Polarization of Steroid Fluorescence HERBERT S. STRICKLER and ROBERT C. GRAUER Department of Research in Endocrinology and Metabolism, The William H. Singer Memorial Research Institute o f the Allegheny General Hospital, Pittsburgh, Pa.
The natural polarizations of 17pestradiol, 17/3,17cu-ethynvIestradiol, estriol, and equilenin w3:e negligible in alcohol, but substantial polarizations were noted in glycerolalcohol. Acid sensitization (Linford method) of 5/3-pregnane-3a,20a-diol gave nonlinear curves of reciprocal polarizations vs.absolute temperature/viscosity, Similar curves were obtained on diluting estrone, treated with acid (Slaunwhite-Engel method), with 3 volumes or more of methanolic H2S04; dilutions less than 3 volumes gave linear curves obeying the Perrin equation for a single oscillator. The interpretation is made that more than one oscillator is present on further dilution. Spectrophotometric data to support this were presented. Dehydroepiandrosterone (Allen reaction) fluoresced with a substantial polarization. Application to Kober-lttrich reaction mixtures offers promise in estimating the estrogen content of crude preparations by noting the ratio of readings with crossed polarizers obtained at two wavelengths of excitation and by utilizing the difference in polarization observed.
A
STUDIES of fluorescence polarization have been made on dyestuffs and more recently on proteins, there has been to our knowledgoe no previous work on the polarization of the fluorescence of steroids. Therefore, research was initiated in this laboratory to ascertain to what extent such measurements would aid in the improvement of methodology for the examination of biological extracts containing steroids. Experiments were first carried out to note the effects of conventional variables (viscosity, temperature, wavelength) on the polarization of fluorescence of pure steroids, and it was found that this class of compounds can be studied conveniently this may. Xlso, examination of solutions of acid-treated estrone containing various amounts of sulfuric acid gave polarization data which were studied for indications of the presence of more than one type of oscillator. The information which we obtained by our study of estrone solutions b y fluorescence polarization helped to further elucidate a fall off of fluorescing
LTHOUGH
1228
ANALYTICAL CHEMISTRY
power on dilution, which had been previously reported from this laboratory (5, 6). Data on acid-treated pregnanediol solutions were also examined. Crude urinary extracts were also examined, after acid treatments, for polarization of fluorescence. EXPERIMENTAL
The Aminco-Bowman spectrophotofluorometer was equipped with Glan or Glan-Thompson prisms, placed a t the entrance of the excitation light to the cuvette and a t the exit of the emitted light from the cuvette. The cuvette holder permitted control of temperature by circulating liquid from an auxiliary bath and pump (EC Apparatus Co.). Most of the work was done with a stable xenon lamp and potted photomultiplier (11’21) tube. Quartz cuvettes, 1 sq. em. in cross section, were used. Calibrations were performed using a mercury arc source, and by using the xenon arc source employing aqueous shellfish glycogen in the cuvette to scatter light. A typical arrangement of slits was: N o . 1, 2, 5 , 6-all inch, No. 7: inch. Readings were made either with a iihotomultiidier microiihotometer or an X-Y recorder (E-I C o . ) . Four sets of readings were taken, corresponding to the four sets of positions (13-1%, 13-E, E-E, E-13) of the prisms. The designation (E) on the prism indicates that the polarizer is oriented to pass only light polarized with the electrical vector perpendicular to the plane formed by the excitation or emission beam. The designation (13) on the prism indicates that the polarizer is oriented to pass only light polarized with the electrical vect,or in the plane formed by the excitation or emission beam. Polarizations ( p ) \yere calculated according to Azumi and XcGIynn ( 1 ) :
=
EE - EB (BE/BB) EE E13 (BE/I313)
+
Usable increase of sensitivity was obtained with the recorder down to 2.5 mv./inch instead of 10 mv./inch as specified by Xminco; this was necessary for some crude urine preparations. The shutter to the cuvette well was closed except during measurement’s to avoid undue exposure of the sample to radiation. Blanks were always run and their values subtracted from the
readings on the steroid preparations. All wavelengths were corrected. For polarization LIS. T / a experiments, the temperature ( T ) of the cuvette holder mas varied. The viscosities were measured by the falling ball technique in a Hoepler viscosimeter whose constant t,emperature jacket’ was connected to the same bath and pump as the cuvette holder. For these measurements, about 40 ml. of a given reaction mixture was prepared, exactly as was done on a smaller scale when treating the steroids with acid prior to fluorescence polarization determination. The natural fluorescence of steroids was examined where possible, but acid sensitization of the steroids to fluorescence was used in most experiments. Usually, the steroid concentrations were about 10-5 molar. For acid treatment,, sulfuric acid and heat were employed in various ways as indicated under the individual experiments, depending on the type of steroid. AIethanolic HzS04was used to dilute Slaunmhite-Engel reaction mixtures. The methanolic H2SO4 was made up from 250 ml. C.P. methanol, chilled in dry-ice-alcohol, plus 231 ml. cone. C.P. H2S04 (Ilul’ont). It was kept in the refrigerator as long as one year without appreciable deterioration. Crude urinary extracts were prepared as follows. T o a few ml. of urine were added 15 volume yoof cone. hydrochloric acid. The mixture was placed in the boiling water bath for 1 hour. On cooling, the material was extracted twice with 2 volumes of ether. Ittrich extracts (3) were treated with anhydrous sodium sulfate, filtered through borosilicate glass woo1, and centrifuged liefore pipetting into the cuvette. RESULTS AND DISCUSSION
Polarization of Natural Fluorescence of Estrogens. lip-Estradiol in absolute alcohol (100 pg./ml.) gave zero polarization with Glan prisms a t 328 m,uj using 285-mp excitation light. However, in glycerol : absolute alcohol 19:1 (v,/v.), the polarization (at 50 pg./ml.) was 0.21, a substantial value. 17p,li~-Ethynylestradiol, estriol, and equilenin also gave a very small polarization (e.g,, 0.02) in alcohol but values comparable to those with Up-estradiol were obtained in glycero1:alcohol. This indicates that steroids with molecular weight of the order of 300, like other small molecular species, will not show
Table I. Fluorescence Polarizations and Viscosities a t Various Temperatures for Dilutions of Acid Treated Estrone with Methanolic H2S04 (Densities ( d ) a t 25' C.) 7,
centi-
Dilution* 1: 1 d = 1.575
p poises t o C. 0,229 21.0 25.0 0 166 14.2 38.1 0.128 9.4 51.7 1:2 0,230 28.3 15.6 d = 1.497 0.215 21.8 22.8 0.207 20.0 25.0 15.6 32.0 0 176 11.7 40.9 0.152 51.8 0 138 8.7 1:3 0,183 18.4 24.5 d = 1.451 0.199 11.8 37.9 0.171 8.7 49.9 1:5 0.195 15.8 24.8 d = 1.417 0.168 10.7 38.9 0.174 7.8 51.4 a Dilution reagent density = 1.372 grams/ml.; viscosity, 13.0 cp. Glan prisms were used.
-,l Figure 1. Nonlinear curve obtained on plotting reciprocal polarization ( I / p ) vs. T / ? (absolute temp. T over viscosity in centipoises) for data on 5P-pregnane-3cu, 20cu-diol treated by Linford method Excitation wavelength, 472 mp; fluorescence, 500 m,u; Glan prisms used
fluorescence polarizations in low viscosity media but do show marked polarization in viscous media. Treatment with Acid to Induce Fluorescence of 5p-Pregnane-3a,2Oadiol was carried out b y t h e method of Linford (4). This involves heating at 57°C. with concentrated H2S04 followed by dilution with an equal volume of ethanol. Figure 1 shows the nonlinear curve obtained on plotting reciprocal polarizations, measured a t 472-mp excitation and 500 mpfluorescence navelengths, us. T / q (absolute temperature over viscosity in centipoises) . The fluorescence of estrone was obtained by heating to 100°C. with 90 wt. % H2SO4in the presence of a small amount of toluene-alcohol (19: 1) according to Slaunwhite-Engel ( 2 ) . A seiies of such preparations, each containing 2 pg./ml. of estrone, were diluted with various amounts of methanolic H,S04. LTseof this diluent was initiated in this laboratory because readings were to be made over a range of temperatures, and the customary diluent, aqueous sulfuiic acid, gives turbid solutions with the Slaunwhitc-Engel reaction mixture except a t elevated temperatures. Densities and viscoGties of these solutions (after dilution) are given in Table I. The viscosities ( q ) adhere closely to the simple equation loglotir = C D I T , where C and D are constants and T i s the absolute temperature. Figure 2 s h o w the linear curve obtained on diluting the SlaunwhiteEngel reaction mixture with two volumes of the methanolic H2S04. GlanThompson prisms were used in this ey2eriment. The procedure was repeated with Glan prisms and dilutions with one volume, 2 volumes (as for Figure 2), 3 volumes, and 5 volumes,
+
Linear curves were obtained for the 1:1 and 1 : 2 dilutions. The data are given in Table I. Note that the polarizations ( p ) for these two dilutions decrease continuously with temperature. This is not noted for the 1:3 and 1:5 dilutions and nonlinear curves like Figure 1 are obtained on plotting l / p US. T/q. A linear plot of l / p us. T/q is presumptive evidence that the classical equation of Perrin: l/P
-
'/3
-
(1
l/3)
rJ
+ tRT/tlV
(1)
is obeyed. Here, po is the value of the polarization as T / T + 0-Le., where there is no longer depolarization by molecular rotation, t the life of the ex6.0I
I
1 . / 1
i5
I
20
1
- - l /
=
WPO
cited state, R the gas constant, I' the volume, and T the absolute temperature. Weber (8) has considered the case of more than one molecular species or one molecular species with more than one oscillator present. The observed polarization is given as the geometric mean p by the relation
25
I
-r
33
I 35
I 43
I
#S
53
?.
Figure 2. Linear curve obtained on plotting reciprocal polarizations ( I / p ) vs. T / ? (absolute temp. T over viscosity in centipoises) for data on estrone treated b y Slaunwhite-Engel method and diluted with 2 vols. of methanolic HzS04 Excitation wavelength, 435 mp; fluorescence, 490 mp; Glan-Thompson prisms used
3 --
1
E--i pi -
(2)
fi '/3
where p , are the polarizations of the components and ft are the corresponding weighting factors. While Weber was concerned with macromolecules (proteins), his equation might apply if Perrin's equation (Equation 1) applied to an analogous simple case (e.g., Figure 2). How ever, the fluorescing power of estrone a t 435 or 475/490 mp falls off very slowly a t the concentration of acid present after dilution 1: 2 in these experiments, but more rapidly a t lower concentrations of acid (5) and simultaneously another absorbance and fluorescence region appears (cf. Figure 3). We postulate that another oscillator in estrone does not become functional until the acid concentration is lowered. I n strong acid solutions, therefore, the linear relation (Equation 1) for a single oscillator would be observed. The low acid curves, however, are convex to the T/q axis, like Figure 1 for pregnanediol. Another case where presumably only one excited molecular species is present but perhaps two oscillators is that of dehydroepiandrosterone treated with aqueous ethanolic H2SO4 and heat (Allen reaction). Csing this well known colorimetric process we prepared soluVOL. 37, NO. 10, SEPTEMBER 1965
1229
tions which on scanning showed a fluorescence band a t 521 mp on excitation at 482 mp. The polarization was initially 0.28. Overnight the fluorescing power decreased but the polarization was 0.30, representing only a slight change. (The concentration used here was 20 pg./ml.) Ittrich (3) was able to obtain considerable purification of crude preparations containing estrogens by extracting the Kober acid reaction products into 2% p-nitrophenol in 1% alcoholic l,l,2,2-tetrabromethane [cf. also ref. ( 7 ) ] . I n our laboratory, polarizations of the fluorescence of such extracts were measured a t 558 mp with various excitation wavelengths. These excitation scans indicated selection of the excitation wavelengths 488 mp and 543 mp. Some data on experiments with nonpregnancy urine, nonpregnancy urine plus estriol, and pregnancy urine, in addition to data obtained by using the crude ether extracts of nonpregnancy urine alone or with added estriol are presented in Table 11. ITse of crude nonpregnancy urine, 0.1 or 0.2 ml., required full sensitivity of the photomultiplier microphotometer plus use of the recorder a t inputs 5 or 2.5 mv./inch, respectively, to give appreciable response. Use of ether extracts of 3 or more ml. of acidhydrolyzed urine also required this amplification. I n Table I1 is first given a description of the sample, then the factor f,which is the ratio of the readings a t polarizer setting BE/BB [cf. ref. ( I ) ] . This ratio is lower (0.40) when a forbidden polarization value of 0.61 is obtained (maximum theoretical value is 0.5) for the second sample. This probably is due to scattering of light by the large
Table
II.
0.7,
0.6
