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Interactions of steroids with human plasma proteins
ARCHIVES
OF
BIOCHEMISTRY
AND
Interactions W. ROY
486492
100,
of Steroids
SLAUNWHITE, From
BIOPHYSICS
with
JR., HANNAH the Roswell
Park
Human
ROSEKTHAL
Memorial
Received
(1963)
Institute,
October
Plasma AND Bu$alo,
Proteins’ AVERY New
A. SANDBERG
York
23, 1961
By means of equilibrium dialyses and, to a lesser extent, ultrafiltration at 5”, the binding of six steroid hormones-estrone, estradiol, testosterone, progesterone, corticosterone, and cortisol-to various human plasma proteins was determined. In the case of human serum albumin, the solubility and binding of a larger series of 18 steroids was investigated. Albumin is responsible for most of the binding of unconjugated steroids in plasma, except for cortisol and corticosterone, due to the fact that it is by far the most abundant protein constituent of plasma. At 25” binding is decreased, but in an irregular fashion depending upon the individual steroid. Corticosterone has one site on human serum albumin with an association constant 30 times that of the other 20 sites. Evidence of competition was seen only between corticosterone and cortisol and among a group of androgens. INTRODUCTION
Although Schellman et al. (1) and especially Levedahl and co-workers (2-5) have studied the interaction of testosterone with bovine serum albumin (BSA), there has been no systematic study of the interaction of steroid hormones with human plasma proteins. The results of such a study, which were reported in part some years ago (6), are reported in full here. Because of the difficulty in obt.aining purified proteins, most of the work has been done with human serum albumin (HSA). EXPERIMENTAL MATERIALS The sources of radioactive steroids were as follows : estrone-16-Cl4 and 17p-estradiol-16-C14, 2.7 pc./mg., C. E. Frost and Co.; progesterone4-W and testosterone-4-CY4, 5 pc./mg., ll-deoxycorticosterone-4-C14 acetate, 0.93 wlmg., cortisone-4-CY4, 13.2 pc./mg., 17a-hydroxypregn4-ene-3,17-dione-4-C’*, 33.3 pc./mg., androst-4. ene-3,17-dione-4-CY4, 7.6 pc./mg., and 3p-hydroxyandrost-5-ene-17.one-7-H3, 2.56 New PC.IPbZ., 1 This work was supported (A-1240) from the National U. S. Public Healt,h Service.
in part Institutes
by a grant of Health, 486
England Nuclear Corporation; corticosterone4-C!14, 9.1 pc./mg., cortisol-4-C14, 3.7 rc./mg., llghydroxyandrost-4-ene-3,17-dione-4-04, 5 Fc./mg., pregnane-3,11,20-trione-4-C14, 17ol,21-dihydroxypregnane-3,11,20-trione-4-C14 al-acetate, 301,17a, 21.trihydroxypregnane-11,20-dione-4-04 al-acetate, aldosterone-Ha, 87 rc./mg., and cortisone4-C14 acetate, Endocrinology St,udy Section of the National Inst,itutes of Health; estriol-16-C14, 22 pc./mg., Dr. Mortimer Levitz; 17cu-hydroxy-6olmethyl-CY4-pregn+ene-3,20-dione acetate, 1.76 and 6ol-methyl-C14-prednisolone, 3.2 w./w., rc./mg., The Upjohn Company. Etiocholanolone4-C14 and androsterone-4-C’4 were isolated from urine after administration of testosterone-4-C14; androst+ene-3,11, 17.trione-4-C14 was prepared from lip-hydroxyandrost-4-ene-3,17-dione-4-C’d by chromic acid oxidation. Paper chromatography showed each steroid to be at least 99% pure. Carrier steroids of U.S.P. quality, obtained from commercial sources, were used as supplied. Plasma fractions were generously supplied by Dr. Harry Antoniades of the Protein Foundation, Inc. and HSA by the American Red Cross as a 257% solution stabilized with 0.02 M sodium acetyltryptophanate and caprylate. Electrophoresis of the HSA in a Tiselius apparatus (Perkin-Elmer model 38) showed only one component at pH 8.6, p = 0.1. The HSA was used without prior dialysis to eliminate the preservatives except in two instances.
STEROID-PLASMA
PROTEIN
TECHNIQUES Equilibrium dialyses were performed as previously described (6). In most cases the protein solution was placed inside, the radioactive steroids outside the casing, but, many of the values sums marized in Tables II-IV wereobtainedfrom dialysiin both directions. In addition, a few determinations were made using the ultrafiltration technique of Lavities (7) as previously described (6). Although the data obtained by ultrafiltration agreed well wit,h the data obtained by dialysis, the met,hod was poorly suited to a large-scale investigation. Protein solutions were extracted three times with 3 vol. of freshly distilled chloroform, and nonprotein solutions t,hree times with equal volumes of chloroform. All extracts were evaporated to dryness at 50” under a stream of air. Solubility was determined by shaking an excess of nonradioactive steroid with the appropriate solvent for at least 72 hr. The shaker used was that employed in dialyses and was sufficiently gentle to prevent denaturation of HSA. Undissolved steroid was removed by filtration of the suspensions through Whatman No. 1 filter paper. Extracts were obtained as described above. Measurement of radioactivity was made in a gas-flow counter as previously described (8). Nonradioactive steroids were measured by ultraviolet spectrophotometry where applicable. 17cu,21-Dihydroxypregnane-3,11,20-trione and 301,17~,21trihydroxypregnane-11,20-dione were determined by the Porter-Silber reaction (9) and pregnanetrione by reaction with thiosemicarbazide (10). Competition experiments were performed in two ways. In the first series radioactive steroid competed with a second nonradioactive steroid present in the following concentrations (based on a total volume of 10 ml. inside and 30 ml. outside the casing): estrone, 1.39 X 10m5 M; estradiol, 4.14 X low5 M; progesterone, 2.39 X 1O-5 M; testosterone, X.tiF X 10d5 ‘$1; corticosterone, 5.05 X 1OV 111;cortisol, 6.21 X 10e4 M; androst-4ene-3,17-dione, 7.87 X 10e5 J!!; androst-4.ene3,11, li-trione, 1.50 X 1OV IV; llp-hydroxyandrost4-ene-3,17-dione, 5.89 X 1O-4 IV. At t,he start of the experiment the radioactive steroid was in solution, the nonradioactive one was not. After 72 hr. there was usually no solid left. In the second series, the only radioactive steroids used were corticosterone and cortisol. These were made 1.95 X 10e5 M and 5.59 X 10m5M, respectively, by addition of the appropriate carrier. The concentrations of competing steroid were 0.7 of t,hose given in the preceding paragraph. Both the HSA and saline solutions were pre-equilibrated with steroids so that all the steroid was in solution at, thestart of the dialysis. The radioactive st,eroid
INTERACTIONS
487
was not added until the commencement of the dialysis. For the determination of the number of binding sites on HSA for corticosterone, equilibrium dialyses were performed in a slightly different manner. Ethanolic solutions of a mixture of carrier and radioactive corticosterone were prepared in various proportions. The concentration of the radioactive steroid was kept constant. Suitable portions were added to ca. 1 g.“A HSA solutions and to 0.155 A4 sodium chloride solutions so that the ethanol concentration never exceeded 2%. Equilibrium dialysis was performed in 1.6 X 13 cm. vials with 4.0 ml. of HSA solution inside a x-in. Nojax cellulose casing and 17.0 ml. of a saline solution outside the casing. Each vial contained about 0.03 pt./ml. Due to the smaller diameter of the casing, equilibrium was attained in 24 hr.; but for convenience, equilibration was continued for another night. The radioactivity of l&ml. portions of both solutions was assayed in an automatic liquid scintillation spectrometer after dilution to 20 ml. with a scintillation solvent containing dioxane and 2-et,hoxyethanol (11). At least 190,000 counts were accumulated during five lo-min. counting periods. Quenching was corrected by using the ratio of count,s in a 10-100-v. window to that in a 35-100-v. window (12). RESULTS
ANI)
DISCUSSION
RECOVERY
Recovery of the trace amounts of radioactive steroids used in these experiments has been nearly quantitative with the exception of progesterone. Our cust,omary extraction (three times with 3 vol. chloroform) removed only 35 %Jof added progesterone-4-C14from plasma diluted 1: 5 with saline and 82 % from 1% HSA. A second extraction removed less than would be expected by the laws of extraction (14 and 5%, respectively), and continuous extraction with ether also failed to remove all the radioactivity (83 and 97 %, respectively). These data indicate that progesterone is bound more strongly to plasma proteins than are the other steroids tested and that HS4 is only partially responsible for this greater avidity. Difficulties in quantitative extraction were also encountered with 17a-acetoxy-6amethylprogesterone and 17~hydroxyprogesterone, but to a decreasingly smaller degree than exhibited by progesterone. Results reported here were obtained with
488
SLAUNWHTTE,
ROSENTHAL
AND
TABLE SOLUBILITY
OF STEROIDS
IN 0.155
SANDBERG
I M NaCl
AND IN 1%
Solubilitya NaCl (A) w/ml.
E&one 176.Estradiol Estriol Testosterone Androst+ene-3,17-dione
AT 5" Dispe;lzgApower
1.0% HSA (B) PM
1.6 2.7 13.9 16.1 20.2
HSA
5.9 9.9 48.2 55.8 70.6
w/ml. 7.6 30.0 27.7 39.4 30.3
PM 27.9 110 96.1 137 106 820
w/ml. 6.0 27.3 13.8 23.3 10.1
PA+4 22.0 loo 47.9 80.0 35.4
B-A A
3.8 10 1 1.5 0.5
lip-Hydroxyandrost-4-ene-3,17dione Androst-4-ene-3,11,17-trione Progesterone 1701.Hydroxypregn-4-ene-3,20-dione 17a-Acetoxy-6a-methylpregn-4-ene3,20-dione
181 43.8 3.6 6.1 1.2
147 11.4 18.5 3.1
46.8 14.3 9.0 2.3
157 45.5 27.3 6.0
3.0 10.7 2.9 1.1
10.0 34.1 8.8 2.9
0.07 3.0 0.5 0.9
11-Deoxycorticosterone Pregnane-3,11,20-trione Corticosterone Cortisone 17a,21-Dihydroxypregnane-3,11,20trione
32.5 40.8 118 182 30.9
98 124 342 506 85.3
128 55.7 247 219 24.4
388 169 741 609 67.4
95.5 14.9 129 37 0
290 45.1 372 lU3 0
2.9 0.4 1.1 G.2 0
301,17~~,21-Trihydroxypregnane11,20-dione Cortisol llp,l7a,21-Trihydroxy-6a-methylpregna-1,4-diene-3,20-dione
216
594
231
635
15
41.2
0.1
197 55.1
544 147
220 74.0
607 198
23 18.9
63 50.5
0.1 0.3
1~The
average
st,andard
601
247
66
219
0.4
____~
deviation
was 7.5oj,.
the customary extraction technique. Hence, values obtained using radioactive progesterone are minimal values for Fractions I through IV-4 since the loss due to adsorption to the protein was not taken into account. In the case of Fraction V (albumin), the loss was ascribed to protein binding and calculated as such. SOLUBILITY
It is difficult to generalize concerning the effect of structure on solubility of steroids (Table I). While hydroxyl groups generally confer greater solubility in water than do ketone groups, exceptions do occur; e.g., testosterone is slightly less soluble than androstenedione. Also unexpected was the fact that pregnanetrione, which is isomeric with deoxycorticosterone, is more soluble than the latter. Although albumin binds most anions and many cations, it, is much more specific in
binding uncharged steroid molecules. The last column in Table I, which represents the fractional increase in solubility conferred by HSA, shows that HSA has very little capacity for most of the steroids examined. It may not be fortuitous that all the steroids but one, which exhibit at least a 100% increase in solubility in 1 YC HSA and relative insolubility in water, are hormones. On the other hand, hormones which have a low affinity for HSA, such as llp-hydroxyandrostenedione and cortisol, possess relatively high water solubility. Very few data are available for comparison. According to Bischoff and Katherman (13), the solubility of estrone in isotonic saline is 5 X 1OP molar at 37”. Eik-Nes et al. (14) found testosterone and cortisone to be somewhat more soluble in buffer solution at 37” than we did at 5’; the value given by them for estradiol was one order of magnitude lower. Although Macek et al. (15)
STEROID-PLASMA
PROTEIK TABLE
PER
CENT
OF VARIOUS
STEROIDS
BOUND
II
TO HUMAN PLASMA AT pH 7.0 AND 50a Plasma
Steroid
1.2 g. 70 II
Q Standard three values. 1) Calculations
23 (21, 25) 63 j 22
(51,
74)
(15-29)
~ 33 (29, 2(i
3b)
~ 23
27)
(18,
deviations were
CEST
lhsor,ve~~
IN 0.155
0.4 a.7~ IV-1
29
71 75
+
3.4
11 (5, 16)
f
6.1
38
44
=!I 13.1
3G (34-40) 42 33
(23-44)
31 (24, 38 7 (3-10) 7 (5-12)
are given corrected
four
a partial
(37,
BIXDISG
TO
33) 39)
4G 5 (l-11)
- ,
where
using
1
(25,
5 (4,
42
1 65
h +
73 85
f
volume
of 0.73.
The biuding of six steroids to the proteiu fractions obtained by Method 6 of Cohn et al. (16) and the bindiug of several addit’ional steroids to HSA were studied. The results of the former group of steroids are given in Table II, those of the latter group of steroids in Table III. The concentration of radioactive steroid used in the determinatiou of binding was of the order of lo-’ dl and varied with the specific activity of the steroid. Per cent binding was calculated (6) on the assumption that any endogenous steroid, if present, would not appreciably affect the results. Since the concentration in plasma of most of these steroids is immeasurable by conventional microchemical techniques and since a large quautity of any steroid is removed during precipitation of HSA with 40 57’ethanol (l(i), this assumption appears to be valid. The results shown in Table II were obtained by ultrafiltration and dialysis in both directions. Due to lack of material we were urlable to repeat determinations ou Fract,ions J, II, and III-O. Extreme care
95.4 95.8
82
specific
PLASMA
96 & 3.2
I+I G.4
were
6.7 3.7
CENT
b
0.7 3.0 2.5 3.0
3.2
88.7
IIZ 4.5
66
3.8
75.6
f
36
&
averaged;
a range
8.91
in
the
case
of
III
OF \'.4~1orrs
HSA
0.4 1.5'
f f
TABLE I'ER
2.4
f
YIZ 0.5
f
values
f
96.4
99.0
f zt
88
v
6.6,
76
2.0
0.8 g.“c
81 zk 3.1 77 8i
or more
FRACTIONS
f
,
+g.%vb
0.4 g.70 IV-4
4.F 6.2
74 * 4G + 5)
M TZaC1
fraction
0.4 g. “h III-O --_
have reported determinations of solubility in HSA, evaluation of their results is impossible since it is not known whether they corrected for the partial specific volume and hydration of the albumin. It was also unfortunate that they chose to use a 25 5%solution and an ccluilibration time of only 1 hr. J’ER
PROTEINS
-
(
Estrone Estradiol Testjosterone Progesterone Corticosterone Cortisol
489
IIV’TERACTIOIW
STEROIDS AT
BOL-ND
TO
5” .__
Steroid
Etiocholanolone Androsterone 3&Hydrosyandrost-5-cue-17-one Androst-4-ene-3,17-dione Androst,-4.ene-3,11,17-trione 110 Hydroryandrost 4 ene dionc ll-Deoxycorticosterone ll-1)eoaycorticostcrone acetate Xldosterone Pregnane-3,11,20-t rione 17a-Hydror;ypregn-~-ene-3,20-dione 17ol-Hydrosy-k-methylpregn-4.ene 3,20-dione acetate 17cu,21-1~ihydr0s~pregna~~e-3,11,20triune 21.acetate 301, licv, 21 TriII?-drosypregnAne __ ^^ . ll,XUmdlone Y&acetate tia-Metllylprednisolltne Cortisone Cortkone acetate Estriol
g.‘% HSA
0.X
4
i-4
91 95
86 87 i(i 32
3, li
97 90 87
24
(i-l
7.3 77
92 94 (j>d
ai 58 59
96
GO
85
36
'75
56
91
36
63 ifi
43 32
(23
89
76 80:
must be exercised in handling Fraction III-O since storage alone is sufficient to cause extensive denaturation. d sample obtained from outdated plasma and then stored at - 5” for several months gave v&es comparable to those of 1% HSA. The protein concentrations employed are
490
SLAUNWHITE,
ROSENTHAL
comparable to those found in normal human plasma, and hence Table II gives an approximation of the distribution of steroids in plasma if one assumes these results can be transferred to conditions in vivo. The effect of temperature on binding to HSA is discussed later. Fractions IV and V are responsible for most of the binding. Indeed, the binding to 4 g. % Fraction V (predominantly HSA) was so high that experiments were repeated at a lower concentration in order to emphasize differences among the steroids and protein fractions. In regard to steroids, there appears to be a rough inverse correlation between solubility and affinity for HSA. The binding of several additional steroids to HSA only is given in Table III. Androsterone has a greater affinity for albumin
AND
SANDBERG
than its C5 isomer, etiocholanolone, or one of its precursors in the body, androstenedione. Introduction of an oxygen at Cll, as in 1 l/3-hydroxyandrost-4-ene-3,17-dione, markedly weakens this affinity. None of the CZ106 steroids possesses much affinity for albumin. CZ103 steroids exhibited binding intermediate between that of progesterone (Table II) and the CzlO, steroids. NUMBER
OF SITES ON CORTICOSTERONE
HSA
FOR
The binding of radioactive corticosterone to HSA was determined in the presence of increasing amounts of carrier steroid. Four two with experimeits were performed: dialyzed (vs. saline) HSA and two with nondialyzed HSA. The pH of the HSA
(+M-’ 0.00 7 0 -I 0
oooz-
o.ooi-
0
I 0.2
I 04
I 0.6
I OS
I 12
I IO
r
I 14
I 16
I IS
I 20
r
1. The binding of corticosterone to HSA at pH 7 and 5”. Four series of experiments are presented: 0,0.7 g. 7cdialyzed HSA, pH 6.85; A, 0.7 g. y0 dialyzed HSA, pH 7.0; l , 1.0 g. % nondialyzed HSA, pH 6.45; A, 1.0 g. 70nondialyzed HSA, pH 6.35. The curve obeys the equation
FIG.
n1 k&s) T = i+kl(S)
ns k*(S) + 1 +
k*(S)
where r is the moles of corticosterone bound per mole of HSA, (8) is the molar concentration of unbound corticosterone, n1 = 1, kl = 0.0036 pM-1, n2 = 20, kz = 0.00012 JW1. As (S) --f 0, r/(S) --) n&l + W& As (S) + m, T + 1~1 + 7t2.
STEROID-PLASMA
PROTEIN
solution at equilibrium was 6.85 and 7.00 in the former case and 6.45 and 6.35 in the latter. The protein concentrations were 0.7 and 1.0 g. 94, respectively. The data were plotted as r/(X) vs. r in which r is the moles of steroid bound per mole of albumin and (S) is the molar concentration of unbound steroid (Fig. 1). The four series of experiments gave similar results within the limits of error, and they were, therefore, treated colIectiveIy for the estimation of association constams (li) and number of binding sites (n>. If all the sites on a protein possessed the same equilibrium constant for a given ligand (homogeneity of binding sites), the plot of r/(S) vs. r would be a straight line. Since the data in Fig. 1 does not follow a straight line, there must be multiple sites with two or more association constants, assuming no interaction between sites. For this purpose we have used the equation n1 k,(S)
n2 h-z(S)
491
INTERACTIONS TABLE
IV
THE EFFECT OF TEMPERATURE OS Brxnrxc 0.8 go/; HSA AT pH 7.0 Per cent bound 5" 25' ---__~__
-~--.-~-__
Estrone Estradiol Testosterone Progesterone Corticosteronr Cortisol
TO
88 96 i3 89 66 36
It f & k f f
2.4 3 2 0.7 3.0 2.5 3.0
82 88 68 83 36 24
(82, 82) f 2.1 zt 9.9 f 14.9 (34, 38) + 5.6
As expected, there was a decrease in binding in every case, but the decrease was not, uniform. While there was a marked decrease in cortisol and especially corticosterone binding, the decrease in binding of the two estrogens was quite modest. In the case of testosterone and progesterone, t’he decrease was not statistically significant. COMPETITIOK
To determine if two steroids are bound at " = 1+ h:,(S) + 1 + k&s) the same site, the determination of binding This equation fits the experimental data of radioactive steroid was performed in the reasonably well when 1~~ = 1, n2 = 20, presence of a uearly saturated solution of a kl = 0.0036 pA-* and kz = 0.00012 ~Ar-l. second, nonradioactive steroid. 1~1 a second Changing nz by 5 or k,/k~ by 10 gives a series a small amount (sufficient to occupy curve that clearly does not represent all the 20%; of the sites) of carrier st,eroid was also experimental data. The equilibrium constant included in the dialysis. In neither series of association then becomes K = n& + was there evidence of competition among n&z = 0.006 pil$-I. The poor dispersing e&one, estradiol, estriol, testosterone, and power which albumin has for corticosterone progesterone. The conclusion that competi(Table I) is a reflection of the smallness of tion does not exist among these steroids this constant. does not follow, however, for the insolubility These results are at variance with those of these compounds in saline prevented even reported previously (6) (n = 2). The differself-inhibition of binding. Addition of ence is probably ascribable to the improved carrier estrone did not produce a significaut accuracy inherent in liquid scintillation decrease in the binding of estrone-C14, counting. This technique, which eliminates carrier estradiol did not compete with several steps from the gas-flow method used estradiol-CP, and similarly for the other previously and in the other parts of this steroids. On the other hand, cortisol proreport, has improved both the accuracy and duced a 68 and 61% inhibition of binding of the precision of counting. corticosterone of high and low specific activity, respectively. Corticosterone pro%FECT OF TEMPERATURE duced a similar inhibition of the biuding of Although most determinations were made cortisol. Evidence of interaction was also at 5” in order to minimize protein denaturaseen among the group androst--l-em-3 , Iition, a few experiments were conducted at dione, nndrost--L-et&~, 11,17-trione, and 25” in order to determine the effect of tem- 1l,&hydroxyandrost--Lene-3,17-dione. Sone perature. The results are shown in Table 11~. of t.hese three compounds inhihit.ed the
492
SLAUNWHITE,
binding of testosterone to HSh conditions of our experiments.
ROSENTHAL under
the
ACKNOWLEDGMENTS The authors wish to thank Drs. Harry N. Antoniades and Robert B. Pennell of the Protein Foundation, Inc. for their generosity in supplying protein fractions and the American Red Cross and especially Dr. J. N. Ashworth for supplies of protein fractions and human serum albumin. We are grateful to Drs. A. Nisonoff, G. Tritsch, and D. Pressman for their advice during the course of this study. The able assistance of Mrs. Lucille Zablotny, Mrs. Maria Karasy, Mr. Lawrence Beecher, and Mr. Elek Karsay is gratefully acknowledged. REFERENCES 1. SCHELLMAN, J. A., LUMRY, R., AND SAMUEI,S, L. T., J. Am. Chem. Sot. 76, 3808 (1954). 2. LEVEDA~L, B. H., AND BERNSTEIN, H., Arch. Bioch.em. Biophys. 62, 353 (1954). 3. LEVEDAHL, B. H., Arch. Biochem. Biophys. 69, 300 (1955). 4. LEVEDAHL, B. H., AND PERLWUTTER, R., Arch. Biochem. Biophys. 61,442 (1956).
AND SANDBERG
5. OYAKAWA, E. K., AND LEVEDAHL, B. H., Arch. Biochem. Biophys. 74, 17 (1958). 6. SANDBERG, A. A., SLAUNWHITE, W. R., JR., AND ANTONIADES, H. N., Rec. Prog. Hormone Res. 13, 209 (1957). 7. LAVITIES, P. H., ,I. Biol. Chem. 120,267 (1937). 8. SANDBERG, A. A., AND SLAUNWHITE, W. R., JR., J. Clin. Invest. 36,1331 (1956). 9. PETERSON, R. E., KARRER, A., AND GUERRA, S. L., Anal. Chem. 29, 144 (1957). 10. BUSH, I. E., Federation Proc. 12,186 (1953). 11. BRUNO, G. A., AND CHRISTIAN, J. E., Anal. Chem. 33,1216 (1961). 12. BRUNO, G. A., AND CHRISTIAN, J. E., Anal. Chem., 33, 650 (1961). 13. BISCHOFF, F., AND KATHERMAN, R. E., Federation Proc. 11, 188 (1952). 14. EIK-NES, K., SCHELLMAN, J. A., LUMRY, Et., AND SAMUELS, L. T., I. BioZ. Chem. 206, 411
(1954). 15. MACEK, T. J., BAADE, W. BACHER, F. A., Science 16. COHN, E. J., STRONG, L. JR., MIJLFORD, D. J., NELIN, Chem.
H., BORNN, A., AND 116, 399 (1952). E., HUGHES, W. L.,
ASHWORTH, M., AND TAYLOR, H. I., Sot. 66, 459 (1946).
J.
N.,
J. =Im.
OF
BIOCHEMISTRY
AND
Interactions W. ROY
486492
100,
of Steroids
SLAUNWHITE, From
BIOPHYSICS
with
JR., HANNAH the Roswell
Park
Human
ROSEKTHAL
Memorial
Received
(1963)
Institute,
October
Plasma AND Bu$alo,
Proteins’ AVERY New
A. SANDBERG
York
23, 1961
By means of equilibrium dialyses and, to a lesser extent, ultrafiltration at 5”, the binding of six steroid hormones-estrone, estradiol, testosterone, progesterone, corticosterone, and cortisol-to various human plasma proteins was determined. In the case of human serum albumin, the solubility and binding of a larger series of 18 steroids was investigated. Albumin is responsible for most of the binding of unconjugated steroids in plasma, except for cortisol and corticosterone, due to the fact that it is by far the most abundant protein constituent of plasma. At 25” binding is decreased, but in an irregular fashion depending upon the individual steroid. Corticosterone has one site on human serum albumin with an association constant 30 times that of the other 20 sites. Evidence of competition was seen only between corticosterone and cortisol and among a group of androgens. INTRODUCTION
Although Schellman et al. (1) and especially Levedahl and co-workers (2-5) have studied the interaction of testosterone with bovine serum albumin (BSA), there has been no systematic study of the interaction of steroid hormones with human plasma proteins. The results of such a study, which were reported in part some years ago (6), are reported in full here. Because of the difficulty in obt.aining purified proteins, most of the work has been done with human serum albumin (HSA). EXPERIMENTAL MATERIALS The sources of radioactive steroids were as follows : estrone-16-Cl4 and 17p-estradiol-16-C14, 2.7 pc./mg., C. E. Frost and Co.; progesterone4-W and testosterone-4-CY4, 5 pc./mg., ll-deoxycorticosterone-4-C14 acetate, 0.93 wlmg., cortisone-4-CY4, 13.2 pc./mg., 17a-hydroxypregn4-ene-3,17-dione-4-C’*, 33.3 pc./mg., androst-4. ene-3,17-dione-4-CY4, 7.6 pc./mg., and 3p-hydroxyandrost-5-ene-17.one-7-H3, 2.56 New PC.IPbZ., 1 This work was supported (A-1240) from the National U. S. Public Healt,h Service.
in part Institutes
by a grant of Health, 486
England Nuclear Corporation; corticosterone4-C!14, 9.1 pc./mg., cortisol-4-C14, 3.7 rc./mg., llghydroxyandrost-4-ene-3,17-dione-4-04, 5 Fc./mg., pregnane-3,11,20-trione-4-C14, 17ol,21-dihydroxypregnane-3,11,20-trione-4-C14 al-acetate, 301,17a, 21.trihydroxypregnane-11,20-dione-4-04 al-acetate, aldosterone-Ha, 87 rc./mg., and cortisone4-C14 acetate, Endocrinology St,udy Section of the National Inst,itutes of Health; estriol-16-C14, 22 pc./mg., Dr. Mortimer Levitz; 17cu-hydroxy-6olmethyl-CY4-pregn+ene-3,20-dione acetate, 1.76 and 6ol-methyl-C14-prednisolone, 3.2 w./w., rc./mg., The Upjohn Company. Etiocholanolone4-C14 and androsterone-4-C’4 were isolated from urine after administration of testosterone-4-C14; androst+ene-3,11, 17.trione-4-C14 was prepared from lip-hydroxyandrost-4-ene-3,17-dione-4-C’d by chromic acid oxidation. Paper chromatography showed each steroid to be at least 99% pure. Carrier steroids of U.S.P. quality, obtained from commercial sources, were used as supplied. Plasma fractions were generously supplied by Dr. Harry Antoniades of the Protein Foundation, Inc. and HSA by the American Red Cross as a 257% solution stabilized with 0.02 M sodium acetyltryptophanate and caprylate. Electrophoresis of the HSA in a Tiselius apparatus (Perkin-Elmer model 38) showed only one component at pH 8.6, p = 0.1. The HSA was used without prior dialysis to eliminate the preservatives except in two instances.
STEROID-PLASMA
PROTEIN
TECHNIQUES Equilibrium dialyses were performed as previously described (6). In most cases the protein solution was placed inside, the radioactive steroids outside the casing, but, many of the values sums marized in Tables II-IV wereobtainedfrom dialysiin both directions. In addition, a few determinations were made using the ultrafiltration technique of Lavities (7) as previously described (6). Although the data obtained by ultrafiltration agreed well wit,h the data obtained by dialysis, the met,hod was poorly suited to a large-scale investigation. Protein solutions were extracted three times with 3 vol. of freshly distilled chloroform, and nonprotein solutions t,hree times with equal volumes of chloroform. All extracts were evaporated to dryness at 50” under a stream of air. Solubility was determined by shaking an excess of nonradioactive steroid with the appropriate solvent for at least 72 hr. The shaker used was that employed in dialyses and was sufficiently gentle to prevent denaturation of HSA. Undissolved steroid was removed by filtration of the suspensions through Whatman No. 1 filter paper. Extracts were obtained as described above. Measurement of radioactivity was made in a gas-flow counter as previously described (8). Nonradioactive steroids were measured by ultraviolet spectrophotometry where applicable. 17cu,21-Dihydroxypregnane-3,11,20-trione and 301,17~,21trihydroxypregnane-11,20-dione were determined by the Porter-Silber reaction (9) and pregnanetrione by reaction with thiosemicarbazide (10). Competition experiments were performed in two ways. In the first series radioactive steroid competed with a second nonradioactive steroid present in the following concentrations (based on a total volume of 10 ml. inside and 30 ml. outside the casing): estrone, 1.39 X 10m5 M; estradiol, 4.14 X low5 M; progesterone, 2.39 X 1O-5 M; testosterone, X.tiF X 10d5 ‘$1; corticosterone, 5.05 X 1OV 111;cortisol, 6.21 X 10e4 M; androst-4ene-3,17-dione, 7.87 X 10e5 J!!; androst-4.ene3,11, li-trione, 1.50 X 1OV IV; llp-hydroxyandrost4-ene-3,17-dione, 5.89 X 1O-4 IV. At t,he start of the experiment the radioactive steroid was in solution, the nonradioactive one was not. After 72 hr. there was usually no solid left. In the second series, the only radioactive steroids used were corticosterone and cortisol. These were made 1.95 X 10e5 M and 5.59 X 10m5M, respectively, by addition of the appropriate carrier. The concentrations of competing steroid were 0.7 of t,hose given in the preceding paragraph. Both the HSA and saline solutions were pre-equilibrated with steroids so that all the steroid was in solution at, thestart of the dialysis. The radioactive st,eroid
INTERACTIONS
487
was not added until the commencement of the dialysis. For the determination of the number of binding sites on HSA for corticosterone, equilibrium dialyses were performed in a slightly different manner. Ethanolic solutions of a mixture of carrier and radioactive corticosterone were prepared in various proportions. The concentration of the radioactive steroid was kept constant. Suitable portions were added to ca. 1 g.“A HSA solutions and to 0.155 A4 sodium chloride solutions so that the ethanol concentration never exceeded 2%. Equilibrium dialysis was performed in 1.6 X 13 cm. vials with 4.0 ml. of HSA solution inside a x-in. Nojax cellulose casing and 17.0 ml. of a saline solution outside the casing. Each vial contained about 0.03 pt./ml. Due to the smaller diameter of the casing, equilibrium was attained in 24 hr.; but for convenience, equilibration was continued for another night. The radioactivity of l&ml. portions of both solutions was assayed in an automatic liquid scintillation spectrometer after dilution to 20 ml. with a scintillation solvent containing dioxane and 2-et,hoxyethanol (11). At least 190,000 counts were accumulated during five lo-min. counting periods. Quenching was corrected by using the ratio of count,s in a 10-100-v. window to that in a 35-100-v. window (12). RESULTS
ANI)
DISCUSSION
RECOVERY
Recovery of the trace amounts of radioactive steroids used in these experiments has been nearly quantitative with the exception of progesterone. Our cust,omary extraction (three times with 3 vol. chloroform) removed only 35 %Jof added progesterone-4-C14from plasma diluted 1: 5 with saline and 82 % from 1% HSA. A second extraction removed less than would be expected by the laws of extraction (14 and 5%, respectively), and continuous extraction with ether also failed to remove all the radioactivity (83 and 97 %, respectively). These data indicate that progesterone is bound more strongly to plasma proteins than are the other steroids tested and that HS4 is only partially responsible for this greater avidity. Difficulties in quantitative extraction were also encountered with 17a-acetoxy-6amethylprogesterone and 17~hydroxyprogesterone, but to a decreasingly smaller degree than exhibited by progesterone. Results reported here were obtained with
488
SLAUNWHTTE,
ROSENTHAL
AND
TABLE SOLUBILITY
OF STEROIDS
IN 0.155
SANDBERG
I M NaCl
AND IN 1%
Solubilitya NaCl (A) w/ml.
E&one 176.Estradiol Estriol Testosterone Androst+ene-3,17-dione
AT 5" Dispe;lzgApower
1.0% HSA (B) PM
1.6 2.7 13.9 16.1 20.2
HSA
5.9 9.9 48.2 55.8 70.6
w/ml. 7.6 30.0 27.7 39.4 30.3
PM 27.9 110 96.1 137 106 820
w/ml. 6.0 27.3 13.8 23.3 10.1
PA+4 22.0 loo 47.9 80.0 35.4
B-A A
3.8 10 1 1.5 0.5
lip-Hydroxyandrost-4-ene-3,17dione Androst-4-ene-3,11,17-trione Progesterone 1701.Hydroxypregn-4-ene-3,20-dione 17a-Acetoxy-6a-methylpregn-4-ene3,20-dione
181 43.8 3.6 6.1 1.2
147 11.4 18.5 3.1
46.8 14.3 9.0 2.3
157 45.5 27.3 6.0
3.0 10.7 2.9 1.1
10.0 34.1 8.8 2.9
0.07 3.0 0.5 0.9
11-Deoxycorticosterone Pregnane-3,11,20-trione Corticosterone Cortisone 17a,21-Dihydroxypregnane-3,11,20trione
32.5 40.8 118 182 30.9
98 124 342 506 85.3
128 55.7 247 219 24.4
388 169 741 609 67.4
95.5 14.9 129 37 0
290 45.1 372 lU3 0
2.9 0.4 1.1 G.2 0
301,17~~,21-Trihydroxypregnane11,20-dione Cortisol llp,l7a,21-Trihydroxy-6a-methylpregna-1,4-diene-3,20-dione
216
594
231
635
15
41.2
0.1
197 55.1
544 147
220 74.0
607 198
23 18.9
63 50.5
0.1 0.3
1~The
average
st,andard
601
247
66
219
0.4
____~
deviation
was 7.5oj,.
the customary extraction technique. Hence, values obtained using radioactive progesterone are minimal values for Fractions I through IV-4 since the loss due to adsorption to the protein was not taken into account. In the case of Fraction V (albumin), the loss was ascribed to protein binding and calculated as such. SOLUBILITY
It is difficult to generalize concerning the effect of structure on solubility of steroids (Table I). While hydroxyl groups generally confer greater solubility in water than do ketone groups, exceptions do occur; e.g., testosterone is slightly less soluble than androstenedione. Also unexpected was the fact that pregnanetrione, which is isomeric with deoxycorticosterone, is more soluble than the latter. Although albumin binds most anions and many cations, it, is much more specific in
binding uncharged steroid molecules. The last column in Table I, which represents the fractional increase in solubility conferred by HSA, shows that HSA has very little capacity for most of the steroids examined. It may not be fortuitous that all the steroids but one, which exhibit at least a 100% increase in solubility in 1 YC HSA and relative insolubility in water, are hormones. On the other hand, hormones which have a low affinity for HSA, such as llp-hydroxyandrostenedione and cortisol, possess relatively high water solubility. Very few data are available for comparison. According to Bischoff and Katherman (13), the solubility of estrone in isotonic saline is 5 X 1OP molar at 37”. Eik-Nes et al. (14) found testosterone and cortisone to be somewhat more soluble in buffer solution at 37” than we did at 5’; the value given by them for estradiol was one order of magnitude lower. Although Macek et al. (15)
STEROID-PLASMA
PROTEIK TABLE
PER
CENT
OF VARIOUS
STEROIDS
BOUND
II
TO HUMAN PLASMA AT pH 7.0 AND 50a Plasma
Steroid
1.2 g. 70 II
Q Standard three values. 1) Calculations
23 (21, 25) 63 j 22
(51,
74)
(15-29)
~ 33 (29, 2(i
3b)
~ 23
27)
(18,
deviations were
CEST
lhsor,ve~~
IN 0.155
0.4 a.7~ IV-1
29
71 75
+
3.4
11 (5, 16)
f
6.1
38
44
=!I 13.1
3G (34-40) 42 33
(23-44)
31 (24, 38 7 (3-10) 7 (5-12)
are given corrected
four
a partial
(37,
BIXDISG
TO
33) 39)
4G 5 (l-11)
- ,
where
using
1
(25,
5 (4,
42
1 65
h +
73 85
f
volume
of 0.73.
The biuding of six steroids to the proteiu fractions obtained by Method 6 of Cohn et al. (16) and the bindiug of several addit’ional steroids to HSA were studied. The results of the former group of steroids are given in Table II, those of the latter group of steroids in Table III. The concentration of radioactive steroid used in the determinatiou of binding was of the order of lo-’ dl and varied with the specific activity of the steroid. Per cent binding was calculated (6) on the assumption that any endogenous steroid, if present, would not appreciably affect the results. Since the concentration in plasma of most of these steroids is immeasurable by conventional microchemical techniques and since a large quautity of any steroid is removed during precipitation of HSA with 40 57’ethanol (l(i), this assumption appears to be valid. The results shown in Table II were obtained by ultrafiltration and dialysis in both directions. Due to lack of material we were urlable to repeat determinations ou Fract,ions J, II, and III-O. Extreme care
95.4 95.8
82
specific
PLASMA
96 & 3.2
I+I G.4
were
6.7 3.7
CENT
b
0.7 3.0 2.5 3.0
3.2
88.7
IIZ 4.5
66
3.8
75.6
f
36
&
averaged;
a range
8.91
in
the
case
of
III
OF \'.4~1orrs
HSA
0.4 1.5'
f f
TABLE I'ER
2.4
f
YIZ 0.5
f
values
f
96.4
99.0
f zt
88
v
6.6,
76
2.0
0.8 g.“c
81 zk 3.1 77 8i
or more
FRACTIONS
f
,
+g.%vb
0.4 g.70 IV-4
4.F 6.2
74 * 4G + 5)
M TZaC1
fraction
0.4 g. “h III-O --_
have reported determinations of solubility in HSA, evaluation of their results is impossible since it is not known whether they corrected for the partial specific volume and hydration of the albumin. It was also unfortunate that they chose to use a 25 5%solution and an ccluilibration time of only 1 hr. J’ER
PROTEINS
-
(
Estrone Estradiol Testjosterone Progesterone Corticosterone Cortisol
489
IIV’TERACTIOIW
STEROIDS AT
BOL-ND
TO
5” .__
Steroid
Etiocholanolone Androsterone 3&Hydrosyandrost-5-cue-17-one Androst-4-ene-3,17-dione Androst,-4.ene-3,11,17-trione 110 Hydroryandrost 4 ene dionc ll-Deoxycorticosterone ll-1)eoaycorticostcrone acetate Xldosterone Pregnane-3,11,20-t rione 17a-Hydror;ypregn-~-ene-3,20-dione 17ol-Hydrosy-k-methylpregn-4.ene 3,20-dione acetate 17cu,21-1~ihydr0s~pregna~~e-3,11,20triune 21.acetate 301, licv, 21 TriII?-drosypregnAne __ ^^ . ll,XUmdlone Y&acetate tia-Metllylprednisolltne Cortisone Cortkone acetate Estriol
g.‘% HSA
0.X
4
i-4
91 95
86 87 i(i 32
3, li
97 90 87
24
(i-l
7.3 77
92 94 (j>d
ai 58 59
96
GO
85
36
'75
56
91
36
63 ifi
43 32
(23
89
76 80:
must be exercised in handling Fraction III-O since storage alone is sufficient to cause extensive denaturation. d sample obtained from outdated plasma and then stored at - 5” for several months gave v&es comparable to those of 1% HSA. The protein concentrations employed are
490
SLAUNWHITE,
ROSENTHAL
comparable to those found in normal human plasma, and hence Table II gives an approximation of the distribution of steroids in plasma if one assumes these results can be transferred to conditions in vivo. The effect of temperature on binding to HSA is discussed later. Fractions IV and V are responsible for most of the binding. Indeed, the binding to 4 g. % Fraction V (predominantly HSA) was so high that experiments were repeated at a lower concentration in order to emphasize differences among the steroids and protein fractions. In regard to steroids, there appears to be a rough inverse correlation between solubility and affinity for HSA. The binding of several additional steroids to HSA only is given in Table III. Androsterone has a greater affinity for albumin
AND
SANDBERG
than its C5 isomer, etiocholanolone, or one of its precursors in the body, androstenedione. Introduction of an oxygen at Cll, as in 1 l/3-hydroxyandrost-4-ene-3,17-dione, markedly weakens this affinity. None of the CZ106 steroids possesses much affinity for albumin. CZ103 steroids exhibited binding intermediate between that of progesterone (Table II) and the CzlO, steroids. NUMBER
OF SITES ON CORTICOSTERONE
HSA
FOR
The binding of radioactive corticosterone to HSA was determined in the presence of increasing amounts of carrier steroid. Four two with experimeits were performed: dialyzed (vs. saline) HSA and two with nondialyzed HSA. The pH of the HSA
(+M-’ 0.00 7 0 -I 0
oooz-
o.ooi-
0
I 0.2
I 04
I 0.6
I OS
I 12
I IO
r
I 14
I 16
I IS
I 20
r
1. The binding of corticosterone to HSA at pH 7 and 5”. Four series of experiments are presented: 0,0.7 g. 7cdialyzed HSA, pH 6.85; A, 0.7 g. y0 dialyzed HSA, pH 7.0; l , 1.0 g. % nondialyzed HSA, pH 6.45; A, 1.0 g. 70nondialyzed HSA, pH 6.35. The curve obeys the equation
FIG.
n1 k&s) T = i+kl(S)
ns k*(S) + 1 +
k*(S)
where r is the moles of corticosterone bound per mole of HSA, (8) is the molar concentration of unbound corticosterone, n1 = 1, kl = 0.0036 pM-1, n2 = 20, kz = 0.00012 JW1. As (S) --f 0, r/(S) --) n&l + W& As (S) + m, T + 1~1 + 7t2.
STEROID-PLASMA
PROTEIN
solution at equilibrium was 6.85 and 7.00 in the former case and 6.45 and 6.35 in the latter. The protein concentrations were 0.7 and 1.0 g. 94, respectively. The data were plotted as r/(X) vs. r in which r is the moles of steroid bound per mole of albumin and (S) is the molar concentration of unbound steroid (Fig. 1). The four series of experiments gave similar results within the limits of error, and they were, therefore, treated colIectiveIy for the estimation of association constams (li) and number of binding sites (n>. If all the sites on a protein possessed the same equilibrium constant for a given ligand (homogeneity of binding sites), the plot of r/(S) vs. r would be a straight line. Since the data in Fig. 1 does not follow a straight line, there must be multiple sites with two or more association constants, assuming no interaction between sites. For this purpose we have used the equation n1 k,(S)
n2 h-z(S)
491
INTERACTIONS TABLE
IV
THE EFFECT OF TEMPERATURE OS Brxnrxc 0.8 go/; HSA AT pH 7.0 Per cent bound 5" 25' ---__~__
-~--.-~-__
Estrone Estradiol Testosterone Progesterone Corticosteronr Cortisol
TO
88 96 i3 89 66 36
It f & k f f
2.4 3 2 0.7 3.0 2.5 3.0
82 88 68 83 36 24
(82, 82) f 2.1 zt 9.9 f 14.9 (34, 38) + 5.6
As expected, there was a decrease in binding in every case, but the decrease was not, uniform. While there was a marked decrease in cortisol and especially corticosterone binding, the decrease in binding of the two estrogens was quite modest. In the case of testosterone and progesterone, t’he decrease was not statistically significant. COMPETITIOK
To determine if two steroids are bound at " = 1+ h:,(S) + 1 + k&s) the same site, the determination of binding This equation fits the experimental data of radioactive steroid was performed in the reasonably well when 1~~ = 1, n2 = 20, presence of a uearly saturated solution of a kl = 0.0036 pA-* and kz = 0.00012 ~Ar-l. second, nonradioactive steroid. 1~1 a second Changing nz by 5 or k,/k~ by 10 gives a series a small amount (sufficient to occupy curve that clearly does not represent all the 20%; of the sites) of carrier st,eroid was also experimental data. The equilibrium constant included in the dialysis. In neither series of association then becomes K = n& + was there evidence of competition among n&z = 0.006 pil$-I. The poor dispersing e&one, estradiol, estriol, testosterone, and power which albumin has for corticosterone progesterone. The conclusion that competi(Table I) is a reflection of the smallness of tion does not exist among these steroids this constant. does not follow, however, for the insolubility These results are at variance with those of these compounds in saline prevented even reported previously (6) (n = 2). The differself-inhibition of binding. Addition of ence is probably ascribable to the improved carrier estrone did not produce a significaut accuracy inherent in liquid scintillation decrease in the binding of estrone-C14, counting. This technique, which eliminates carrier estradiol did not compete with several steps from the gas-flow method used estradiol-CP, and similarly for the other previously and in the other parts of this steroids. On the other hand, cortisol proreport, has improved both the accuracy and duced a 68 and 61% inhibition of binding of the precision of counting. corticosterone of high and low specific activity, respectively. Corticosterone pro%FECT OF TEMPERATURE duced a similar inhibition of the biuding of Although most determinations were made cortisol. Evidence of interaction was also at 5” in order to minimize protein denaturaseen among the group androst--l-em-3 , Iition, a few experiments were conducted at dione, nndrost--L-et&~, 11,17-trione, and 25” in order to determine the effect of tem- 1l,&hydroxyandrost--Lene-3,17-dione. Sone perature. The results are shown in Table 11~. of t.hese three compounds inhihit.ed the
492
SLAUNWHITE,
binding of testosterone to HSh conditions of our experiments.
ROSENTHAL under
the
ACKNOWLEDGMENTS The authors wish to thank Drs. Harry N. Antoniades and Robert B. Pennell of the Protein Foundation, Inc. for their generosity in supplying protein fractions and the American Red Cross and especially Dr. J. N. Ashworth for supplies of protein fractions and human serum albumin. We are grateful to Drs. A. Nisonoff, G. Tritsch, and D. Pressman for their advice during the course of this study. The able assistance of Mrs. Lucille Zablotny, Mrs. Maria Karasy, Mr. Lawrence Beecher, and Mr. Elek Karsay is gratefully acknowledged. REFERENCES 1. SCHELLMAN, J. A., LUMRY, R., AND SAMUEI,S, L. T., J. Am. Chem. Sot. 76, 3808 (1954). 2. LEVEDA~L, B. H., AND BERNSTEIN, H., Arch. Bioch.em. Biophys. 62, 353 (1954). 3. LEVEDAHL, B. H., Arch. Biochem. Biophys. 69, 300 (1955). 4. LEVEDAHL, B. H., AND PERLWUTTER, R., Arch. Biochem. Biophys. 61,442 (1956).
AND SANDBERG
5. OYAKAWA, E. K., AND LEVEDAHL, B. H., Arch. Biochem. Biophys. 74, 17 (1958). 6. SANDBERG, A. A., SLAUNWHITE, W. R., JR., AND ANTONIADES, H. N., Rec. Prog. Hormone Res. 13, 209 (1957). 7. LAVITIES, P. H., ,I. Biol. Chem. 120,267 (1937). 8. SANDBERG, A. A., AND SLAUNWHITE, W. R., JR., J. Clin. Invest. 36,1331 (1956). 9. PETERSON, R. E., KARRER, A., AND GUERRA, S. L., Anal. Chem. 29, 144 (1957). 10. BUSH, I. E., Federation Proc. 12,186 (1953). 11. BRUNO, G. A., AND CHRISTIAN, J. E., Anal. Chem. 33,1216 (1961). 12. BRUNO, G. A., AND CHRISTIAN, J. E., Anal. Chem., 33, 650 (1961). 13. BISCHOFF, F., AND KATHERMAN, R. E., Federation Proc. 11, 188 (1952). 14. EIK-NES, K., SCHELLMAN, J. A., LUMRY, Et., AND SAMUELS, L. T., I. BioZ. Chem. 206, 411
(1954). 15. MACEK, T. J., BAADE, W. BACHER, F. A., Science 16. COHN, E. J., STRONG, L. JR., MIJLFORD, D. J., NELIN, Chem.
H., BORNN, A., AND 116, 399 (1952). E., HUGHES, W. L.,
ASHWORTH, M., AND TAYLOR, H. I., Sot. 66, 459 (1946).
J.
N.,
J. =Im.