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Examination of steroid protein interaction by ultraviolet difference spectrophotometry
ARCHIVES
OF
RIOCHEMISTRY
Examination
AND
BIOPHYSICS
407-417
126,
of Steroid
Protein
Difference
Spectrophotometry’
MICHAEL Department
(1968)
of Biochemistry, Received
Facully October
Interaction
by Ultraviolet
T. RYAN
of Medicine, 24, 1967;
accepted
University February
of Ottawa,
Ottawa,
Canada
6, 1968
The phenomenon of steroid-protein interaction has been examined in terms of ultraviolet difference spectra. In the case of interactions of serum albumins and some a&unsaturated ketosteroids two spectral effects are discernible, one attributed to the perturbation of the steroid absorption band by a hydrophobic environment at a binding site and the other to perturbation of aromatic amino acid bands. Interaction with androstenolone, which does not have the a#-unsaturated ketone group, revealsdifference troughs at approximately235,282, and 288 rnp which have been attributed to a red shift of aromatic absorption bands. Interaction of steroids with several enzyme uroteins exhibits. under the conditions used, poorly defined difference spectra which are unlike the ones for serum albumins.
The phenomenon of protein-small molecule interaction has been intensively investigated for a number of years (1). As noted by Eik-Nes et al. (2), steroids represent a particularly suitable class of small molecule for such study, being uncharged and possessedof a wide range of structural variety impressed on a fairly rigid hydrocarbon framework. This has further acquired heightened biological significance not only in terms of the nonspecific and specific transport properties of serum proteins (3-9) but as providing a conceptual model (10) with which to approach the problem of hormone action at the molecular level. Toft and Gorski (11) have claimed to have isolated a receptor protein from uterine tissue. Experimental demonstration of the reversible combination of steroids and proteins has been provided by a number of techniques, such as equilibrium dialysis (5), solubility (Z), electrophoresis (12), and ultrafiltration (1.3); and a fairly extensive list of binding parameters has been compiled (3-5) for the common plasma proteins. Westphal (6) has furthermore demonstrated that there 1 This wo:rk was supported by grant MA-1616 from the Medical Research Council of Canada. 407
is a depression of the absorbance of A4-3ketosteroids in the presence of proteins and Westphal et al. have shown that there is a correlation between the value of AA at x maxand the binding affinity of the steroid for serum albumin (14). The single wavelength parameter has been employed in an attempt to study the relationship between steroid structure and binding afhnity in the case of a large number of steroids (6-9), an approach which, as the present paper tends to show, may not always be valid. In spite of these extensive investigations and except for the special case of the steroid isomerase studied by Wang et al. (15) and the evidence of the general “polarity rule” 2 (4) it can be said that very little is known about the precise nature of the phenomenon (see ref. 1, p. 282). Moreover, the configurational adaptability which serum al2 The term “polarity” is used to imply the number of polar groups (carbonyl, hydroxyl) present in the steroid molecule. It has been demonstrated by Eik-Nes et al. (2) and by Chen et al. (13) that the binding affinity of steroids for serum albumins is inversely related to the number of such groups in the molecule. Westphal (7) refers to this phenomenon as the polarity rule.
408
RYAN
bumin apparently also displays in interaction with cortisol (16) renders the concept of binding site somewhat complex. In studying the binding phenomenon we have reexamined the spectrophotometric mebhod of Westphal (6), extending considerably the range of measurement and expressing the results as difference spectra. This approach has been exploited successfully in other protein studies (17). EXPERIMENTAL
PROCEDURE
Materials 6, 1701-Dimethyl-6-dehydro-progesterone3 (1) was a gift;4 it melted at 146-147’ (Fisher-Johns). All other st)eroids were obtained either from the Sigma Chemical Co. or Mann Research Laboratories and were recrystallized from aqueous alcohol when necessary. Their uncorrected melting points were 128-129” (progesterone), ‘211.5~213.5” (cortisol), 138-139” (deoxycorticosterone), 154155” (testosterone), 152-153” (androstenolone), lg&190” (16-dehydroprogesterone), 183-184” (androsterone). Cryst,alline BSA was a product of Armour and Co. Crystalline HSA was obtained from Pentex Corp. Two samples of chymotrypsin were used: Mann Research Laboratories (3 X crystallized, salt-free) and Worthington Biochemicals (chromatographically pure). Trypsin was a product of Mann Research Laborat,ories (2 X crystallized). Ljrsozyme was obtained from Worthington Biochemicals (2 X crystallized, saIt-free). The &hydroxysteroid dehydrogenase (3-p-hy,droxysteroid: NAD (P) oxidoreductase, EC 1.1. 1.51) was a crude extract of dried cells of P. testosteroni which were obtained from Worthington BiochemicaIs. Tris base was obtained from the Sigma Chemical Co. Distilled deionized water was used for making aqueous solutions and for washing glassware.
Procedure The experimental approach consisted of attempt to observe perturbations of ultraviolet absorbing bands present either in the steroid protein or both, which might arise as a result
an or of
3 The following abbreviations are used: I, 6,17cu-dimethyl-6-dehydro-progesterone; 16-dehydroprogesterone, pregna-4,16, -dien-3,20-dione; androstenolone, 3P-hydroxy-androst-5-en-17-one; HSA, human serum albumin; BSA, bovine serum albumin. 4 Courtesy of Dr. Sam Solomon, McGill University.
interaction. Difference spectra rather than actual perturbed spectra were studied since these present a sharper picture of such perturbations (17), especially under conditions in which total absorbance is constitut,ed of several types of overlapping bands. This represents the first systematic application of this technique to this problem. The results were obtained as two separate sets of spectra measured on a Beckman DU Spectrophotometer: (A) in Tris chloride buffer pH 8.0, r/2 0.1 and (B) in presence of proteins dissolved in this same buffer. After applying corrections to some values of set (B) (see below) difference spectra were computed by subtraction. Later results in these investigations were obtained using the tandem cell technique of Herskovits and Laskowski (18) on a Cary Model 15. Some spectral measurements have also been made on solutions of steroids in absolute redistilled ethanol. The measurements on the DU were made in rect,anguIar cells of IO-mm light path and using the photomultiplier, deuterium lamp, and thermospacers with water circulating at 25 f 1”. Cylindrical tandem cells, each with a total light path of 20 mm (Pyrocell Mfg. Co., Westwood, New Jersey) were used in the Cary. Ceils were stored in nitric-sulphuric acid (19) and washed with water, deionized water, and alcohol, a,nd dried in a vacuum oven. In using the Beckman t,he effective band width varied from 0.4 to 1.8 m/l. The possibility of error due to light scattering was eliminated by extending some measurement,s to 400 mp. When using tandem-cell technique in the Cary, the 100% line on the chart was trimmed with the cells filled with alcohol. The cells were then emptied and dried in a vacuum oven. The reference cell was filled with the mixture of protein and steroid in one chamber and Tris in the other. The sample cell was filled with steroid in Tris in one chamber and protein in Tris in the other, each at the same concentration as in the mixture in the other cell. Records were obtained at dynode settings 2-4 and on both the 1.0 and 0.1 absorbance range. Difference spectra are expressed in terms of AA; in the 280-300 rnp region in Fig. 4 values for Ae protein are also provided. Stock solutions of steroids in Tris were prepared by evaporating the appropriate volume of a 1 mM or 2 rnM solution in ethanol under a st.ream of nitrogen in an Erlenmeyer flask. The fine film so obtained was dried in vacua, dissolved with shaking in Tris, and filtered through a medium-porosity, sintered glass filter. The actual concentrations were determined in a manner similar to that of Westphal (6) from extinction values measured at lower steroid concentrations in 2% alcohol in Tris. In most instances the actual concentrations
STEROID were close to the by these dilul;ions.5 tained by mixing tions in Tris with 192 y.l~ solution of
PROTEIN
nominal concentration sought Working solutions were ob5 ml of the stock aqueous solueither 1 ml of Tris or 1 ml of a the protein in Tris.
Stray Ligh.t Since values below 240 rnp seemed of interest, an attempt was made to measure stray light arising in this range in 32 LAM protein solutions (21,22) and to apply corrections for it. The method consisted of measiuring the transmittance of the protein solution in a Corex (Pyrex) cell, using the protein solution in a silica cell as 100% T. Since Corex cuts off at about 260 rnp such values should represent stray light originating above this wavelength and hence correspond to the source of error in protein solutions. Values for the stray light component (Ts) so measured for the proteins under investigation were of the order of 357, at 235 rnp for 32 ~GM albumin and were used in applying corrections to the observed 2’7, (Tabs) using the assumption that in the expression of Saidel et al. (21) I,, := I,. Under these conditions Tcorrect = T abs - T,/lOO - T,, providing that the stray light component does not change on interaction of steroid and1 protein. The determination of the concentration of androstenolone in stock solution in Tris was performed using the method of Talalay (23). Only the nominal concentration of androsterone is shown in Fig. 3 since the crude a-hydroxysteroid dehydrogenase (3.a-hydroxysteroid: NAD (P) oxidoreductase, EC 1.1.1.50) proved inactive in Tris buffer. RESULTS
Difference spectra, which were obtained using the single beam instrument for the interaction of four A4-3-ketosteroids and androstenolone with 32 j.&M BSA, are presented in Fig. 2 and are characterized by a difference peak at 259 rnp for the a,@ unsaturated steroids and a difference trough 5 Results rleported here are based on measurements using steroid stock solutions in Tris buffer stored at room temperature in stoppered flasks and which were used usually within 7-10 days. Checking the absorbance at A,,,,, for the steroid revealed virtlually no change for periods up to 6 weeks, unlike the results reported by Zimmering et al. (20) for phosphate buffer. In one instance a solution of testosterone in Tris buffer which had been stored for 5 months exhibited a 1670 increase in extinction value.
INTERACTION
409
at about 235 rnp for androstenolone. Spectra for the interaction both of these same steroids and also androsterone, I and 16dehydro-progesterone with 32 ~ccy HSA are presented in Fig. 3 where it can be seen that the spectral picture is similar to that for BSA. Figure 3 also contains values for the interaction of progesterone and HSA at wavelengths below 240 rnp which were obtained with t’he aid of the stray-light corrections described above and which are represented by a dotted line. Similar results have been obtained below 240 rnp for the interaction of other A4-3-ketosteroids not only with HSA but also with BSA. However, for reasons given below, they have been excluded from these figures. The validity of the results represented by solid lines in Figs. 2 and 3 is confirmed by the results of a study of the interaction of progesterone and androstenolone with 32 pc~1l1 HSA using tandem cell technique in t#he Cary (Fig. 4), although the trough for androstenolone is shifted slightly. This figure also provides additional detail in the 28&300 rnp region. The artifactitious nature of the peak in the 235 rnp region represented by dotted lines and registered both by the single-beam (Fig. 3) and double-beam instrument (Fig. 4) is revealed by study of the interaction at lower protein concentrations. In the case of progesterone, valid difference measurements in the 23&240 mp region can only be obtained at HSA concentrations of 8 PM or lower. The artifact peak has been observed in the case of other A4-3-ketosteroids but not in the case of androstenolone where the shape of the curve for androstenolone-HSA interaction is only slightly dependent on protein concentration. The artifact may possibly be attributed to fluorescence quenching at protein concentrations and in a wavelength range where a significant portion of the transmitted beam consists of fluorescent energy originating in the protein. Measurements referred to earlier suggest that this may be of the order of 35% at 235 rnp in 32 PM HSA. Fluorescence quenching has been shown to occur in the binding of 19-nor-testosterone to the steroid isomerase (15) and has been reported briefly in the case of serum albumins (24).
410
RYAN
The location of the androstenolone absorption band in ethanol, water, and Tris, as judged by absorbance measurements at the long wavelength edge on dilutions prepared from a common stock solution in alcohol, is reported in Table I. The absorption characteristics of several CX,~ unsaturated ketosteroids in ethanol and Tris are presented in Table II together with the characteristics of the difference spectra which have been graphically computed from these. A plotted spectrum and difference spectrum for the case of cortisol in ethanol and Tris is given in Fig. 1 and illustrates the
spectral effect that might be anticipated should an equivalent change in chromophore environment arise in the binding of a steroid to a protein. Difference spectra for the interaction of some steroids with trypsin, chymotrypsin, and lysozyme are presented in Fig. 5 where it can be seen that well-defined features, such as are seen in the case of serum albumins, are not evident. Table III contains a comparison of the percentage depression values at 259 rnp and 249 rnp with those of Westphal (6, 9) for the interaction of a steroid series of increasing polarity with several proteins.
TABLE
I
EXTINCTIONVALUESFORANDROSTENOLONEINETHANOL,WATER, 0.1~ NACL,ANDTRIS~HS.O,~/~O.~, MEASURED IN 10 mm SILICA CELLS.~ORHING SOLUTIONS WERE PREPARED FROM A COMMON STOCK SOLUTION IN ALCOHOL AND MADE TO A CONCENTRATION OF 40 MM (mr)
Ethall01
water
NaCl
(md
Ethanol
water
NaCl
Tris
210 211 212 213 214 215 216 217 218
1,850 1,450 1,175 875 675 500 350 250 175
1,900 1,650 1,425 1,175 1,025 875 700 600 475
2,063 1,788 1,550 1,313 1,150 975 813 700 575
219 220 221 222 223 224
150 125
425 350 275 200 150 125
465 400 315 265 215 175
450 400 338 300 238 200
TABLE
II
SPECTRAL VALUES FOR SOLUTIONS OF STEROIDS IN TRIS AND IN ETHANOL bnax b/d
10-a6
10-3(Af)msx
Progesterone Alcohol= Trisc Deoxycorticosterone AlcohoP Trisc Cortisol Alcohol,= Trisc Testosterone Alcohol5 Tris I Alcohol Tris
240 248
16.4 16.0
-7.3
(260 rnp)b
+4.9
(235 rnw)
240 249
16.0 16.0
-8.2
(260 mp)
$3.8
(233 mB)
241 248
15.1 15.8
-5.5
(260 mp)
+2.3
(233 mp)
240 248
18.9 15.7
-6.2
(260 rnk)
+7.3
(234 rnp)
289 297-299
23.4
a Reported values of A,,, and emsI may be found in Dusza et al. (40) and also Ulrich (41). b Values in parentheses refer to the wavelength of (Ae)msx. c Values for X,,, and smaL in Tris are in good agreement with those reported by Westphal phosphate buffer.
(6, 9) for
STEROID
PROTEIN
INTERACTION
411
make only minor contribution here. The extinction values for androstenolone in ethanol, Tris and in water to be found in 17. 16. -8 Table I reveal that there is an apparent red (....., shift of the band in aqueous solution as com-7 --6 pared to ethanol, demonstrable by the fact that not only are the extinction values at a given wavelength higher in water but the tail of the band clearly extends into longer wavelengths. This is in keeping with the known red shift of double bond bands of other steroids in going from hexane to ethanol (2527). It will be noted that the extinction values above 220 m~.1 in water are very low and it is difficult to conceive of any mechanism which could move this band any further to the red so as to thus account 2Ifor the difference spectra for androstenolone in protein solution (Figs. 2, 3, 4). The posi220 230 240 250 260 270 200 tion of the main absorption band of the A4-3WAVELENGTH (mp) ketosteroids is also solvent-dependent (28), FIG. 1. Spectra and difference spectrum of the red shift associated with increasing cortisol in ethanol and Tris pH 8.0, I’/2 0.1. A, solvent polarity being due to increased staethanol. B, Tris. C, difference spectrum. Measurebilization of the dipolar excited state (30).6 ments made in l-cm silica cells in a Beckman DU Such a lateral shift can conveniently be wit,h photomultiplier. Difference spectrum obtained by subtraction of values for B from values represented by a difference spectrum such for A. as that in Fig. 1 for the caseof cortisol in the solvents Tris and ethanol. This curve exDISCUSSION hibits a difference peak and trough whose Steroid spectra and solvent efects. The locations are fairly constant for the four ultraviolet absorbance of monounsaturated steroids progesterone, cortisol, deoxycortisteroids and saturated steroid ketones has costerone, and testosterone (Table II) with been studied in recent years by means of respect to the same solvent pair, i.e., at 260 high performance instruments (25-27) and rnp and 233-235 rnp respectively. Spectral perturbations in protein solutions. despite the variation in the reported values Spectral values obtained from steroid soluof ~~~~~~ and. emaxfor such steroids (27) the dependence of these values on the location tions in the presence and absence of proand degree of substitution of the isolated teins may also be expressed as difference double bond seems well established (28). spectra. In the case of bovine and human While the rspectrum of androstenolone has serum albumin for example (Figs. 2, 3) not apparently been examined in modern distinctive difference spectra are seen in the instruments it would be expected to have a case of the A4-3-ketosteroids where the peak at 191-195 rnp, similar to that of height of the peak seemsto be related to the cholesterol (27). Bladon et al. (29) have polarity of the steroids. The difference shown that the long wavelength edge of this spectra as seen with the aid of the tandem band (>205 rnp) is accessible to measure- cells and the Cary show additional detail ment with a quartz spectrophotometer. in the 28&295 ~QJregion, as exemplified by difference spectra for progesterone and This spectrum is complicated by contributions from the carbonyl absorption but 6 In this, the red shift due to increase in dit,hese bands are deeper in the ultraviolet electric constant apparently dominates a blue (25) and of much lower intensity (25-27) shift to be expected from the decrease in refracthan the double bond band and should tive index. 19. 18.
412
RYAN TABLE IIEPRESSION
OF
MOLAR
A~RSoRPTI~~ITY EXPRESSED
Steroid
Bovine Concn
(/AM) 25')
Progesterone Deoxycorticosterone Testosterone Cortisol
15.1
serum
m/l
III OF
STERoiDS
IX
PRESENCE
OF
albumin 249
mp
16.1
25.6 21.2
13.0 10.0
10.5 16.9
27.6 0.5
15.2 0.7
(2.2 mg/ml) 249
mg
Human Concn
serum
albumin
(2.1 m&ml)
249 Inp
249 mp*
(jA.4 259 mp
16.5 16.0
32.3 23.2
12.2 11.2
13.0(6)
9.8(G)
4.0(6)
17.5 16.6
25.3 3.1
12.6 1.9
13.2(9) 4.5(6)
11.8(6)
Trypsin (1.15 mg/ml)
Progesteronec Deoxycorticosterone Testosterone Cortisolc
PROTEINS,
AS PERCEXTAGE~
12.5(6)
Chymotrypsin (0.7 mg/ml)
15.6 16.0
5.3 +1.5
4.0 +12
5.1(9) 4.9(9)
15.5 16.0
o.oc 0.0
1.7 1.5
10.8(9) 12.7(9)
17.7 16.7
f1.9 5.5
0.7 4.0
5.2(9)
17.7 16.4
2.3c 2.oc
2.1 0.4
12.9(9)
Lysozyme (0.16 n&ml)
Progesterone Deoxycorticosterone Testosterone Cortisol
15.5 16.0
1.0 +2.9
1.6 +0.4
11.6(g) 12.8(9)
17.7 16.7
t-4.3 2.5
+2.9 0.4
15.5(9)
4 Positive sign indicates apparent increases in the absorptivity. * Values of Westphal and co-workers for higher protein concentrations references given in parentheses. c Solutions contained Ca++ at a concentration of of 0.02 M.
androstenolone in Fig. 4. Westphal et al. (14) have noted an inverse relationship between AAz49 and the polarity of the steroid but it is clear that the complete difference spectrum presents a more meaningful picture. The difference peaks may arise out of perturbation of either the protein or steroid chromophores. It must be noted however that the difference peak at 259 rnp (Figs. 2, 3) does not conform either in shape or position to those perturbations of tyrosine and tryptophan (twin peaks in the 270-295 m,u range) most frequently seen for proteins under a variety of perturbing conditions (3134). Perturbation of the phenylalanine absorption spectrum may yield a diff erence peak in the 250-270 mp region (33) where the fine structureof theunperturbed bandis, however, carried over into the difference spectrum. Because of this and the very much lower contribution of phenylalanine to protein absorption in this region it is unlikely that this amino
in phosphate
pH
7.6 in the
acid can be implicated in bhe difference peak observed. The possibility of the aromatic amino acid chromophores contributing to this peak is more convincingly eliminated by means of a study of the diflerence spectrum produced by I, in which the electron transfer band of the steroid is shifted to the other side of that due to the aromatic amino acid absorption. This was the only such steroid available to us and it has a very much lower solubility in aqueous solution than desirable. In the difference spectrum for the interaction of I with 32 I.IM HSA (Fig. 3) the peak at 314-316 rnN can only be attributed to perturbation of t’he steroid absorption band at 297-299 rnp. It will be noted that in addition a broad trough appears at 280 rnp which, by analogy with Fig. 1, would be expected from a lateral shift of the absorption band toward shorter wavelengths. However, this may be com-
STEROID
PROTEIN
plicated by contributions from aromatic chromophores in this region (see below). From the foregoing observations and the demonstrated effect of solvents on steroid spectra it is postulated, as has indeed been suggested by Wang et al. (15), that the difference peak at 259 rnp for interaction of A4-3-ketosteroids with serum albumin and at 314-316 rnp for I may be attributed to the lowering of the effective dielectric constant associated with the enveloping of the steroid chromophore in a hydrophobic region of t,he protein molecule. It will be noted that the location of the difference peak is close to that for the transfer of the steroids from Tris to ethanol (Fig. 1 and Table 11)-a situation which should approximate the postulated binding process. It may be questioned that the spectrum of cortisol, the most polar steroid, is in fact perturbed in view of the very low difference values and the absence of an actual peak at 259 rnp. Preliminary examination of the cortisol-albumin interaction in the Cary shows no acbual difference values in this region whereas the corticosterone-albumin interaction exhibits an actual difference peak with a value of -0.010 for AA&. This may be taken as evidence that cortisol is bound at a site in the molecule quite different from that (or those) for the other steroids and is in keeping with the published results of competition studies (3). A simple lateral shift in the absorption band should also however produce a trough which, by analogy with Fig. 1, would be located at approximately 233-235 rnp. Examination of the progesterone-albumin interaction at a concentration of 8.0 pM HSA shows a cross-over point at 242 rnp with increasingly positive values down to 230 rnp but without the appearance of an actual trough (Fig. 4, curve C). This failure of the anticipated trough to appear suggests t’hat an additional spectral effect may be operative and this finds appreciable support in closer examination of the results in the 280-,300 rnk region for progesterone (Fig. 4) and in both the 235 rnp and 28CL300 rnp regions for androstenolone (Figs. 2, 3, 4). In the case of androstenolone positive dif7 Unpublished
results.
413
INTERACTION
WAVELENGTH
(mp)
FIG. 2. Perturbation of ultraviolet spectra arising out of the interaction of steroids with BSA at a concentration of 32 PM (2.2 mg/ml) in Tris PI-I 8.0, I’@ 0.1. Expressed as difference spectra from measurements made in IO-mm silica cells in a Beckman DU with photomultiplier, at 25 Z!Z 1”. Stray-light corrections have been applied below 240 rnp for E (see Experimental). A, 15.1 PM progesterone. B, 16.9 PM cortisol. C, 16.5 PM testosterone. I), lG.l PM deoxycorticosterone. E, 60.0 PM androstenolone. Algebraic sign is relative to the unperturbed state of steroid and protein in Tris.
ference values appear over the entire spectral range, rising to increasingly more positive values below 240 rnp in the form of a broad trough which, though centered at approximately 235 rnp, clearly extends beyond the range of measurement. Such a trough is also exhibited in the interaction of androsterone (Fig. 3) with albumin. Since androstenolone exhibits virtually no absorption in Tris above 225 rnp (Table I) the spectral effect in this case, and presumably also that of androsterone, can only be attributed to the protein. Examination of the androstenolone-albumin interaction by means of tandem-cell technique in the extended absorbance range of the Cary reveals that there are also smaller troughs located at 282 and 288 rnp (Fig. 4). Such spectral values for the interaction of
414
RYAN
235 rnp region which are approximat,ely 5 times those at 282 rnp and 288 rnp. The direction of the spectral shift is, however, toward the red, unlike not only the denaturation shift but also the blue shift which occurs in the binding of such substances as octanol and dodecyl sulphate to albumin (36). In this connection Ray et al. (36) have quest’ioned that the most usual explanation for the blue shift-the exposure of hidden aromatic residues to the aqueous solventis necessarily correct since they could not, using solvent-perturbation technique, demonstrate any such unmasking. It is possible that the red shift produced by the steroid alcohol could be accounted for by a small local conformational change affecting the charge environment of a particular tyrosine residue as has been suggested by Bigelow and Sonenberg (37) for the blue shift in the dodecyl sulphat,e-albumin interaction. In this context it is of interest that Alfsen (38) has observed changes in optical activity on interaction of testosterone with serum albumin which she has attributed to an in.08 crease in helical content of the protein. In 235 240 245 250 255 260 265 270 275 280 285 the study of the interaction of progesterone WAVELENGTH (my) with 32 PM HSA by means of tandem-cell technique (Fig. 4) it will be noted that there FIG. 3. Perturbation of ultraviolet spectra is a small but clearly discernible trough arising out of the interaction of steroids with with two minima located at approximately HSA at a concentration of 32 SM (2.1 mg/ml) in 286 rnp and 292 rnp. If these, too, can be Tris pH 8.0, p/2 0.1. Expressed as difference spectra from measurements made in lo-mm silica attributed to aromatic chromophore perturcells in a Beckman DU with photomultiplier, at bation then a contribution of approximately 25 f 1”. Stray-light corrections have been applied 5-7 times these values would be anticipated below 240 mp for A, F, and E (see Eqnerimental). in the 235 rnp region. The latter, when A, 16.5 PM progesterone; B, 16.6 PM cortisol; C, superimposed on a steroid perturbation 17.5 PM testosterone; D, 16.0 PM deoxycorticosspectrum, might account for the shape of terone; E, 60.3 JAM androstenolone; F, 66.5 PM curve C, Fig. 4, between 230 rnp and 240 rnp. androsterone; G, 13.4 PM 16-dehydroprogesterone; The fact that interaction of the A4-3H, 5.5 PM I; J, curve for E uncorrected. ketosteroids with serum albumins produces a spectral difference peak at 259 rnp suggests androstenolone with HSA seem to correthat difference values measured at t,he peak spond to aromatic chromophore perturbawavelength represent a more sensitive and tion analogous to what has been suggested (31) as a partial explanation for the denatu- more accurate measure of Ae for the steroid than those at 249 rm.~.In Table III there is ration blue shift seen at 235 rnp in the case presented a comparison of our percentage of several proteins (32-34). Edsall et al. (35) depression values at 259 rnp and 249 rnp wit,h have noted in the study of denaturation those of Westphal (6-9) at 249 rnp for not of carbonic anhydrases that the values of Ae2s6were 5-7 times those at 282 rnp and only the serum albumins but also several en291 rnw. The interaction of androstenolone zyme proteins. Here it can be seen that the with HSA produces difference values in the values at 249 rnp agree very well with those
STEROID
PROTEIY
INTERACTIOY
41ii
OA p. : I
O.lO0.09 ooe-
j : j
: ‘i :
0070.06
WAVELENGTH
(mp)
FIG. 4. Perturb&ion of ultraviolet spectra arising out of 1he interaction of 14.1 PM progesterone and 60.3 pM androstenolone with HSA in Tris pH 8.0, r/2 0.1. (A) progesterone in 32 PM HSA (2.1 mg/ml) ; (B) progesterone in 16 PM HSA; (C) progesterone in 8 PM HSA; (D) androstenolone in 9.7 pM HSA; (E) androstenolone in 32 PM HSA. Expressed as difference spectra, and measured by means of tandem-cell technique in a Cary 15.
of WestphaJ in the case of interaction of progesterone, deoxycorticosterone, and testosterone with albumins and that the values at 259 rnp are approximately twice those at 249 rnp. However, there is significant discrepancy in the values for cortisol where ouI;‘s are consideribly lower than those of Westphal. This discrepancy may possibly be attributed to the different buffer conditions in our experiments. The difference spectra given in Fig. 5 for the enzyme proteins present a contrast with the results for the serum albumins in that distinctive absorption band perturbations are not evident. Furthermore an examination of T&le III with respect to these proteins reveats further discrepancies between our results and those of Westphal and coworkers (6, 9), especially in the case of chymotrypsin and lysozyme. This may be partly due’ to the fact that we used much lower concentrations of these proteins, which have much higher absorptivity in the
ultraviolet than the serum albumins.* Furthermore, with the exception of lysozyme, most of our determinations were made on these proteins in the presence of Ca++. Thus one does not see, even in the case of lysozyme, the correlation claimed by Westphal and Ashley (7-9) between At249and the polarity of the steroid or evidence for the operation of an inverse polarity rules in the case of these enzyme proteins as suggested by these authors. This, together with the fact that there is no characteristic spectral structure in the plot of difference values against wavelength, suggests that AEZ~S * For example, at 0.2 mg/ml; A278 albumin = 0.125, A 281lysozyme = 0.522, A 282chymotrypsin = 0.372, A2T8 trypsin = 0.282. At the concentration of 1.72 mg/ml used by these authors for lysozyme Azn3 = 2.25. Stray light may have introduced some error into the values observed by them. 9 That is to say that, unlike interactions with serllm albumins, the binding affinity would increase with increasing polarity of the steroid.
416
RYAN
an
dues which may be directly implicated in the binding process are as yet unknown although the present results suggest that there may be hydrophobic residues near the binding site for some steroids in the serum albumins.
LYSOZYME 003
-
REFERENCES 1.
STEINHARDT, J., AND BEYCHOK, S., in “The Proteins” edition.
6.
(H. Neurath, ed.), Vol. 2, 2nd Academic Press, New York (1964). EIK-NES, K., SCHELLMAN, J. A., LUMRY, R., AND SAMUELS, L., J. Biol. Chem. 206, 411 (1953). SANDBERG, A. A., SLAUNWHITE, W. R., JR., AND ANTONIADES, H. A., Recent Progr. in Hormone Res., 13, 209 (1957). DAUGHADAY, W. H., Physiol. Rev. 39, 885 (1959). SLAUNWHITE, W. R., JR., ROSENTHAL, H., AND SANDBERG, A. A., Arch. Biochem. Biophys. 100, 486 (1963). WESTPHAL, U., Arch. Biochem. Biophys. 66, 71
7.
(1957). WESTPHAL, U., in “Mechanism
2.
003-
I
TRYPSIN
I
3.
002 ..
0.0, i.r 0.01
..-.-. J
- ._,_._._._._._,_.
*.-.-.-.-.-.-‘-’
4. 5.
WAVELENGTH
(mp)
FIG. 5. Perturbation of ultraviolet spectra arising out of the interaction of steroids with 32 PM chymotrypsin (0.7 mg/ml), 48 @M trypsin (1.15 mg/ml), and 32 PM lysozyme (0.46 mg/ml) in Tris pH 8.0, I’@ 0.1. Expressed as difference spectra from measurements in lo-mm silica cells in a Beckman DU (see Figs. 2, 3). A, 15.6 PM progesterone; B, 16.7 p~ cortisol; C, 17.7 PM testosterone; D, 16.0 PM deoxycorticosterone; E, 60.3 PM androstenolone. Stray-light corrections have been applied below 236 rnp. Results for A and E in trypsin and A and B in chymotrypsin were obtained in the presence of 0.02 M Cat+.
may not be a valid measure of binding in the case of these proteins. However, these interactions are now being re-examined in the Cary, especially with reference to the influence of Ca++. The results presented for the albumins, which represent the first systematic examination by difference spectroscopy, are thus explainable in terms of environmental effects arising out of the mutual interaction of steroid and protein. The appearance of a tyrosine-like perturbation spectrum in the case of androstenolone and the suggestion of it in the case of other steroids is of interest in view of the suggested involvement of tyrosine in steroid binding (39). However, this is probably not a direct involvement. The specific amino acid resi-
of Action of Steroid Hormones” (L. Engel and C. A. Villee, eds.), p. 33. Macmillan (Pergamon), New York (1961). 8. WESTPHAL, U., AND ASHLEY, B. D., J. Biol. Chem. 237, 2763 (1962). 9. WESTPHAL, U., AND ASHLEY, B. D., J. Biol. Chem. 233, 57 (1958). 10. MONOD, J., CHANGEUX, P., AND JACOB, F. J. Mol. Biol., 6, 306 (1963). 11. TOFT, D., AND GORSKI, J., Proc. N&Z. Acad. Sci. U. S. 66, 1574 (1966). 12. SLAUNWHITE, W. R., JR., AND SANDBERG, A. A., J. Clin. Invest. 38,384 (1959). 13. CHEN, P. S., MILLS, I. H., AND BARTTER, F. C., J. Endocrinol. 23, 129 (1961). 14. WESTPHAL, U., ASHLEY, B. D., AND SELDEN, G. L., J. Am. Chem. Sot. 80, 5135 (1958). 15. WANG, S., KAWAHARA, F. S., AND TALALAY, P., J. Biol. Chem. 238, 576 (1963). 16. BRUNKHORST, W. K., AND HESS, E. L., Arch. B&hem. Biophys. 111, 54 (1965). Structure,” p. 17. SCHERAGA, H. A., “Protein 217. Academic Press, New York (1961). 38. HERSKOVITS, T. T., AND LASKOWSIU, M. J., J. Biol. Chem. 23’7, 2481 (1962). ~~.~SCOTT, J. F., in “Physical Techniques in Biological Research” (G. Oster and A. W. Pollister, eds.), Vol 1. Academic Press, New York (1955). 20. ZIMMERING, P. E., LIEBERMAN, S., AND ERLANGER, B. F., Biochemistry 6, 154 (1967).
STEROID
PROTEIN
21. SAIDEL, L. J., GOLDFARR, A. H., AND KALT, W. B., Science 113, 683 (1951). 22. WETLAUFE~R, D. B., Advances in Protein Chem. 17, 303 (1962). 23. TALALAY, P., in “Methods of Biochemical Analysis” (1~. Glick, ed.), Vol. 8. p. 119. Wiley (Interscience) New York (1960). 24. LATA, G. IT., AND ATALLAH, N. A., Federation Proc. 24., 2234 (1965). 25. MITCHELL]:, 11. A., AND APPLEX~HITE, T. H., J. Org. Chem. 27, 345 (1962). 26. ELLIXGTON, P. S., AND MEAKINS, G. D., J. Chem. Sot. 697 (1960). 27. CHAPMAN, J. H., AND PARKEA, A. C., J. Chem. Sot. 2076 (1961). 28. Scow, A. I., “Interpretation of the Ultraviolet Spectra of Natural Products,” p. 23. Macmillan (Pergamon), New York (1964). 29. BLADON, P., HENREST, H. B., AND WOOD, G. W., or. Chem. Sot. 2737 (1952). 30. JAFF~, H. II., AND ORCHIN, M., “Theory and Applicaiions of Ultraviolet Spectroscopy,” p. 192. Wiley, New York (1962). 31. EISENBERI;, 1). S., AXD EUSALL, J. T., Science 142, 50 (1963).
INTERACTION
417
32. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem. 236, PC43 (1960). 33. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem. 236, 2942 (1961). 34. YANARI, S., AND BOVEY, F. A., J. Biol. Chem. 236, 2818 (1960). 35. EDS~LL, J. T., MEHTA, S., MYERS, D. LT., AND ARMSTROXG, J. McD., Biochem. Z. 346, 9 (1966). 36. NAY, A., REYNOLDS, J. A., POLETF, H., AND STEINHARDT, 37. BIGELO\V, C.
J., Biochemistry, 6,2606 (1966). C., AND SOXENBERG, Il., Bio-
chemistry 1, 197 (1962). 38. ALFSEN, A., Compt. Rend. Trav. Lab. Carlsberg 33, 415 (1963). 39. OYAKAVA, E. K., AND LEVEDAHL, B. H., Arch. Biochem. Biophys. 74, 17 (1958). 40. D~SZA, J. P., HELLER, M., AND BERNSTEIN, S., in “Physical Properties of Steroid Hormones” (L. L. Engel, Ed.), p. 69. Macmillan, New York (1963). 41. ULRICH, W. F., Develop. Appl. Speck. 2, 131~ (1962).
OF
RIOCHEMISTRY
Examination
AND
BIOPHYSICS
407-417
126,
of Steroid
Protein
Difference
Spectrophotometry’
MICHAEL Department
(1968)
of Biochemistry, Received
Facully October
Interaction
by Ultraviolet
T. RYAN
of Medicine, 24, 1967;
accepted
University February
of Ottawa,
Ottawa,
Canada
6, 1968
The phenomenon of steroid-protein interaction has been examined in terms of ultraviolet difference spectra. In the case of interactions of serum albumins and some a&unsaturated ketosteroids two spectral effects are discernible, one attributed to the perturbation of the steroid absorption band by a hydrophobic environment at a binding site and the other to perturbation of aromatic amino acid bands. Interaction with androstenolone, which does not have the a#-unsaturated ketone group, revealsdifference troughs at approximately235,282, and 288 rnp which have been attributed to a red shift of aromatic absorption bands. Interaction of steroids with several enzyme uroteins exhibits. under the conditions used, poorly defined difference spectra which are unlike the ones for serum albumins.
The phenomenon of protein-small molecule interaction has been intensively investigated for a number of years (1). As noted by Eik-Nes et al. (2), steroids represent a particularly suitable class of small molecule for such study, being uncharged and possessedof a wide range of structural variety impressed on a fairly rigid hydrocarbon framework. This has further acquired heightened biological significance not only in terms of the nonspecific and specific transport properties of serum proteins (3-9) but as providing a conceptual model (10) with which to approach the problem of hormone action at the molecular level. Toft and Gorski (11) have claimed to have isolated a receptor protein from uterine tissue. Experimental demonstration of the reversible combination of steroids and proteins has been provided by a number of techniques, such as equilibrium dialysis (5), solubility (Z), electrophoresis (12), and ultrafiltration (1.3); and a fairly extensive list of binding parameters has been compiled (3-5) for the common plasma proteins. Westphal (6) has furthermore demonstrated that there 1 This wo:rk was supported by grant MA-1616 from the Medical Research Council of Canada. 407
is a depression of the absorbance of A4-3ketosteroids in the presence of proteins and Westphal et al. have shown that there is a correlation between the value of AA at x maxand the binding affinity of the steroid for serum albumin (14). The single wavelength parameter has been employed in an attempt to study the relationship between steroid structure and binding afhnity in the case of a large number of steroids (6-9), an approach which, as the present paper tends to show, may not always be valid. In spite of these extensive investigations and except for the special case of the steroid isomerase studied by Wang et al. (15) and the evidence of the general “polarity rule” 2 (4) it can be said that very little is known about the precise nature of the phenomenon (see ref. 1, p. 282). Moreover, the configurational adaptability which serum al2 The term “polarity” is used to imply the number of polar groups (carbonyl, hydroxyl) present in the steroid molecule. It has been demonstrated by Eik-Nes et al. (2) and by Chen et al. (13) that the binding affinity of steroids for serum albumins is inversely related to the number of such groups in the molecule. Westphal (7) refers to this phenomenon as the polarity rule.
408
RYAN
bumin apparently also displays in interaction with cortisol (16) renders the concept of binding site somewhat complex. In studying the binding phenomenon we have reexamined the spectrophotometric mebhod of Westphal (6), extending considerably the range of measurement and expressing the results as difference spectra. This approach has been exploited successfully in other protein studies (17). EXPERIMENTAL
PROCEDURE
Materials 6, 1701-Dimethyl-6-dehydro-progesterone3 (1) was a gift;4 it melted at 146-147’ (Fisher-Johns). All other st)eroids were obtained either from the Sigma Chemical Co. or Mann Research Laboratories and were recrystallized from aqueous alcohol when necessary. Their uncorrected melting points were 128-129” (progesterone), ‘211.5~213.5” (cortisol), 138-139” (deoxycorticosterone), 154155” (testosterone), 152-153” (androstenolone), lg&190” (16-dehydroprogesterone), 183-184” (androsterone). Cryst,alline BSA was a product of Armour and Co. Crystalline HSA was obtained from Pentex Corp. Two samples of chymotrypsin were used: Mann Research Laboratories (3 X crystallized, salt-free) and Worthington Biochemicals (chromatographically pure). Trypsin was a product of Mann Research Laborat,ories (2 X crystallized). Ljrsozyme was obtained from Worthington Biochemicals (2 X crystallized, saIt-free). The &hydroxysteroid dehydrogenase (3-p-hy,droxysteroid: NAD (P) oxidoreductase, EC 1.1. 1.51) was a crude extract of dried cells of P. testosteroni which were obtained from Worthington BiochemicaIs. Tris base was obtained from the Sigma Chemical Co. Distilled deionized water was used for making aqueous solutions and for washing glassware.
Procedure The experimental approach consisted of attempt to observe perturbations of ultraviolet absorbing bands present either in the steroid protein or both, which might arise as a result
an or of
3 The following abbreviations are used: I, 6,17cu-dimethyl-6-dehydro-progesterone; 16-dehydroprogesterone, pregna-4,16, -dien-3,20-dione; androstenolone, 3P-hydroxy-androst-5-en-17-one; HSA, human serum albumin; BSA, bovine serum albumin. 4 Courtesy of Dr. Sam Solomon, McGill University.
interaction. Difference spectra rather than actual perturbed spectra were studied since these present a sharper picture of such perturbations (17), especially under conditions in which total absorbance is constitut,ed of several types of overlapping bands. This represents the first systematic application of this technique to this problem. The results were obtained as two separate sets of spectra measured on a Beckman DU Spectrophotometer: (A) in Tris chloride buffer pH 8.0, r/2 0.1 and (B) in presence of proteins dissolved in this same buffer. After applying corrections to some values of set (B) (see below) difference spectra were computed by subtraction. Later results in these investigations were obtained using the tandem cell technique of Herskovits and Laskowski (18) on a Cary Model 15. Some spectral measurements have also been made on solutions of steroids in absolute redistilled ethanol. The measurements on the DU were made in rect,anguIar cells of IO-mm light path and using the photomultiplier, deuterium lamp, and thermospacers with water circulating at 25 f 1”. Cylindrical tandem cells, each with a total light path of 20 mm (Pyrocell Mfg. Co., Westwood, New Jersey) were used in the Cary. Ceils were stored in nitric-sulphuric acid (19) and washed with water, deionized water, and alcohol, a,nd dried in a vacuum oven. In using the Beckman t,he effective band width varied from 0.4 to 1.8 m/l. The possibility of error due to light scattering was eliminated by extending some measurement,s to 400 mp. When using tandem-cell technique in the Cary, the 100% line on the chart was trimmed with the cells filled with alcohol. The cells were then emptied and dried in a vacuum oven. The reference cell was filled with the mixture of protein and steroid in one chamber and Tris in the other. The sample cell was filled with steroid in Tris in one chamber and protein in Tris in the other, each at the same concentration as in the mixture in the other cell. Records were obtained at dynode settings 2-4 and on both the 1.0 and 0.1 absorbance range. Difference spectra are expressed in terms of AA; in the 280-300 rnp region in Fig. 4 values for Ae protein are also provided. Stock solutions of steroids in Tris were prepared by evaporating the appropriate volume of a 1 mM or 2 rnM solution in ethanol under a st.ream of nitrogen in an Erlenmeyer flask. The fine film so obtained was dried in vacua, dissolved with shaking in Tris, and filtered through a medium-porosity, sintered glass filter. The actual concentrations were determined in a manner similar to that of Westphal (6) from extinction values measured at lower steroid concentrations in 2% alcohol in Tris. In most instances the actual concentrations
STEROID were close to the by these dilul;ions.5 tained by mixing tions in Tris with 192 y.l~ solution of
PROTEIN
nominal concentration sought Working solutions were ob5 ml of the stock aqueous solueither 1 ml of Tris or 1 ml of a the protein in Tris.
Stray Ligh.t Since values below 240 rnp seemed of interest, an attempt was made to measure stray light arising in this range in 32 LAM protein solutions (21,22) and to apply corrections for it. The method consisted of measiuring the transmittance of the protein solution in a Corex (Pyrex) cell, using the protein solution in a silica cell as 100% T. Since Corex cuts off at about 260 rnp such values should represent stray light originating above this wavelength and hence correspond to the source of error in protein solutions. Values for the stray light component (Ts) so measured for the proteins under investigation were of the order of 357, at 235 rnp for 32 ~GM albumin and were used in applying corrections to the observed 2’7, (Tabs) using the assumption that in the expression of Saidel et al. (21) I,, := I,. Under these conditions Tcorrect = T abs - T,/lOO - T,, providing that the stray light component does not change on interaction of steroid and1 protein. The determination of the concentration of androstenolone in stock solution in Tris was performed using the method of Talalay (23). Only the nominal concentration of androsterone is shown in Fig. 3 since the crude a-hydroxysteroid dehydrogenase (3.a-hydroxysteroid: NAD (P) oxidoreductase, EC 1.1.1.50) proved inactive in Tris buffer. RESULTS
Difference spectra, which were obtained using the single beam instrument for the interaction of four A4-3-ketosteroids and androstenolone with 32 j.&M BSA, are presented in Fig. 2 and are characterized by a difference peak at 259 rnp for the a,@ unsaturated steroids and a difference trough 5 Results rleported here are based on measurements using steroid stock solutions in Tris buffer stored at room temperature in stoppered flasks and which were used usually within 7-10 days. Checking the absorbance at A,,,,, for the steroid revealed virtlually no change for periods up to 6 weeks, unlike the results reported by Zimmering et al. (20) for phosphate buffer. In one instance a solution of testosterone in Tris buffer which had been stored for 5 months exhibited a 1670 increase in extinction value.
INTERACTION
409
at about 235 rnp for androstenolone. Spectra for the interaction both of these same steroids and also androsterone, I and 16dehydro-progesterone with 32 ~ccy HSA are presented in Fig. 3 where it can be seen that the spectral picture is similar to that for BSA. Figure 3 also contains values for the interaction of progesterone and HSA at wavelengths below 240 rnp which were obtained with t’he aid of the stray-light corrections described above and which are represented by a dotted line. Similar results have been obtained below 240 rnp for the interaction of other A4-3-ketosteroids not only with HSA but also with BSA. However, for reasons given below, they have been excluded from these figures. The validity of the results represented by solid lines in Figs. 2 and 3 is confirmed by the results of a study of the interaction of progesterone and androstenolone with 32 pc~1l1 HSA using tandem cell technique in t#he Cary (Fig. 4), although the trough for androstenolone is shifted slightly. This figure also provides additional detail in the 28&300 rnp region. The artifactitious nature of the peak in the 235 rnp region represented by dotted lines and registered both by the single-beam (Fig. 3) and double-beam instrument (Fig. 4) is revealed by study of the interaction at lower protein concentrations. In the case of progesterone, valid difference measurements in the 23&240 mp region can only be obtained at HSA concentrations of 8 PM or lower. The artifact peak has been observed in the case of other A4-3-ketosteroids but not in the case of androstenolone where the shape of the curve for androstenolone-HSA interaction is only slightly dependent on protein concentration. The artifact may possibly be attributed to fluorescence quenching at protein concentrations and in a wavelength range where a significant portion of the transmitted beam consists of fluorescent energy originating in the protein. Measurements referred to earlier suggest that this may be of the order of 35% at 235 rnp in 32 PM HSA. Fluorescence quenching has been shown to occur in the binding of 19-nor-testosterone to the steroid isomerase (15) and has been reported briefly in the case of serum albumins (24).
410
RYAN
The location of the androstenolone absorption band in ethanol, water, and Tris, as judged by absorbance measurements at the long wavelength edge on dilutions prepared from a common stock solution in alcohol, is reported in Table I. The absorption characteristics of several CX,~ unsaturated ketosteroids in ethanol and Tris are presented in Table II together with the characteristics of the difference spectra which have been graphically computed from these. A plotted spectrum and difference spectrum for the case of cortisol in ethanol and Tris is given in Fig. 1 and illustrates the
spectral effect that might be anticipated should an equivalent change in chromophore environment arise in the binding of a steroid to a protein. Difference spectra for the interaction of some steroids with trypsin, chymotrypsin, and lysozyme are presented in Fig. 5 where it can be seen that well-defined features, such as are seen in the case of serum albumins, are not evident. Table III contains a comparison of the percentage depression values at 259 rnp and 249 rnp with those of Westphal (6, 9) for the interaction of a steroid series of increasing polarity with several proteins.
TABLE
I
EXTINCTIONVALUESFORANDROSTENOLONEINETHANOL,WATER, 0.1~ NACL,ANDTRIS~HS.O,~/~O.~, MEASURED IN 10 mm SILICA CELLS.~ORHING SOLUTIONS WERE PREPARED FROM A COMMON STOCK SOLUTION IN ALCOHOL AND MADE TO A CONCENTRATION OF 40 MM (mr)
Ethall01
water
NaCl
(md
Ethanol
water
NaCl
Tris
210 211 212 213 214 215 216 217 218
1,850 1,450 1,175 875 675 500 350 250 175
1,900 1,650 1,425 1,175 1,025 875 700 600 475
2,063 1,788 1,550 1,313 1,150 975 813 700 575
219 220 221 222 223 224
150 125
425 350 275 200 150 125
465 400 315 265 215 175
450 400 338 300 238 200
TABLE
II
SPECTRAL VALUES FOR SOLUTIONS OF STEROIDS IN TRIS AND IN ETHANOL bnax b/d
10-a6
10-3(Af)msx
Progesterone Alcohol= Trisc Deoxycorticosterone AlcohoP Trisc Cortisol Alcohol,= Trisc Testosterone Alcohol5 Tris I Alcohol Tris
240 248
16.4 16.0
-7.3
(260 rnp)b
+4.9
(235 rnw)
240 249
16.0 16.0
-8.2
(260 mp)
$3.8
(233 mB)
241 248
15.1 15.8
-5.5
(260 mp)
+2.3
(233 mp)
240 248
18.9 15.7
-6.2
(260 rnk)
+7.3
(234 rnp)
289 297-299
23.4
a Reported values of A,,, and emsI may be found in Dusza et al. (40) and also Ulrich (41). b Values in parentheses refer to the wavelength of (Ae)msx. c Values for X,,, and smaL in Tris are in good agreement with those reported by Westphal phosphate buffer.
(6, 9) for
STEROID
PROTEIN
INTERACTION
411
make only minor contribution here. The extinction values for androstenolone in ethanol, Tris and in water to be found in 17. 16. -8 Table I reveal that there is an apparent red (....., shift of the band in aqueous solution as com-7 --6 pared to ethanol, demonstrable by the fact that not only are the extinction values at a given wavelength higher in water but the tail of the band clearly extends into longer wavelengths. This is in keeping with the known red shift of double bond bands of other steroids in going from hexane to ethanol (2527). It will be noted that the extinction values above 220 m~.1 in water are very low and it is difficult to conceive of any mechanism which could move this band any further to the red so as to thus account 2Ifor the difference spectra for androstenolone in protein solution (Figs. 2, 3, 4). The posi220 230 240 250 260 270 200 tion of the main absorption band of the A4-3WAVELENGTH (mp) ketosteroids is also solvent-dependent (28), FIG. 1. Spectra and difference spectrum of the red shift associated with increasing cortisol in ethanol and Tris pH 8.0, I’/2 0.1. A, solvent polarity being due to increased staethanol. B, Tris. C, difference spectrum. Measurebilization of the dipolar excited state (30).6 ments made in l-cm silica cells in a Beckman DU Such a lateral shift can conveniently be wit,h photomultiplier. Difference spectrum obtained by subtraction of values for B from values represented by a difference spectrum such for A. as that in Fig. 1 for the caseof cortisol in the solvents Tris and ethanol. This curve exDISCUSSION hibits a difference peak and trough whose Steroid spectra and solvent efects. The locations are fairly constant for the four ultraviolet absorbance of monounsaturated steroids progesterone, cortisol, deoxycortisteroids and saturated steroid ketones has costerone, and testosterone (Table II) with been studied in recent years by means of respect to the same solvent pair, i.e., at 260 high performance instruments (25-27) and rnp and 233-235 rnp respectively. Spectral perturbations in protein solutions. despite the variation in the reported values Spectral values obtained from steroid soluof ~~~~~~ and. emaxfor such steroids (27) the dependence of these values on the location tions in the presence and absence of proand degree of substitution of the isolated teins may also be expressed as difference double bond seems well established (28). spectra. In the case of bovine and human While the rspectrum of androstenolone has serum albumin for example (Figs. 2, 3) not apparently been examined in modern distinctive difference spectra are seen in the instruments it would be expected to have a case of the A4-3-ketosteroids where the peak at 191-195 rnp, similar to that of height of the peak seemsto be related to the cholesterol (27). Bladon et al. (29) have polarity of the steroids. The difference shown that the long wavelength edge of this spectra as seen with the aid of the tandem band (>205 rnp) is accessible to measure- cells and the Cary show additional detail ment with a quartz spectrophotometer. in the 28&295 ~QJregion, as exemplified by difference spectra for progesterone and This spectrum is complicated by contributions from the carbonyl absorption but 6 In this, the red shift due to increase in dit,hese bands are deeper in the ultraviolet electric constant apparently dominates a blue (25) and of much lower intensity (25-27) shift to be expected from the decrease in refracthan the double bond band and should tive index. 19. 18.
412
RYAN TABLE IIEPRESSION
OF
MOLAR
A~RSoRPTI~~ITY EXPRESSED
Steroid
Bovine Concn
(/AM) 25')
Progesterone Deoxycorticosterone Testosterone Cortisol
15.1
serum
m/l
III OF
STERoiDS
IX
PRESENCE
OF
albumin 249
mp
16.1
25.6 21.2
13.0 10.0
10.5 16.9
27.6 0.5
15.2 0.7
(2.2 mg/ml) 249
mg
Human Concn
serum
albumin
(2.1 m&ml)
249 Inp
249 mp*
(jA.4 259 mp
16.5 16.0
32.3 23.2
12.2 11.2
13.0(6)
9.8(G)
4.0(6)
17.5 16.6
25.3 3.1
12.6 1.9
13.2(9) 4.5(6)
11.8(6)
Trypsin (1.15 mg/ml)
Progesteronec Deoxycorticosterone Testosterone Cortisolc
PROTEINS,
AS PERCEXTAGE~
12.5(6)
Chymotrypsin (0.7 mg/ml)
15.6 16.0
5.3 +1.5
4.0 +12
5.1(9) 4.9(9)
15.5 16.0
o.oc 0.0
1.7 1.5
10.8(9) 12.7(9)
17.7 16.7
f1.9 5.5
0.7 4.0
5.2(9)
17.7 16.4
2.3c 2.oc
2.1 0.4
12.9(9)
Lysozyme (0.16 n&ml)
Progesterone Deoxycorticosterone Testosterone Cortisol
15.5 16.0
1.0 +2.9
1.6 +0.4
11.6(g) 12.8(9)
17.7 16.7
t-4.3 2.5
+2.9 0.4
15.5(9)
4 Positive sign indicates apparent increases in the absorptivity. * Values of Westphal and co-workers for higher protein concentrations references given in parentheses. c Solutions contained Ca++ at a concentration of of 0.02 M.
androstenolone in Fig. 4. Westphal et al. (14) have noted an inverse relationship between AAz49 and the polarity of the steroid but it is clear that the complete difference spectrum presents a more meaningful picture. The difference peaks may arise out of perturbation of either the protein or steroid chromophores. It must be noted however that the difference peak at 259 rnp (Figs. 2, 3) does not conform either in shape or position to those perturbations of tyrosine and tryptophan (twin peaks in the 270-295 m,u range) most frequently seen for proteins under a variety of perturbing conditions (3134). Perturbation of the phenylalanine absorption spectrum may yield a diff erence peak in the 250-270 mp region (33) where the fine structureof theunperturbed bandis, however, carried over into the difference spectrum. Because of this and the very much lower contribution of phenylalanine to protein absorption in this region it is unlikely that this amino
in phosphate
pH
7.6 in the
acid can be implicated in bhe difference peak observed. The possibility of the aromatic amino acid chromophores contributing to this peak is more convincingly eliminated by means of a study of the diflerence spectrum produced by I, in which the electron transfer band of the steroid is shifted to the other side of that due to the aromatic amino acid absorption. This was the only such steroid available to us and it has a very much lower solubility in aqueous solution than desirable. In the difference spectrum for the interaction of I with 32 I.IM HSA (Fig. 3) the peak at 314-316 rnN can only be attributed to perturbation of t’he steroid absorption band at 297-299 rnp. It will be noted that in addition a broad trough appears at 280 rnp which, by analogy with Fig. 1, would be expected from a lateral shift of the absorption band toward shorter wavelengths. However, this may be com-
STEROID
PROTEIN
plicated by contributions from aromatic chromophores in this region (see below). From the foregoing observations and the demonstrated effect of solvents on steroid spectra it is postulated, as has indeed been suggested by Wang et al. (15), that the difference peak at 259 rnp for interaction of A4-3-ketosteroids with serum albumin and at 314-316 rnp for I may be attributed to the lowering of the effective dielectric constant associated with the enveloping of the steroid chromophore in a hydrophobic region of t,he protein molecule. It will be noted that the location of the difference peak is close to that for the transfer of the steroids from Tris to ethanol (Fig. 1 and Table 11)-a situation which should approximate the postulated binding process. It may be questioned that the spectrum of cortisol, the most polar steroid, is in fact perturbed in view of the very low difference values and the absence of an actual peak at 259 rnp. Preliminary examination of the cortisol-albumin interaction in the Cary shows no acbual difference values in this region whereas the corticosterone-albumin interaction exhibits an actual difference peak with a value of -0.010 for AA&. This may be taken as evidence that cortisol is bound at a site in the molecule quite different from that (or those) for the other steroids and is in keeping with the published results of competition studies (3). A simple lateral shift in the absorption band should also however produce a trough which, by analogy with Fig. 1, would be located at approximately 233-235 rnp. Examination of the progesterone-albumin interaction at a concentration of 8.0 pM HSA shows a cross-over point at 242 rnp with increasingly positive values down to 230 rnp but without the appearance of an actual trough (Fig. 4, curve C). This failure of the anticipated trough to appear suggests t’hat an additional spectral effect may be operative and this finds appreciable support in closer examination of the results in the 280-,300 rnk region for progesterone (Fig. 4) and in both the 235 rnp and 28CL300 rnp regions for androstenolone (Figs. 2, 3, 4). In the case of androstenolone positive dif7 Unpublished
results.
413
INTERACTION
WAVELENGTH
(mp)
FIG. 2. Perturbation of ultraviolet spectra arising out of the interaction of steroids with BSA at a concentration of 32 PM (2.2 mg/ml) in Tris PI-I 8.0, I’@ 0.1. Expressed as difference spectra from measurements made in IO-mm silica cells in a Beckman DU with photomultiplier, at 25 Z!Z 1”. Stray-light corrections have been applied below 240 rnp for E (see Experimental). A, 15.1 PM progesterone. B, 16.9 PM cortisol. C, 16.5 PM testosterone. I), lG.l PM deoxycorticosterone. E, 60.0 PM androstenolone. Algebraic sign is relative to the unperturbed state of steroid and protein in Tris.
ference values appear over the entire spectral range, rising to increasingly more positive values below 240 rnp in the form of a broad trough which, though centered at approximately 235 rnp, clearly extends beyond the range of measurement. Such a trough is also exhibited in the interaction of androsterone (Fig. 3) with albumin. Since androstenolone exhibits virtually no absorption in Tris above 225 rnp (Table I) the spectral effect in this case, and presumably also that of androsterone, can only be attributed to the protein. Examination of the androstenolone-albumin interaction by means of tandem-cell technique in the extended absorbance range of the Cary reveals that there are also smaller troughs located at 282 and 288 rnp (Fig. 4). Such spectral values for the interaction of
414
RYAN
235 rnp region which are approximat,ely 5 times those at 282 rnp and 288 rnp. The direction of the spectral shift is, however, toward the red, unlike not only the denaturation shift but also the blue shift which occurs in the binding of such substances as octanol and dodecyl sulphate to albumin (36). In this connection Ray et al. (36) have quest’ioned that the most usual explanation for the blue shift-the exposure of hidden aromatic residues to the aqueous solventis necessarily correct since they could not, using solvent-perturbation technique, demonstrate any such unmasking. It is possible that the red shift produced by the steroid alcohol could be accounted for by a small local conformational change affecting the charge environment of a particular tyrosine residue as has been suggested by Bigelow and Sonenberg (37) for the blue shift in the dodecyl sulphat,e-albumin interaction. In this context it is of interest that Alfsen (38) has observed changes in optical activity on interaction of testosterone with serum albumin which she has attributed to an in.08 crease in helical content of the protein. In 235 240 245 250 255 260 265 270 275 280 285 the study of the interaction of progesterone WAVELENGTH (my) with 32 PM HSA by means of tandem-cell technique (Fig. 4) it will be noted that there FIG. 3. Perturbation of ultraviolet spectra is a small but clearly discernible trough arising out of the interaction of steroids with with two minima located at approximately HSA at a concentration of 32 SM (2.1 mg/ml) in 286 rnp and 292 rnp. If these, too, can be Tris pH 8.0, p/2 0.1. Expressed as difference spectra from measurements made in lo-mm silica attributed to aromatic chromophore perturcells in a Beckman DU with photomultiplier, at bation then a contribution of approximately 25 f 1”. Stray-light corrections have been applied 5-7 times these values would be anticipated below 240 mp for A, F, and E (see Eqnerimental). in the 235 rnp region. The latter, when A, 16.5 PM progesterone; B, 16.6 PM cortisol; C, superimposed on a steroid perturbation 17.5 PM testosterone; D, 16.0 PM deoxycorticosspectrum, might account for the shape of terone; E, 60.3 JAM androstenolone; F, 66.5 PM curve C, Fig. 4, between 230 rnp and 240 rnp. androsterone; G, 13.4 PM 16-dehydroprogesterone; The fact that interaction of the A4-3H, 5.5 PM I; J, curve for E uncorrected. ketosteroids with serum albumins produces a spectral difference peak at 259 rnp suggests androstenolone with HSA seem to correthat difference values measured at t,he peak spond to aromatic chromophore perturbawavelength represent a more sensitive and tion analogous to what has been suggested (31) as a partial explanation for the denatu- more accurate measure of Ae for the steroid than those at 249 rm.~.In Table III there is ration blue shift seen at 235 rnp in the case presented a comparison of our percentage of several proteins (32-34). Edsall et al. (35) depression values at 259 rnp and 249 rnp wit,h have noted in the study of denaturation those of Westphal (6-9) at 249 rnp for not of carbonic anhydrases that the values of Ae2s6were 5-7 times those at 282 rnp and only the serum albumins but also several en291 rnw. The interaction of androstenolone zyme proteins. Here it can be seen that the with HSA produces difference values in the values at 249 rnp agree very well with those
STEROID
PROTEIY
INTERACTIOY
41ii
OA p. : I
O.lO0.09 ooe-
j : j
: ‘i :
0070.06
WAVELENGTH
(mp)
FIG. 4. Perturb&ion of ultraviolet spectra arising out of 1he interaction of 14.1 PM progesterone and 60.3 pM androstenolone with HSA in Tris pH 8.0, r/2 0.1. (A) progesterone in 32 PM HSA (2.1 mg/ml) ; (B) progesterone in 16 PM HSA; (C) progesterone in 8 PM HSA; (D) androstenolone in 9.7 pM HSA; (E) androstenolone in 32 PM HSA. Expressed as difference spectra, and measured by means of tandem-cell technique in a Cary 15.
of WestphaJ in the case of interaction of progesterone, deoxycorticosterone, and testosterone with albumins and that the values at 259 rnp are approximately twice those at 249 rnp. However, there is significant discrepancy in the values for cortisol where ouI;‘s are consideribly lower than those of Westphal. This discrepancy may possibly be attributed to the different buffer conditions in our experiments. The difference spectra given in Fig. 5 for the enzyme proteins present a contrast with the results for the serum albumins in that distinctive absorption band perturbations are not evident. Furthermore an examination of T&le III with respect to these proteins reveats further discrepancies between our results and those of Westphal and coworkers (6, 9), especially in the case of chymotrypsin and lysozyme. This may be partly due’ to the fact that we used much lower concentrations of these proteins, which have much higher absorptivity in the
ultraviolet than the serum albumins.* Furthermore, with the exception of lysozyme, most of our determinations were made on these proteins in the presence of Ca++. Thus one does not see, even in the case of lysozyme, the correlation claimed by Westphal and Ashley (7-9) between At249and the polarity of the steroid or evidence for the operation of an inverse polarity rules in the case of these enzyme proteins as suggested by these authors. This, together with the fact that there is no characteristic spectral structure in the plot of difference values against wavelength, suggests that AEZ~S * For example, at 0.2 mg/ml; A278 albumin = 0.125, A 281lysozyme = 0.522, A 282chymotrypsin = 0.372, A2T8 trypsin = 0.282. At the concentration of 1.72 mg/ml used by these authors for lysozyme Azn3 = 2.25. Stray light may have introduced some error into the values observed by them. 9 That is to say that, unlike interactions with serllm albumins, the binding affinity would increase with increasing polarity of the steroid.
416
RYAN
an
dues which may be directly implicated in the binding process are as yet unknown although the present results suggest that there may be hydrophobic residues near the binding site for some steroids in the serum albumins.
LYSOZYME 003
-
REFERENCES 1.
STEINHARDT, J., AND BEYCHOK, S., in “The Proteins” edition.
6.
(H. Neurath, ed.), Vol. 2, 2nd Academic Press, New York (1964). EIK-NES, K., SCHELLMAN, J. A., LUMRY, R., AND SAMUELS, L., J. Biol. Chem. 206, 411 (1953). SANDBERG, A. A., SLAUNWHITE, W. R., JR., AND ANTONIADES, H. A., Recent Progr. in Hormone Res., 13, 209 (1957). DAUGHADAY, W. H., Physiol. Rev. 39, 885 (1959). SLAUNWHITE, W. R., JR., ROSENTHAL, H., AND SANDBERG, A. A., Arch. Biochem. Biophys. 100, 486 (1963). WESTPHAL, U., Arch. Biochem. Biophys. 66, 71
7.
(1957). WESTPHAL, U., in “Mechanism
2.
003-
I
TRYPSIN
I
3.
002 ..
0.0, i.r 0.01
..-.-. J
- ._,_._._._._._,_.
*.-.-.-.-.-.-‘-’
4. 5.
WAVELENGTH
(mp)
FIG. 5. Perturbation of ultraviolet spectra arising out of the interaction of steroids with 32 PM chymotrypsin (0.7 mg/ml), 48 @M trypsin (1.15 mg/ml), and 32 PM lysozyme (0.46 mg/ml) in Tris pH 8.0, I’@ 0.1. Expressed as difference spectra from measurements in lo-mm silica cells in a Beckman DU (see Figs. 2, 3). A, 15.6 PM progesterone; B, 16.7 p~ cortisol; C, 17.7 PM testosterone; D, 16.0 PM deoxycorticosterone; E, 60.3 PM androstenolone. Stray-light corrections have been applied below 236 rnp. Results for A and E in trypsin and A and B in chymotrypsin were obtained in the presence of 0.02 M Cat+.
may not be a valid measure of binding in the case of these proteins. However, these interactions are now being re-examined in the Cary, especially with reference to the influence of Ca++. The results presented for the albumins, which represent the first systematic examination by difference spectroscopy, are thus explainable in terms of environmental effects arising out of the mutual interaction of steroid and protein. The appearance of a tyrosine-like perturbation spectrum in the case of androstenolone and the suggestion of it in the case of other steroids is of interest in view of the suggested involvement of tyrosine in steroid binding (39). However, this is probably not a direct involvement. The specific amino acid resi-
of Action of Steroid Hormones” (L. Engel and C. A. Villee, eds.), p. 33. Macmillan (Pergamon), New York (1961). 8. WESTPHAL, U., AND ASHLEY, B. D., J. Biol. Chem. 237, 2763 (1962). 9. WESTPHAL, U., AND ASHLEY, B. D., J. Biol. Chem. 233, 57 (1958). 10. MONOD, J., CHANGEUX, P., AND JACOB, F. J. Mol. Biol., 6, 306 (1963). 11. TOFT, D., AND GORSKI, J., Proc. N&Z. Acad. Sci. U. S. 66, 1574 (1966). 12. SLAUNWHITE, W. R., JR., AND SANDBERG, A. A., J. Clin. Invest. 38,384 (1959). 13. CHEN, P. S., MILLS, I. H., AND BARTTER, F. C., J. Endocrinol. 23, 129 (1961). 14. WESTPHAL, U., ASHLEY, B. D., AND SELDEN, G. L., J. Am. Chem. Sot. 80, 5135 (1958). 15. WANG, S., KAWAHARA, F. S., AND TALALAY, P., J. Biol. Chem. 238, 576 (1963). 16. BRUNKHORST, W. K., AND HESS, E. L., Arch. B&hem. Biophys. 111, 54 (1965). Structure,” p. 17. SCHERAGA, H. A., “Protein 217. Academic Press, New York (1961). 38. HERSKOVITS, T. T., AND LASKOWSIU, M. J., J. Biol. Chem. 23’7, 2481 (1962). ~~.~SCOTT, J. F., in “Physical Techniques in Biological Research” (G. Oster and A. W. Pollister, eds.), Vol 1. Academic Press, New York (1955). 20. ZIMMERING, P. E., LIEBERMAN, S., AND ERLANGER, B. F., Biochemistry 6, 154 (1967).
STEROID
PROTEIN
21. SAIDEL, L. J., GOLDFARR, A. H., AND KALT, W. B., Science 113, 683 (1951). 22. WETLAUFE~R, D. B., Advances in Protein Chem. 17, 303 (1962). 23. TALALAY, P., in “Methods of Biochemical Analysis” (1~. Glick, ed.), Vol. 8. p. 119. Wiley (Interscience) New York (1960). 24. LATA, G. IT., AND ATALLAH, N. A., Federation Proc. 24., 2234 (1965). 25. MITCHELL]:, 11. A., AND APPLEX~HITE, T. H., J. Org. Chem. 27, 345 (1962). 26. ELLIXGTON, P. S., AND MEAKINS, G. D., J. Chem. Sot. 697 (1960). 27. CHAPMAN, J. H., AND PARKEA, A. C., J. Chem. Sot. 2076 (1961). 28. Scow, A. I., “Interpretation of the Ultraviolet Spectra of Natural Products,” p. 23. Macmillan (Pergamon), New York (1964). 29. BLADON, P., HENREST, H. B., AND WOOD, G. W., or. Chem. Sot. 2737 (1952). 30. JAFF~, H. II., AND ORCHIN, M., “Theory and Applicaiions of Ultraviolet Spectroscopy,” p. 192. Wiley, New York (1962). 31. EISENBERI;, 1). S., AXD EUSALL, J. T., Science 142, 50 (1963).
INTERACTION
417
32. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem. 236, PC43 (1960). 33. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem. 236, 2942 (1961). 34. YANARI, S., AND BOVEY, F. A., J. Biol. Chem. 236, 2818 (1960). 35. EDS~LL, J. T., MEHTA, S., MYERS, D. LT., AND ARMSTROXG, J. McD., Biochem. Z. 346, 9 (1966). 36. NAY, A., REYNOLDS, J. A., POLETF, H., AND STEINHARDT, 37. BIGELO\V, C.
J., Biochemistry, 6,2606 (1966). C., AND SOXENBERG, Il., Bio-
chemistry 1, 197 (1962). 38. ALFSEN, A., Compt. Rend. Trav. Lab. Carlsberg 33, 415 (1963). 39. OYAKAVA, E. K., AND LEVEDAHL, B. H., Arch. Biochem. Biophys. 74, 17 (1958). 40. D~SZA, J. P., HELLER, M., AND BERNSTEIN, S., in “Physical Properties of Steroid Hormones” (L. L. Engel, Ed.), p. 69. Macmillan, New York (1963). 41. ULRICH, W. F., Develop. Appl. Speck. 2, 131~ (1962).