein if two conditions are met. First, the steroid-protein contact in the complex must place the steroid very near a fluorescent group. Second, the steroid must be a suitable acceptor; that is, the steroid must have an absorption band overlapping the protein fluorescence band. If the second condition is met, the magnitude of the quenching offers information concerning the binding site. CONCLUSIONS
The method of fluorescence quenching titration is a reliable and rapid technique of measuring binding site concentrations and assessing affinity constants of PBG with A4-3-ketosteroids. It does not require radiolabeled steroids. The procedure can also be applied to AAG and other steroidbinding proteins if a fluorescent group is present at or near the binding site, and if the steroid ligand is a suitable energy acceptor for the fluorophore. In addition, analysis of the fluorescence quenching provides information on the dimensional relationships at the binding site, and can be used to assess the influence of structural and steric features of the steroid molecules on the interaction with specific proteins. ACKNOWLEDGMENTS This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AM-06369) and a research career award (UWl from the Division of General Medical Sciences (GM-K6-14, 1381 of the United States Public Health Service. Alpha,-acid glycoprotein used in these studies was provided by the American Red Cross National Fractionation Center with the partial support of National Institutes of Health Grant HE 13881 (HEM). The authors wish to thank Mr. George B. Harding and Mrs. Karen Acree for performing the equilibrium dialysis studies.
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STROUPE,
CHENG
REFERENCES 1. DIAMOND, M., RUST, N., AND WESTPHAL, U. (1969) Endocrinology 84, 1143-1151. 2. HEAP, R. B. (1969) J. Reprod. Fert. 18, 546-548. 3. BURTON, R. M., HARDING, G. B., RUST, N., AND WESTPHAL, U. (1971) Steroids 17, l-16. 4. LEA, 0. A. (1973) Biochim. Biophys. Acta 322, 68-74. 5. TAN, S. Y., AND MURPHY, B. E. P. (1974) Endocrinology 94, 122-127. 6. MACLAUGHLIN, D. T., BURTON, R. M., ABOULHOSN, W. A., AND WESTPHAL, U. (1972) Fed. PFOC. 31, 430. 7. ALLOUCH, P., BAULIEU, E. E., AND MILGROM, E. (1972) C. R. Acad. Sci. 275, 1707-1710. 8. MILGROM, E., ALLOUCH, P., ATGER, M., AND BAULIEU, E. E. (1973) J. Biol. Chem. 248, 1106-1114. 9. LEA, 0. A. (1973) Biochim. Biophys. Acta 317, 351-363. 10. BURTON, R. M., HARDING, G. B., ABOUL-HOSN, W. R., MACLAUGHLIN, D. T., AND WESTPHAL, U. (1974) Biochemistry 13, 3554-3561. 11. STROUPE, S. D., CHENG, S. L., AND WESTPHAL, U. (1974) Endocrinology 94, A306. 12. STROUPE, S. D., AND WESTPHAL, U. (1974) Fed. Proc. 33, 1571. 13. GANGULY, M., CARNIGHAN, R. H., AND WESTPHAL, U. (1967) Biochemistry 6,2803-2814. 14. WESTPHAL, U., AND ASHLEY, B. D. (1958) J. Biol. Chem. 233, 57-62. Interac15. WESTPHAL, U. (1971) Steroid-Protein tions, pp. 36-41, 133-163, Springer-Verlag, New York. Assay in 16. UDENFRIEND, S. (1962) Fluorescence Biology and Medicine, p. 16, Academic Press, New York. 17. STEINER, R. F., ROTH, J., AND ROBBINS, J. (1966) J. Biol. Chem. 241. 560-567.
AND WESTPHAL 18. HALFMAN, C. J., AND NISHIDA, T. (1972) Biochemistry 11, 3493-3498. 19. FBRSTER, Th. (1948) Ann. Physik 2, 55-75. 20. F~RSTER, Th. (1959) Discuss. Faraday Sot. 27, 7-17. 21. LAW, S. A., CHEUNG, H. T., AND BLOUT, E. R. (1965) J. Amer. Chem. Sot. 87, 995-1003. 22. STEINBERG, I. Z. (1971) Annu. Reu. Biochem. 40, 83-114. 23. CHEN; R. F., EDELHOCH, H., AND STEINER, R. F. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), Part A, p. 171, Academic Press, New York. 24. CHEN, R. F., EDELHOCH, H., AND STEINER, R. F. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), Part A, p. 206, Academic Press, New York. 25. EISINGER, J., FEUER, B., AND LAMOLA, A. A. (1969) Biochemistry 8, 3908-3915. 26. WESTPHAL, U., BURTON, R. M., AND HARDING, G. B. (1975) Methods Enzymol. 36,91-104. 27. WESTPHAL, U. (1971) Steroid-Protein Interactions, pp. 375-433. Springer-Verlag, New York. 28. SNEDECOR,G. W. (1950) Statistical Methods, 4th ed., pp. 64-65. Iowa State College Press, Ames. 29. WESTPHAL, U. (1957) Arch. Biochem. Biophys. 66, 71-90. 30. FIESER, L. F., AND FIESER, M. (1959) Steroids, pp. 15-16, Reinhold, New York. 31. DORFMAN, L. (1953) Chem. Reu. 53, 47-144. 32. WANG, S. F., KAWAHARA, F. S., AND TALALAY. P. (1963) J. Biol. Chem. 238, 576-585. 33. TALALAY, P. (1965) Annu. Rev. Biochem. 34, 347-380. 34. ATTALLAH, N. A., AND LATA, G. F. (1968) Biochim. Biophys. Acta 168, 321-333. 35. KASHA, M. (1961) in Light and Life (McElroy, W. D. and Glass, B., eds.), pp. 31-64. Johns Hopkins Press, Baltimore.
OF BIOCHEMISTRY
AND
168, 473-482 (1975)
BIOPHYSICS
Steroid-Protein Fluorescence
Quenching
of Progesterone-Binding
Glycoprotein STEPHEN Department
D. STROUPE,
of Biochemistr\:
Interactions’
Upon Binding SU-LI
Ufliuersit.v
CHENG,
of Louisuille.
Globulin
of Steroids AND
ULRICH
School of Medicine,
Received September
and al-Acid
WESTPHAL
Louisuille,
Kentuckv
40201
9, 1974
The influence of progesterone and four other steroids on the intrinsic fluorescence of progesterone-binding globulin was investigated. The corresponding effect of progesterone on a,-acid glycoprotein was also studied. The intrinsic fluorescence of the progesteronebinding globulin and of a,-acid glycoprotein was quenched by about 60 and 17%, respectively, upon forming stoichiometric complexes with progesterone. Graphical analysis of fluorescence quenching titrations with progesterone gave affinity constants at 23°C of 2 x lo9 M- 1 for progesterone-binding globulin and 1 x lo6 M ’ for a,-acid glycoprotein. With progesterone-binding globulin, affinity constants of 1 x lo9 Mu ’ were determined for desoxycorticosterone. 1 x lOa M ’ for testosterone, and 2 x lo6 M ~1 for cortisol. The fluorescence quenching of PBG by 5-pregnen-3@ol-20-one, 5a-pregnanedione, and 5ppregnanedione, steroids lacking the A’-3-keto grouping, was too small to be evaluated; however, binding of the pregnanediones to progesterone-binding globulins was demonstrated when the progesterone-progesterone-binding globulin complex was “unquenched” as a result of competitive displacement of progesterone by addition of the pregnanediones. The quenching phenomenon is assumed to be mainly due to radiationless transfer from protein to the near uv (n - **) absorption band of steroids containing the A’-3-keto chromophore.
Progesterone-binding globulin (PBG) 2 is the specific progesterone-binding macromolecule of pregnant guinea pig plasma. The circulating level of PBG rises to relatively high levels during pregnancy (1, 2); it can be separated from the corticosteroidbinding globulin by chromatographic procedures (3). Binding affinity is high for progestogens and androgens, but low for corticoids (4, 5). PBG is a glycoprotein of very high carbohydrate content (6, 7). Purification of PBG and investigation of its physical and chemical properties have ’ This article is Paper XXX1 in the series, “Steroid-protein Interactions.” Paper XXX is MacLaughlin, D.T., and Westphal, U. (1974) Biochim. Biophys. Acta 365, 3755388. 2 Abbreviations used: AAG, a,-acid glycoprotein or orosomucoid; PBG, progesterone-binding globulin; K,,, association constant. 473 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved
been reported from several laboratories (8-10). The characteristics of this glycoprotein make it an attractive model to study the structural relationships between ligand and binding site in a steroid-binding protein of high affinity and distinct specificity. For rapid quantitation of active PBG, and for a study of the influence of steroid structure on the binding affinity to the glycoprotein, the equilibrium dialysis method has disadvantages. It is time consuming and requires radiolabeled steroids when applied at practicable concentrations. In search of a method that was free of these drawbacks, the effect of steroid binding on PBG fluorescence was explored (11). It was found that the intrinsic fluorescence of PBG was strongly quenched upon binding to progesterone. This afforded the basis
474
STROUPE,
CHENG
for a convenient assay of steroid-PBG interaction. Exploitation of data on the absorption spectrum of progesterone and the fluorescence spectrum of PBG led to information on the protein-steroid interaction at the molecular level. The present report describes the basic phenomena and the procedure of fluorescence quenching titration for the determination of binding site concentration and affinity constants of steroid-protein complexes. MATERIALS
AND
METHODS
PBG was prepared from pooled pregnant guinea pig serum as previously described (12). AAG was obtained from the American Red Cross and further purified (13). The steroids were obtained from commercial sources and recrystallized; the melting points were checked. The progesterone used in the uvabsorption studies was a highly purified (14) preparation. All other chemicals were reagent grade and water was glass redistilled and deionized. Fluorescence measurements were made in an Aminco-Bowman spectrofluorometer equipped with a xenon lamp and a liquid-cooled cell compartment. The temperature of the cell contents was maintained at 23 i 1°C with a Haake circulating water bath. Both activation and emission monochromators were calibrated and the settings verified with a mercury discharge lamp as described in the instrument instruction manual. Activation and emission spectra were corrected only for blank fluorescence. Fluorescence quenching titrations. All protein solutions were prepared at pH 7.4 in either 50 mM phosphate buffer or 10 mM Tris chloride containing 0.1 M NaCl. The titrations were performed by adding with a microliter syringe small aliquots of relatively concentrated steroid solutions (about lo-“-lo-“M in 50% ethanol) to 2.0 or 3.0 ml of the protein solution in the cell. Ethanolic steroid solutions were prepared by weight and the concentrations verified (where applicable) by measuring the ultraviolet absorbance using previously reported extinction coefficients (15). Thorough mixing after each addition was necessary to obtain reproducible results. Excitation was usually at 280 nm and emission was read at 340 nm, i.e., at the respective maxima. The absorbance of the protein solutions was below 0.02 to fulfill conditions for fluorescence changes to be proportional to concentration changes (16). More concentrated solutions were either diluted or they were excited at a wavelength other than that of maximum absorption. The fluorescence corrected for dilution was plotted as a function of added steroid, the data yielding a characteristic curve (see, for example, Fig. 3). The linear portion at excess steroid was extrapolated to zero steroid concentration to correct for nonspecific
AND WESTPHAL quenching due to ethanol in the steroid solvent and inner filter effects. The initial linear portion was extended to intersect the extrapolated baseline thus yielding the equivalence point. At this point, the concentration of ethanol was approximately 0.25%. Since PBG is polydisperse (lo), it is not possible to relate the concentration of binding sites to protein weight. The PBG concentration was therefore operationally defined as the concentration of binding sites. Equilibrium dialysis data evaluated by the Scatchard procedure (10) are in agreement with the assumption that all steroid binding sites in PBG are identical and independent of each other. Furthermore, as seen in Figs. 3 and 5, the change of fluorescence over the range of complete binding is linear demonstrating that the binding sites are fluorometrically the same. Therefore all binding sites are considered independent and identical. The affinity constant was determined from the expression:K. = a/ (1 - a)*P where a is the degree of association as given by the ratio of quenching at the equivalence point over total quenching, and P is the binding site concentation at the equivalence point. The degree of quenching at the equivalence point was obtained by drawing a smooth curve through all data points between the two linear portions (see above), thereby basing the value of a not on a single point, but rather on an averaged value. The data representing the quenching curve between the linear portions can be presented as a Scatchard plot. Since the affinities obtained by both methods of calculation are the same within experimental error, Scatchard analysis was not routinely used. The calculation procedures described by Steiner et al. (17) and Halfman and Nishida (18) were also utilized to calculate affinity constants. However, both methods yielded approximately the same results as those from the above expression; therefore, the calculations were made according to the simpler formula. Ultrauiolet absorption spectra were obtained with a Cary 15 spectrophotometer at room temperature. The absorption spectra of steroids in the presence of PBG (difference spectra) were measured after setting the baseline with the same solution of PBG in both the reference and sample cells. The steroid was then added to the sample cell and an equal volume of solvent to the reference cell. Analysis of the overlap of PBG fluorescence and progesterone absorption spectra was made utilizing Forster’s theory of radiationless energy transfer (19, 20). The corrected equation given by Latt et al. (21) was applied: R B = 0
90001n10K2Q
128r5n’N
J
’
where R, is the distance for equal probability of radiationless energy transfer or fluorescence. K2 is an orientation factor varying from 0 to 4 (22); a value of 4
PROTEIN
FLUORESCENCE
was applied. Q is the donor quantum yield; it was determined relative to tryptophan (23). The value of 0.13 for the quantum yield of tryptophan given in (24) was utilized. The symbol n, the refractive index of the solvent surrounding the fluorophore and chromophore has been assumed to be 1.5 (25); N is Avogadro’s number. J is the overlap integral defined by: J = I c(x) F(h) A’ dX, where F(x) is the donor fluorescence
normalized
(RLm6 l+(RJR)” ’ efficiency
Excitation
Emission
itic. : 2 ;4f ‘G %20. 200
e(x) is the acceptor molar extinction coefficient. From R, one can calculate R, the separation between donor and acceptor, by:
where E is the overall
81
475
BINDING
to 1:
IF(x) dz+ = 1;
E=
AND STEROID
of energy transfer
250
300
350
400
450
FIG. 2. Fluorescence excitation and emission spectra of AAG (solid lines) and AAG-progesterone complex (broken lines). Emission of the excitation spectra was measured at 340 nm; excitation of the emission spectra was at 282 nm. Concentration of both AAG and the complex was 1.3 x 10m5 M, determined by titration of binding sites.
(20). RESULTS
Figures 1 and 2 give the fluorescence excitation and emission spectra of PBG and AAG and of their progesterone complexes. PBG has an excitation maximum at 280 nm and an emission maximum at 340 nm; the excitation of AAG is maximal at 282 nm and its emission at 340 nm. The excitation and emission properties of both proteins are characteristic of polypeptides containing tryptophan. When either protein forms a progesterone complex, the excitation and emission wavelengths are not shifted; however, in both cases the fluorescence is quenched. The PBG-progesterone complex has a fluorescence only 40% that of the free protein; that is, the 2.5
1
200
Excitation
250
Emission
300
350
400
450
FIG. 1. Fluorescence excitation and emission spectra of PBG (solid lines) and PBG-progesterone complex (broken lines). Emission of the excitation spectra was measured at 340 nm; excitation of the emission spectra was at 280 nm. Concentration of both PBG and the complex was 3.3 x lo-’ M, determined by titration of binding sites.
fluorescence of PBG is quenched by about 60% upon binding progesterone. On the other hand, AAG fluorescence is quenched by only about 17% upon forming a stoichiometric complex with progesterone. Figure 3 is a fluorescence quenching titration curve of PBG with progesterone. The initial linear fluorescence decrease is readily extrapolated to the baseline defining the equivalence point at a PBG concentration of 2.70 x 1O-8 M. The fraction dissociated at equivalence yields an affinity constant of 2 x 10s M- I. The inset in Fig. 3 shows the same data in the form of a Scatchard plot giving a K, of 2 x lo9 M- ’ and a concentration of 2.77 x 1Om8M PBG. Note that the data in the Scatchard plot span only about 25% of the total binding range, reflecting the fact that under the conditions used binding is “fluorometritally complete” over the initial portion of the titration curve. In Fig. 4, fluorescence quenching titration of AAG with progesterone is shown. The slope of the baseline is greater than that obtained in Fig. 3. This steeper slope was always observed with AAG and may result from greater exposure of fluorophores to solvent and added steroid than is the case with PBG. Extrapolation of the initial fluorescence decrease to the baseline affords an active AAG concentration of 1.3 x 10m5 M. The degree of dissociation at equivalence corresponds to an affinity con-
STROUPE,
r 0
CHENG
AND WESTPHAL
1.0 1.5 Progesterone/PEG
0.5
2.0
, 2.5
FIG. 3. Fluorescence quenching titration of PBG with progesterone. At the equivalence point, 12.6 ~1 of 4.32 x lo-’ M progesterone had been added to 2.0 ml of PBG solution, yielding a binding site concentration of 2.70 x 1Oe8 M. The solid curve yields a K, of 2 x 10QM- I. The inset gives the data from the nonlinear region as a Scatchard plot. The filled circles correspond to the actual data points whereas the open circles are taken from the solid curve. The Scatchard plot gives a concentration of 2.77 x 10m8 M and a K, of 2.1 x lo9 M-‘.
I 0
1.0
2.0
3.0
Progerfcrone/AAG
FIG. 4. Fluorescence quenching titration of AAG with progesterone. At equivalence, AAG concentration was 1.3 x lo-” M. A K, of 1 x lo6 M-’ was calculated from the solid curve.
stant, K,, of 1 x lo6 M- I. This affinity constant, determined at 23”C, is in agreement with a value of 1.5 x lo6 M-r obtained at 4°C by equilibrium dialysis (26) when corrected by the previously published temperature dependence of AAG-progesterone interaction (13). The AAG binding site concentration of 1.3 x 10m5M and the total protein concentration of 1.2 x IOe5 M (assuming M, = 41,000) give a value of n = 1.1 binding site per molecule AAG, which agrees with the established value determined by equilibrium dialysis (27). Although the signal change is not large,
titration curves with AAG are reproducible and are routinely used in this laboratory. Eleven titrations over a period of several months3 gave an average n value of 0.88 i 0.13 (SD). In tests of the reproducibility of the method, a series ‘of 15 titrations of progesterone vs PBG, performed over a period of several months, afforded an average K, of 2.0 f 0.6 x log M-l (fiducial limits calculated (28) for 99% confidence level = 1.6 to 2.5 x log M-‘). In evaluating the precision of concentration determinations by fluorescence quenching titrations, quadruplicate determinations on the same solution gave a binding site concentration of 2.84 * 0.18 x 1O-8 M. This standard deviation corresponds to an error of 6.4%. In another test of concentration determinations, a different solution was titrated six times over a period of three weeks; between tests the sample was stored frozen. The average binding site concentration was found to be 8.41 h 0.13 x 10e7 M, i.e., the standard deviation was 1.5%. The interaction of other steroids with PBG was studied by the fluorometric method. Figure 5A-C shows the fluorescence quenching titrations of PBG by desoxycorticosterone, cortisol, and testosterone. The results are reported as a function 3 Unpublished
results by Mr. Timothy
Kute.
PROTEIN
FLUORESCENCE
AND STEROID
477
BIiYDIIKG
50
40
30
20 0
0.5
Testosterone/PBG
1.5 1.0 Stemid/PBG
20
25
FIG. 5. Fluorescence quenching titrations of PBG with: A, desoxycorticosterone, K, = 1 x lo9 M- ‘, concentration at equivalence = 1.57 x lo-’ M; B, cortisol, K, = 2 x lo6 M-‘, concentration at equivalence = 8.37 x 10m7 M; C, testosterone, K, = 1 x 10’ Mm’, concentration at equivalence _ 8.50 x 10m7 M; D, pregnenolone and progesterone, concentration at equivalence for both = 8.42 x lo-’ M.
of the steroid to protein ratio; the concentration of active PBG was determined separately by titration with progesterone. Duplicate determinations with desoxycorticosterone gave an average K, of 1 x lo9 M-i, four determinations with testosterone an average K, of 1 x 10s M-l. A single determination with cortisol corresponded to a K, of 2 x lo6 M- ‘. Note that different protein concentrations were utilized for the titrations in Fig. 5. All values are in agreement with previously published studies using a competitive equilibrium dialysis method (4). Figure 5D gives the titration of two identical PBG samples with progesterone pregnenolone (5-pregnen-3@01-20and one). Even though pregnenolone has been reported to bind to PBG with an affinity constant of 3 x lo7 M-i (4) (an order of magnitude greater than cortisol) fluorescence of PBG is quenched only 6% by pregnenolone compared to 26% by cortisol and 62% by progesterone. To explore the reasons for lack of quenching by pregnenolone, uv absorption spectra of progesterone and pregnenolone, both complexed with PBG were obtained (Fig. 6). The spectra are reported as molar absorptivities based on moles of complex. The progesterone-PBG spectrum in Fig. 6
-4
i i 1 ,I meJ ‘j 220
240
260 “In
280
300
FIG. 6. Difference spectra of PBG complexes with progesterone (solid line) and pregnenolone (broken line). The baseline was recorded with the same PBG solution in reference and sample cuvettes. Steroid solution was then added to the sample cuvette with an equal volume of solvent added to the reference cuvette and the difference spectrum recorded. Concentration of both complexes was 1.7 x 10 6 M.
has a broad maximum near 245 nm and a sharp peak at 233 nm. The longer wavelength peak is assigned to the major transition of progesterone whose maximum is at 249 nm in water, but is shifted to shorter wavelengths in nonpolar solvents and when bound to proteins (29). The peak at 233 nm
478
STROUPE.
CHENG
AND WESTPHAL
is not due to the progesterone chromopoint results in the expected fluorescence phore; rather it must arise as a difference decrease; however, when 5a-pregnanedione spectrum between the liganded and unlior 5fi-pregnanedione are added to the ganded protein. This follows from the preg- quenched complex, the fluorescence innenolone curve which does not show ab- creases again and approaches the initial sorption at 245 nm because the A4-3-keto fluorescence at higher concentrations of group is absent; however, there is a sharp competing ligand. Progesterone is being maximum at 232 nm. Since pregnenolone displaced; the curves show that 5cY-pregitself does not absorb at 232 nm the signal nanedione is bound more firmly than 5pmust again be due to a difference in pregnanedione, in agreement with pubabsorption between liganded and unlilished results (4). The inset in Fig. 7 gives ganded PBG. Both progesterone (a A4-3- the titration of unliganded PBG with 5pketosteroid) and pregnenolone (lacking pregnanedione; no quenching is observed. this chromophore) appear to induce a simiFurther insight into the mechanism of lar conformational change in PBG resultthe fluorescence quenching by A4-3-ketoing in the difference signal at 232 or 233 steroids is offered in Fig. 8. The near-uv nm. Therefore the absence of quenching absorption spectra of progesterone in hepseen in Fig. 5D cannot result from lack of tane, methanol, and water with 10% ethainteraction between PBG and pregnenonol, representing a wide range in polarity, lone. are given. Changes in solvent polarity are To demonstrate that both A4-3-ketosterwell known to alter the wavelength of lacking the (Y,& maximum absorption of A4-3-ketosteroids oids and steroids unsaturated keto grouping bind at the (30); however, such changes also result in same site on PBG, displacement experisignificant differences in the near-uv specments were performed using 5/3-preg- tra of A4-3-ketosteroids. Of particular innane-3,20-dione (5@-pregnanedione) and terest is the spectrum in heptane which 5-a-pregnane-3,20-dione (5a-pregnanediexhibits vibrational fine structure. As seen one); in both steroids the A4-3-keto chro- for progesterone in Fig. 8, there are well mophore is absent. In Fig. 7, .titration of defined maxima at 337 nm (t =39), 323 nm PBG with progesterone to the equivalence (t = 40), and 296-297 nm (E= 44). Shoulders are evident at 365-370 nm, 350 nm, 307 nm, and 288 nm. The spectrum in methanol is smooth with a single broad maximum at 292-293 nm (t = 106). In water containing 10% ethanol there is no discernible maximum in the near-uv region; however, a shoulder is observed around 290-300 nm with t -200 (not shown). These molecular extinction coefficients are of the same magnitude as those observed in the near ultraviolet for other A’-3-ketosteroids (31). Also presented in Fig. 8 is the normalized fluorescence of PBG illustrating the overFIG. 7. Fluorescence quenching titration of PBG lap of PBG fluorescence and progesterone with progesterone (open circles and triangles) followed by addition of Sa-pregnanedione (filled circles) absorption. The quantum yield of PBG or 5 P-pregnanedione (filled triangles). At the end of fluorescence was 72% that of tryptophan; the progesterone titrations, both samples were 4.95 x utilization of the value of 0.13 (24) results 10 7 M in progesterone binding sites and 5.35 x 10 ’ M on 0.094 as the quantum yield of PBG. The in progesterone. When the competing steroids were product t(x) F(X) x4 used to calculate the added, the PBG samples were “unquenched” by 50% critical distance R, in Fijrster’s radiationat 1.0 x 10e6 M 5a-pregnanedione and at 3.0 x 10m6M less energy transfer theory is also shown for 5/Spregnanedione. The inset shows the fluorescence the absorption spectrum in 10% ethanol. titration of an identical PBG sample with 5/3-pregTable I gives the values of the overlap nanedione.
PROTEIN
FLUORESCENCE
160
60
0 260
260
300
320
340 "ml
360
360
400
42:
FIG. 8. Ultraviolet absorption spectra of progesterone in water containing 10% ethanol (-. -. ). methanol (---I and heptane (. .) illustrating the overlap with PBG fluorescence (-0-l. PBG fluorescence was normalized as explained in the text. The overlap product c(h) F(h) h’ t-Cl-1 is given for the absorption spectrum in 10% ethanol. TABLE
I
OVERLAP INTEGRALS, J, AND CRITICAL DISTANCES, R,, FOR PBG-PROGESTERONE COMPLEX Solvent
J(cm6/mmoll
90% Water + 10% ethanol Methanol Heptane
6.49 x lO-‘¶ 6.64 x lo-‘” 5.90 x 10-I”
R, (Aja 5.9 5.9 5.8
“The value for n is assumed to be 1.5; K* is taken to be 4.
integral, J, for all three solvents and the critical distance, R,, for 50% energy transfer. Note that in spite of wide differences in the absorption spectra, all solvents give nearly the same value of J which results in similar values for R, no matter what solvent best approximates the near-uv spectrum of progesterone bound to PBG. DISCUSSION
Fluorescence quenching has been utilized in the study of many protein-ligand systems; a recent review (22) covers some of them. Concerning protein-steroid interactions, binding of 19-nortestosterone to A5-3-ketosteroid isomerase quenched the protein intrinsic fluorescence by 75% (32). The quenching was ascribed to a proteinto-steroid energy transfer, and it was sug-
AND STEROID
BINDING
479
gested that tyrosine residues are involved in the steroid binding site (33). The interaction between bovine serum albumin and various androstane derivatives has also been investigated fluorometrically by determining “formation constants” and stoichiometries for various steroids (34). No details have been reported on the energy transfer in fluorescence studies involving steroids. The decrease of intrinsic fluorescence exhibited by PBG upon interaction with A4-3-ketosteroids is of interest for two reasons. Fluorescence quenching titrations can be utilized to quantitate the steroid binding protein in solution and to measure the affinity of the interactions. Secondly, the quenching phenomenon can be evaluated to afford information on the nature of the steroid-protein interaction at the molecular level. The study of the influence of progesterone on PBG fluorescence was undertaken in an attempt to develop a convenient method of measuring the concentration of active PBG and its affinity constant in one experiment without resorting to equilibrium dialysis. Therefore emphasis was placed on obtaining stoichiometric binding over a large part of the titration to provide a reliable measure of PBG binding sites upon which to base the calculation of the affinity constants. In testing the precision of concentration determinations by this method, standard deviations of 6.4% at 2.84 x 1Om8M and 1.5% at 8.41 x 10e7 M were obtained. In equilibrium dialysis and Scatchard analysis, five replications of a 1:200 dilution of the same pool of pregnant guinea pig serum gave a binding site concentration of 4.2 i 0.2 x 10e8 M with a standard deviation of 7%. Therefore, both methods are considered comparable in precision. However, fluorescence quenching titrations generally gave concentrations lo-25% lower than equilibrium dialysis. In a direct comparison of four different PBG solutions the concentrations determined by fluorescence quenching titration were on the average 23% lower than those obtained by equilibrium dialysis. This deviation is outside the experimental error found for the two methods. Since no such discrep-
480
STROUPE,
CHENG
ancy was observed for AAG, the difference may be specific for PBG. Concerning the affinity constants, 15 titrations gave a K, value of 2.0 f 0.6 x log M-l at 23°C for PBG-progesterone interaction. A comparable series of equilibrium dialysis studies on both diluted pregnant guinea pig serum and purified PBG gave a K, of 2.1 f 0.4 x log M-’ at 4°C in 16 separate experiments. However, the affinity constants by the two methods were obtained at different temperatures; three determinations by equilibrium dialysis at 23°C gave an average K, value of 1.0 x log M-’ (fiducial limits (28) at 99% confidence level 0.7-1.3 x log M-l). Thus fluorescence titrations are seen to overestimate the equilibrium dialysis affinity constant by more than experimental error. Comparison of the two methods for accuracy is difficult because of the relatively wide range of values actually obtained with the fluorescence quenching method. For the 15 titrations mentioned above the range was 1.1 to 3.0 x log M-l. Similar variations (up to twofold) were observed when the method was applied to measurements on a larger series of steroids.’ Therefore, to obtain acceptable precision, affinity constants must be based on at least quadruplicate determinations. If such an average is taken, the fluorometric method gives the correct order of magnitude for the affinity constants for the various steroids and correct relationships of the affinities to each other. The method of fluorescence quenching titration has the advantage of generally using less binding protein (typically 0.1 nmol PBG per experiment) and being much more rapid than equilibrium dialysis. It does not require radiolabeled steroids. However, the protein must be in purified form and the steroid must have the appropriate ultraviolet absorption. Analysis of fluorescence quenching can also be used in assaying for PBG activity during purification procedures without introducing radiosteroids: the fluorescence of the relevant fractions read with and without a large excess of progesterone affords a mea’ Unpublished
results by Mr. A. T. Blanford.
AND WESTPHAL
sure of the progesterone-induced quenching which is proportional to PBG purity. For the second aspect mentioned above, the quenching phenomenon can offer information concerning the molecular basis of the PBG-progesterone interaction. Fiirster (20) has listed four mechanisms by which energy can be transferred from an excited state: (1) nontrivial transfer [resonant transfer], (2) reabsorption, (3) complexing, (4) encounter. The quenching is not due to reabsorption because the optical densities are kept low, and the percent quench is invariant at 60% over a hundred-fold concentration range from 10e6 to lo-* M PBG. Complexing, with an attendant alteration in the absorption spectrum is unlikely to be the major source of quenching. A small difference spectrum (not shown in Fig. 6) is induced between 280 and 300 nm upon steroid binding, but the change is less than 5% of the total absorption, too small to account for the large decrease in fluorescence. Encounter quenching will arise if there is a conformational difference between PBG and the PBG-steroid complex and if the PBG fluorophores in the complex are more exposed to solvent. This can be rejected on two accounts. First, pregnenolone, 5a-pregnanedione, and 5P-pregnanedione all form complexes with PBG, but without significant quenching. Second, in a comparison between a titration in buffer with and without 20% sucrose, the quenching at equivalence was 60% in both cases; less quenching would be expected in the more viscous medium if quenching resulted from increased encounter transfer in the complex. The remaining transfer mechanism is resonant transfer which classically is proven by enhanced acceptor fluorescence upon donor excitation. If a A4-3-ketosteroid is the acceptor, this method of proof is unavailable because molecules with an n + S* transition as their lowest energy transition are nonfluorescent (35). The absorption spectrum of progesterone overlapping the fluorescence emission spectrum of PBG makes resonant transfer possible, but does not prove it. In accordance, we did not observe quenching by steroids lacking the overlapping absorption. Furthermore,
PROTEIN
FLUORESCENCE
when a quenched complex was backtitrated with a nonquenching steroid, the complex fluorescence increased to that of unliganded PBG. These two results prove that merely forming a complex between PBG and a steroid is not sufficient to quench fluorescence; rather the steroid must possess an overlapping absorption band to accept energy from the PBG excited electronic state. Resonant transfer is the only mechanism of energy transfer consistent with these observations. Although important information is lacking for a quantitative interpretation of the overlap integrals (Table I), the following comments are made in an attempt to understand the mechanism of the interaction. Using the simplest case for energy transfer, we assumed only one of several tryptophan residues to be fluorescent. The calculations utilize K* = 4, the most favorable case for transfer with resultant distances being maximum values. With an R, value of 5.9 A and the 60% quench observed with progesterone, a maximum separation of 5.5 A is calculated. This small separation is predicated by the low extinction coefficient of the steroid in the fluorescence overlap region. Since testosterone and desoxycorticosterone quench PBG fluorescence to about the same extent as progesterone, it is most likely that these steroids are bound at the same site and in the same mode as progesterone. In contrast, cortisol quenches the fluorescence of PBG by only 26% at saturation. The overlap integral of cortisol (and indeed of other A*-3-ketosteroids) ought to be approximately the same as that of progesterone as long as the similarity of the steroid chromophore system in ring A is maintained. It can be calculated, therefore, that cortisol is located up to 6.9 A from the critical tryptophan in the complex. A detailed physical interpretation of separation distances smaller than the molecules themselves is meaningless. However, in the case of cortisol, since the value for J should be the same as for progesterone, the diminished quenching must result either from an orientation less favorable to transfer (change in K’), or an increase in separation (change in R,), or both. Whatever change there is in
AND STEROID
BINDING
481
the PBG-cortisol complex, it results in a stability three orders of magnitude lower than that of the PBG-progesterone complex. The proposed mechanism of fluorescence quenching by energy transfer from protein fluorophores to the nAr* absorption band of a conjugated carbonyl group in the steroid is general. The mechanism implies that any protein-steroid interaction will quench the intrinsic fluorescence of the protein if two conditions are met. First, the steroid-protein contact in the complex must place the steroid very near a fluorescent group. Second, the steroid must be a suitable acceptor; that is, the steroid must have an absorption band overlapping the protein fluorescence band. If the second condition is met, the magnitude of the quenching offers information concerning the binding site. CONCLUSIONS
The method of fluorescence quenching titration is a reliable and rapid technique of measuring binding site concentrations and assessing affinity constants of PBG with A4-3-ketosteroids. It does not require radiolabeled steroids. The procedure can also be applied to AAG and other steroidbinding proteins if a fluorescent group is present at or near the binding site, and if the steroid ligand is a suitable energy acceptor for the fluorophore. In addition, analysis of the fluorescence quenching provides information on the dimensional relationships at the binding site, and can be used to assess the influence of structural and steric features of the steroid molecules on the interaction with specific proteins. ACKNOWLEDGMENTS This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AM-06369) and a research career award (UWl from the Division of General Medical Sciences (GM-K6-14, 1381 of the United States Public Health Service. Alpha,-acid glycoprotein used in these studies was provided by the American Red Cross National Fractionation Center with the partial support of National Institutes of Health Grant HE 13881 (HEM). The authors wish to thank Mr. George B. Harding and Mrs. Karen Acree for performing the equilibrium dialysis studies.
482
STROUPE,
CHENG
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