STEROID-PROTEIN CONJUGATES

I. PREPARATION AND CHARACTERIZATION OF CONJUGATES OF BOVINE SERUM ALBUMIN WITH TESTOSTERONE

AND WITH CORTISONE*

BY BERNARD F. ERLANGER, FELIX BOREK, SAM M. BEISER, AND SEYMOUR LIEBERMAN

(From the Departments of Microbiology, Biochemistry, Obstetrics, and Gynecology, College of Physicians and Surgeons, Columbia

University, New York, New York)

(Received for publication, March l&1957)

Antihormonal principles which could counteract the physiological ef- fects of endogenously produced hormones would be of great importance to many phases of endocrinology. In addition to the possible fortuitous discovery of such inhibitory factors, two rational approaches may be considered. The first, similar to that which has been successful with antivitamins, is to prepare compounds with structures analogous to those of the steroids. This approach, although pursued only to a limited extent, has been unpromising, and it is unlikely that much effort will be expended in this direction until more is known about the mode of action of the steroid hormones. A second approach is the formation of specific anti- bodies which could neutralize endogenously formed steroid hormones. That various protein hormones are antigenic and can evoke the formation of antihormones has been well established. Thus, refractoriness has been reported after the prolonged use of some of the hypophyseal hormones (gonadotropins, thyrotropin, etc.), parathyroid hormone, and insulin (1). Maddock et al. (2) and Leathem (3) have shown that the neutralizing antibodies present in the serum of patients receiving purified hog follicle- stimulating hormone are not species-specific, since they were active against human pituitary follicle-stimulating hormone as well as human chorionic gonadotropin.

Although there have been reports (4-6) that some steroid hormones are allergenic, there is at present no evidence of acquired tolerance to chemi- cally pure estrogens, androgens, progesterone, or adrenal cortical hormones. The possibility of rendering low molecular weight steroids antigenic by coupling them to proteins suggested itself from the pioneering investigations of Landsteiner. This hapten principle has been applied by Clutton et al.

* The authors gratefully acknowledge the partial support given them by research grants from the National Science Foundation (NSF No. G-2315) and from the Popu- lation Council, Inc.

713

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714 STEROID-PROTEIN CONJUGATES. I

(7), who prepared thyroxyl derivatives of proteins and then demonstrated that these elicited antisera capable of inhibiting the physiological action of thyroglobulin. Previous attempts to apply this principle to steroids either have been unsuccessful (8) or have been reported only in abstract form with insufficient detail to permit evaluation of the results (9). Mooser and Grilichess (8) failed to obtain antibodies against conjugated proteins prepared from AS-androstene-3, 17-diol-17-(p-amino-o-sulfobenzoate) and A6-androstene-3, 17-diol-3-(p-amino-o-sulfobenzoate) by coupling the diazo- tized derivatives of these steroids with tyrosine residues in horse serum, bovine serum albumin, casein, or egg albumin.

This paper reports the preparation and properties of three steroid- protein conjugates derived from bovine serum albumin and testosterone or cortisone. The steroids in these conjugates are linked by amide bonds to the lysine residues of the protein. The 59 lysine residues of bovine

0

OAC, OH

odP pP I II

f :: ~H20G(CH2)2C.OH

c=o

tH2-COOH

FIG. 1. Steroid derivatives used for the preparation of steroid-protein conjugates

serum albumin, as contrasted with only 21 tyrosine residues, provide sites sufficient in number to produce conjugates with highly specific antigenic properties. The steroid derivatives used for coupling were compounds whose structural differences from the parent steroids were minimal.

Fig. 1 shows the structural formulas of the three coupling derivatives. These are as follows: testosterone 17-chlorocarbonate (I), prepared by the reaction of testosterone with phosgene (I), testosterone 3-(O-car- boxymethyl)oxime (II), prepared by the reaction of testosterone with 0-(carboxymethyl)hydroxylamine (H2NO.CH2.COOH) (lo), and corti- sone 21-hemisuccinate (III). Coupling was effected, in the case of the chlo- rocarbonate, by a simple Schotten-Baumann reaction and with the car- boxylic acid derivatives, by the mixed anhydride technique with use of isobutyl chlorocarbonate (11).

The three steroid-protein conjugates described in this paper are antigenic in rabbits, the antibodies being specific for the conjugates and not for bovine serum albumin.

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ERLASGER, BOREK, BEISER, ASI) LIEBERMAN 715

EXPERIMENTAL’

Preparation of Steroid-Protein Conjugates

l’-17-BXA-A solution of 2 gm. (5.7 mmoles) of testosterone l’i-chloro- carbonate (12) in 126 ml. of dioxane was added with vigorous stirring and cooling (ice bath) over a period of 20 minutes to a solution of 8.75 gm. (about 0.125 mmole) of bovine serum albumin2 in 187.5 ml. of water, 7 ml. of x NaOH, and 63 ml. of dioxane. Subsequently, an additional 3 ml. of N NaOH mere added dropwise, at which point the solution was some- what turbid. After the reaction had proceeded for 30 minutes, the further addition of 2.6 ml. of N NaOH resulted in a clear solution. Stirring and cooling were continued for a total of 4 hours. The solution was dialyzed for 18 hours and brought to a pH of 4.6 with 5 ml. of s HCI. The conju- gate precipitated and, after storage in the cold for 6 hours, was collect,ed by centrifugation. After being washed twice with water, it was redissolved in water by bringing the pH to 3.5 with dilute HCl. The clear solution was lyophilized, yielding 9.3 gm. of product, [o(]z5 -25.0” f 2” (10.0 mg. in 5.0 ml. of 0.05 M Tris (tris(hydroxymethyl)aminomethane) buffer, pH 8.5). The specific rotation of BSA was found to be -59.4” f 1” (25.1 mg. in 5.0 ml. of 0.05 M Tris buffer, pH 8.5).

In an analysis for BSA, the literature values (13) were NHZ-N, 1.38; total N, 16.07. Calculated for T-17-BSA (assumed molecular weight 78,530 and twenty-seven of 60 KHz groups substituted): NH,-N, 0.59; total N, 14.2; ratio3 of NH,-K to total N, 0.041; found, NH,-n’, 0.54: total N, 13.3; moisture, 6.0; ratio of NH*-N to total N, 0.041.

In one preparation, a crystalline product separated during the dialysis and was collected by centrifugation. A photomicrograph of the crystals is shown in Fig. 2.

In order to ascertain whether the conjugate contained any unbound or adsorbed steroid, three techniques were employed : (a) dialysis according

1 The following abbreviations are used in this paper: BSA = bovine serum albumin; T-17.BSA = the conjugate derived from the reaction of testosterone 17.chlorocar- bonate and bovine serum albumin; T-3-B&4 = the conjugate derived from testos- terone 3-(0-carboxymethyl)oxime and bovine serum albumin; C-21.BSA = the conjugate derived from cortisone 21.hemisuccinate and bovine serum albumin; FI>NB = I-fluoro-2,4-dinitrobenzene; DNI’ = 2,4-dinitrophenyl.

2 Armour and Company, Fraction V, powder. 3 Analytical figures are corrected for moisture content, determined by drying at

78” for 4 hours, a procedure which does not remove all the moisture. The significant data are the ratios of amino to total nitrogen, which are unaffected by moisture content. The calculated values are derived from data given in Table 7-B of Brand (13), from \vhich a table was constructed containing the figures for the total nitrogens, amino nitrogens, and molecular weights of conjugates containing 0 to 60 steroid substituents.

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716 STEROID-PROTEIN CONJUGATES. I

to the procedure of Zaffaroni (14), (b) continuous extraction of the solid conjugate with chloroform, and (c) hand extraction of an aqueous suspen- sion of the conjugate with ether.

(a) A cellophane casing containing a solution of 48 mg. of T-17-BSA in 100 ml. of Tris buffer (pH 8.5) and 50 ml. of methanol was placed in a glass cylinder with 200 ml. of 40 per cent aqueous methanol and 50 ml. of chloroform. The cylinder was stoppered and shaken with a longitudinal motion for 20 hours. After withdrawal of the dialysis tube from the cyl- inder, the chloroform phase of the dialysate was separated and the aqueous

FIG. 2. Photomicrograph of crystalline T-17-BSA

phase extracted with three 50 ml. portions of chloroform. The combined chloroform fractions were washed with water, dried over sodium sulfate, and evaporated to dryness. The residue, shown by its infrared spectrum (determined with Perkin-Elmer model No. 21 spectrometer) to be testos- terone, was estimated by ultraviolet spectrometry to comprise not more than 3 per cent of the weight of the conjugate.

(b) A sample of 24 mg. of T-17-BSA was continuously extracted with chloroform for 18 hours. The extract was filtered and evaporated to dryness. The amount of extracted testosterone determined spectrophoto- metrically was found to be 0.4 mg., or 2 per cent of the weight of the conjugate.

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ERLANGER, BOREK, BEISER, AND LIEBERMAN 717

(c) A sample of 7 mg. of T-17-BSA was suspended in 6 ml. of water and extracted in a separatory funnel with three 20 ml. portions of ether. The combined extracts were dried over sodium sulfate and evaporated to dry- ness. The amount of extracted testosterone determined spectrophoto- metrically was found to be 0.27 mg., or 4 per cent of the weight of the conjugate.

Since the foregoing experiments revealed the presence of unbound testosterone in T-17-BSA to the extent of 2 to 4 per cent, the following method was employed to remove this contaminant from the bulk of the conjugate: 450 ml. of ice-cold acetone were added gradually to a cooled, stirred solution of 800 mg. of T-17-BSA in 75 ml. of 1 per cent sodium bicarbonate. The resulting precipitate was collected by centrifugation. This treatment was repeated twice. After the third precipitation the solid was dissolved in 1 per cent NaHC03, dialyzed against running water, and lyophilized. The three acetone-containing supernatant liquids were evaporated to dryness, and the residues dissolved in ethanol, and examined spectrophotometrically for testosterone. The supernatant liquid from the first precipitation was found to contain 30 mg. of testosterone and that from the second precipitation 2 mg., the total being equivalent to 4 per cent of the weight of the conjugate. The supernatant liquid from the third precipitation contained no detectable testosterone. The product after acetone treatment was more difficultly soluble than the original conjugate in aqueous solvents.

T-S-BSA : Testosterone S-(0-Carboxymethyl)oxime-A solution of 0.98 gm. (3.4 mmoles) of testosterone and 1.07 gm. (8.4 mmoles) of (O-carboxy- methyl)hydroxylamine (10) in 200 ml. of ethanol was made alkaline by the addition of 20 ml. of 5 per cent NaOH and heated at a reflux for 1.5 hours. The solution was reduced to a small volume, diluted with water, and extracted with ether. The alkaline aqueous phase was acidified with concentrated hydrochloric acid. The resulting precipitate was extracted with ether, and the ether extract washed with water, dried over sodium sul- fate, and evaporated to dryness. The crude product weighed 0.84 gm. (68 per cent), m.p. 167-185’. Three recrystallizations from benzene- ligroin yielded 0.27 gm., m.p. 179-181”; [a] z5 f143.8” f 2” (19.5 mg. in 1.5 ml. of ethanol); +140.2” f 2” (26.2 mg. in 5.0 ml. of 0.05 M Tris buffer, pH 8.5). The ultraviolet spectrum of the solution in 0.05 M Tris buffer (pH 8.5) showed a maximum at 251 to 252 rnb (e 15,360). The infrared spectrum (Nujol) showed a band at 1730 cm.-l (C=O of carboxyl) and no absorption in the region 1670 to 1640 cm.-’ (conjugated C=O). Calcu- lated (C21H~I0,N). Neutral equivalent 361; C, 69.81; H, 8.59; N, 3.87; found, neutral equivalent 368; C, 69.72; H, 8.68; N, 3.92.

Preparation of Conjugates-l.1 gm. (3.05 mmoles) of testosterone 3-(0- carboxymethyl)oxime and 0.75 ml. (0.60 gm., 3.05 mmoles) of tri-n-butyl-

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718 STEROID-PROTEIN CONJUGATES. I

amine were dissolved in 30 ml. of dioxane and, after the solution was cooled to lo”, 0.40 ml. (0.45 gm., 3.05 mmoles) of isobutyl chlorocarbonate was added. The reaction was allowed to proceed for 20 minutes at 4”, after which the mixture was added in one portion to a well stirred, cooled solu- tion of 4.2 gm. (0.06 mmole) of BSA in 220 ml. of 1: 1 water-dioxane and 4.2 ml. of N NaOH. Gas evolution was apparent and after 1 hour an additional 2 ml. of N NaOH were added. Stirring and cooling were con- tinued for a total of 4 hours. The solution was dialyzed against running water for 18 hours and brought to pH 4.5 with N HCl. The product precipitated and, after storage in the cold for 4 days, was collected by centrifugation. It was suspended in 100 ml. of water and was redissolved by adding a minimal quantity of NaHC03. The clear solution was lyophilized, yielding 4.1 gm. of conjugate, [oL]:~ -14.3” f 2” (25.5 mg. in 5.0 ml. of 0.05 M Tris buffer, pH 8.5). Calculated (T-3-BSA) (assumed molecular weight 80,350; thirty of 60 NH2 groups substituted), NH*-N, 0.52; total N, 14.52; ratio3 of NHS-N to total N, 0.036; found, NHz-N, 0.45; total N, 12.56; moisture 9.0; ratio of NHZ-N to total N, 0.036.

Dialysis and extraction experiments showed that the amount of unbound steroid in this preparation was insignificant.

C-21-BXA-0.69 gm. (1.5 mmoles) of cortisone Zl-hemisuccinate4 in 15 ml. of dioxane, 0.35 ml. (0.28 gm., 1.5 mmoles) of tri-n-butylamine, and 0.19 ml. (0.21 gm., 1.5 mmoles) of isobutyl chlorocarbonate were allowed to react to form the mixed anhydride as described for T-3-BSA. The mixture was added in one portion to a stirred, cooled solution of 2.5 gm. (0.03 mmole) of BSA in 130 ml. of 1: 1 water-dioxane and 2.5 ml. of N

NaOH. The addition was followed by the appearance of slight turbidity and gas evolution. The pH fell to 7.5 and the solution became clear. After 1 hour another 1 ml. of N NaOH was added, the pH rising to 8.5. Stirring and cooling were continued for a total of 4 hours. The solution was dialyzed overnight against running water and was brought to a pH of 4.5 with N HCl. The resulting precipitate was stored in the cold overnight and then was collected by centrifugation. It was redissolved by bringing the pH to 5.5 with NaHC03, and the solution was dialyzed against running water for 4 hours. Lyophilization gave 1.6 gm. of conjugate, [o(]i5 -21.6” f 2” (26.3 mg. in 5.0 ml. of 0.05 M Tris buffer, pH 8.5). Calculated (C-Zl-BSA) (assumed molecular weight 81,490; twenty-six of 60 NH, groups substituted). NHZ-N, 0.58; total N, 13.79; ratio3 of NHZ-N to total N, 0.042; found, NHZ-N, 0.56; total N, 13.32; moisture, 9.0; ratio of NHZ-N to total N, 0.042.

Dialysis and extraction experiments showed that the amount of unbound

4 We wish to thank Merck and Company, Inc., for a generous supply of this com- pound.

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ERLANGER, BORER, BEISER, AND LIEBERMAN 719

steroid was equivalent to 2 to 3 per cent of the weight of the conjugate. The infrared spectrum of the residue obtained by evaporation of the dialysate corresponded to that of a mixture of cortisone and its 21-hemisuc- cinate.

A solution of 370 mg. of C-21-BSA in 60 ml. of 1 per cent NaHC03 was subjected to treatment with acetone, according to the procedure described for T-17-BSA. The first extract contained 8 mg. of steroid, and the second and third 1 mg. each. The total amount of steroid extracted was equiva- lent to 2.5 per cent of the weight of the conjugate. Similarly, as in the case of T-17-BSA, the acetone treatment decreased the solubility of C-21- BSA.

The BSA used for dinitrophenylation, electrophoresis, and ultraviolet spectroscopy was first subjected to experimental conditions similar to those under which the conjugates were prepared. In this way any effects due to alteration of the protein by solvents or other physical factors would be accounted for. The solubility of the treated BSA, as well as its electro- phoretic mobility and serological properties, was identical with those of the original BSA.

Characterixation of Steroid-Protein Conjugates

Ultraviolet Spectra-Fig. 3 shows the ultraviolet spectra of the three conjugates and the corresponding steroid derivatives,5 each accompanied by a spectrum of BSA. Testosterone 17-hemisuccinate4 exhibited an absorption maximum at 248 rnp in Tris buffer, and its molar extinction coefficient is 15,650 at this wave length. The absorbance of the steroid moiety of the T-17-BSA was determined by subtracting the absorption curve of BSA from that of T-17-BSA and was found to be maximal at 241 to 242 mp. In a similar way the steroid contributions to the spectra of T-3-BSA and C-21-BSA were derived and were maximal at 252 and 244 rnp, respectively.

The absorption curves were used to calculate the number of steroid residues in the conjugates (Table I). For these calculations the assump- tion was made that the extinction coefficients of the conjugated steroids are similar to those of the steroid derivatives from which they were pre- pared. A sample calculation follows: The optical density of T-17-BSA at 242 mp at a concentration of 45 mg. per liter is 0.288. The absorbance of BSA at the same concentration and wave length is 0.047; the difference attributed to testosterone 17-oxycarbonyl residues, therefore, is 0.241. Hence, the concentration of steroid residues is 0.241/15,650 liters per mole = 1.54 X lop5 mole per liter. Since the residue weight of testosterone 17-

5 Testosterone 17.hemisuccinate was substituted for testosterone 17-chlorocar- bonate because of its greater solubility in water.

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720 STEROID-PROTEIN CONJUGATES. I

oxycarbonyl is 315 gm. per mole, its concentration is 1.54 X lo@ mole per liter X 315 gm. per mole, or 4.9 mg. per liter. By subtracting the concentration of the steroid residues from that of T-17-BSA, the concen-

O.D. 600 A B

T- /7-b’s T-3- CM0

230 250 220 240 260 my my

I I I I I I I I I I

230 230 250 250 270 270 mp mp

FIG. 3. Ultraviolet spectra of conjugates and related substances in 0.05 M Tris buffer, pH 8.4. The curves composed of broken lines represent values obtained by subtracting the absorption of BSA from that of the conjugates. Concentration of BSA and conjugates = 6 X 10M7 M; other derivatives = 3.5 X 1O-6 M. Abbrevia- tions : T-17.HS = testosterone 17.hemisuccinate; T-3-CM0 = testosterone 3- (0-carboxymethyl)oxime; C-21-HS = cortisone 21.hemisuccinate.

TABLE I

Number of Steroid Residues per Molecule of Conjugate

Conjugate N&-N Ratio N Ultraviolet spectra Dinitrophenylation*

T-17-BSA. 27 27 36 T-3-BSA. 30 41 36 C-PI-BSA. 26 22 34

* These figures are mean values of at least four determinations.

tration of the BSA constituent is obtained (40.1 mg. per liter). If the molecular weight of BSA is assumed to be 70,000 (13), the number of moles per liter of BSA in the sample is 40.1 X 10s3 gm. per liter divided by 70,000 gm. per mole = 5.73 X 10W7.

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Therefore, the number of moles of t#estosterone per mole of BSA = 1.54 x 10-5/5.73 x 1o-7 = 26.9.

Analogous calculations provided the values for the other conjugates listed in Table I.6

Determina.tion oj Number of Steroid Residues by Dinitrophenylation-The experimental conditions of the coupling reactions between the st,eroid derivatives and BSA were chosen to favor the formation of amide linkages with the free amino groups of BSA. The reaction medium was maintained at an alkaline pH and the ratio of reactants was such that an excess of amino groups existed throughout the coupling. These conditions should deter the formation of est’er bonds between the steroid coupling agents and the phenolic or hydroxyl groups of BSA. BSA contains 59 lysine residues (13) and one N-terminal amino acid (15-17). There are, there- fore, 60 free amino groups available for attachment to the steroid reactants. If the number of free amino groups in the conjugates is compared to the number in BSA, the extent of steroid substitution in t’he conjugates can be estimated. This figure would be a minimal value since it would not include substitutions t,hat, despite the precautions, might have taken place on sites other than the amino groups.

In order to make this estimate, the conjugates and BSA mere dinitro- phenylated by the method of Sanger (18) and were hydrolyzed, and the amount of t-DNP lysine was estimated spectrophotometrically. No attempt was made to determine whether attachment to the terminal amino group occurred, since the error introduced by this simplification of t,he procedure is 1 part in 60, or less t,han 2 per cent. The dinitrophenyl- ated proteins (about 10 mg.) were hydrolyzed in a sealed tube in 6 N HCl (1 ml.) at 110” for 24 hours. The hydrolysates were extracted five times with 3 ml. portions of ether, and the aqueous layer was brought to a 50 ml. volume with N HCl and est’imated as E-DNP lysine spectrophotometri- tally at 390 rnp (19). It has been shown (20) that E-DNP lysine can be estimated directly under these conditions. Corrections were made for the destruction of E-DNP lysine during hydrolysis. The following is a sample calculation as applied to an hydrolysate of DiYP T-17-BSA: The corrected value for the number of moles of E-DNP lysine in 9.20 mg. of the DNP T-17-BSA is determined as 2.71 X 10p6. Since the residue weight, of dinitrophenyl is 167, there are 167 X 2.71 X 10m6, or 0.46 mg., of these residues in 9.20 mg. of T-17-BSA. Hence, the weight of the conjugate is 9.20 - 0.46, or 8.74 mg. Assuming a molecular weight of 80,000 for T-17-BSA (approximated from ultraviolet spectral data), the number of moles of conjugat’es is 1.09 X lo@. Therefore the number of moles of

6 The molar extinction coefficient of cortisone 21-hemisuccinate (A,,, 247.5 mN) is 16,400.

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722 STEROID-PROTEIN CONJUGATES. I

dinitrophenyl residues per mole of T-17-BSA is 2.71 X 10+/1.09 X lo-’ = 25.

The BSA control run with this sample was found to contain 61 E-amino

TABLE II Electrophoretic Mob&ties of Conjugates

Protein I

Mobility X 106, Mobility X 106, pH = 5.5, p = 0.2

I pH = 7.4, p = 0.2

cm.2 sozr’ sec.-’ cm.* v0u-l Sec.-’

BSA. ........................... 3.05 4.85 T-17-BSA. ...................... 4.60 7.66* T-3-BSA. ....................... 7.36 C-21-BSA. ...................... 5.41 7.47

*p = 0.1.

FIG. 4. Top, T-17-BSA (right) and BSA at pH 7.4. M/15 phosphate buffer, 1920 seconds (ascending). Bottom, C-PI-BSA (right) and BSA at pH 7.4, M/15

phosphate buffer, 2820 seconds (ascending).

groups. The number of blocked E-amino groups in T-17-BSA is therefore 61 - 25 = 36 and represents the extent of substitution by steroid residues.

Similar calculations were carried out with the hydrolysates of DNP T3-BSA and DNP C-21-BSA. The DNP derivatives of T-17-BSA and C-21-BSA were prepared from the conjugates before as well as after acetone treatment, the latter procedure being found to have no influence on the results. Table I shows the number of steroid residues in each of the three

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ERLANGER, BOREK, BEISER, AND LIEBERMAN 723

conjugates, each value being the mean of at least four dinitrophenylation experiments.

Electrophoretic Mobil&es-Table II lists the mobilities of BSA and the conjugates in 0.02 M phosphate buffer, pH 7.4, and 0.05 M acetate buffer, pH 5.5. As shown in Fig. 4, mixtures of either T-17-BSA or C-21-BSA with BSA were easily resolved electrophoretically. The conjugate T-3-

TABLE III

Comparison of Infrared Spectral Characteristics of Conjugates with Those of Related Steroids*

Conjugates Related steroids

T-I?-BSA Testosterone 17.chlorocarbonate

cm.-1 cm.-’

1018 Weak 1020 Weak 944 “ 950, 942 Medium 869 “ 865, 875 “

T-3-BSA Testosterone 3.(O-carboxymethyl)oxime

1078 Medium 1083, 1072 Medium 1027 Weak 1026 Strong

870 “ 880 “ 853 “ 855 “ 841 ‘I 833 “

C-Zl-BSA

1135 Weak 1048 “ 1055 (Shoulder)

925 Weak 868 “

Cortisone Zl-hemisuccinate -

1135 Strong 1042 “ 1050 (Shoulder)

925 Strong 868 Medium

* These absorption bands are absent from the spectrum of BSA.

BSA, because of its low solubility at pH 5.5, was investigated only at pH 7.4. At this pH its electrophoretic boundary was broad and low, and consequently its mobility could not be determined with accuracy. The electrophoretic mobilities of all three conjugates were similar at pH 7.4, being 1.5 times greater than that of BSA itself: At pH 5.5, the mobility of C-21-BSA was 1.8 times greater than that of BSA, whereas the ratios of the mobilities of T-17-BSA and BSA at this pH and at pH 7.4 were the same. The mobilities of the three conjugates were unaffected by acetone treatment.

Calculation of the number of steroid moieties per mole of conjugate

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724 STEROID-PROTEIN CONJUGATES. I

from the electrophoretic mobilities (with a value of 0.2 X 10-b mobility unit per charge (21)) gave results considerably lower than those obtained from the ultraviolet spectra and dinitrophenylation. Such calculations, however, are not reliable since they rest upon the assumption that the frictional properties of the conjugates are the same as those of BSA. Saroff et al. (22), working with methylated BSA, observed the same dis- crepancy and as a result of ultracentrifugal studies suggested that this conjugate possessed different frictional properties from those of BSA. It might be expected that such differences would be magnified in the steroid conjugates as a result of the greater complexity of the addition products.

Infrared Spectra-The infrared spectra of the free conjugates, determined as mulls in Nujol, provided an additional means of qualitative characteri- zation. Since the absorption band corresponding to the stretching fre- quencies of the A4-3-keto group of the steroid moieties occurs at the same wave length as the strong peptide absorption of the protein, characteristic bands could be determined only in the fingerprint region of the spectrum.

Table III lists the frequencies of the absorption bands present in the spectra of the conjugates and absent from the spectra of BSA. Given also for comparison are the frequencies of the absorption bands of the related steroid derivatives.

DISCUSSION

The determination of the number of steroid residues by hydrolytic procedures, both acidic and alkaline, was deemed impractical because control experiments had demonstrated that at most 50 per cent of the steroids could be recovered after being heated with acid or alkali in the presence of protein. AS a result the indirect methods described above were utilized.

The data presented in Tables I and II are consistent for conjugates composed of steroid residues covalently linked to bovine serum albumin. In addition, the data show that each molecule of conjugate contains at least twenty-two steroid moieties. Because of uncertainties inherent in the available analytical methods, the exact number of residues cannot be determined. For example, the accuracy of the ratio of amino to total nitrogen depends upon the determination of a small quantity of amino nitrogen. An error in this determination may be magnified in the ratio and may in extreme cases be as high as 15 per cent.

The estimates based upon ultraviolet spectroscopy may be too low because the calculations depend upon an assumption which may not be completely valid, i.e. that the extinction coefficient of the combined steroid moieties is the same as that of the simple steroid derivatives in aqueous buffer solution. The nature of the curves in Fig. 3 indicates that this

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ERLANGER, BOREK, BEISER, AND LIEBERMAN 725

assumption may not be true. The curve representing the contribution of the steroid residues in T-17-BSA has a maximum at a lower wave length than that of testosterone 17-succinate. This hypochromic shift resembles the findings of Westphal (23)) who noted a similar effect in protein solu- tions of steroids. His data also show an accompanying decrease (about 10 per cent) of extinction coefficients. If the spectral shift observed with steroid-protein conjugates is accompanied by an analogous diminution of absorbance, then the number of steroid residues would be underestimated. It is interesting to note that the spectral shift occurs with T-17-BSA and C-21-BSA but not with T-3-BSA. In the latter the chromophoric cw,p- unsaturated group is covalently linked to the point of attachment to BSA and is not free to associate with other parts of the protein molecules as it may in the two other conjugates. The spectral shift, therefore, may be the result of a non-covalent interaction (probably hydrogen bonding) between the a,/?-unsaturated system and bovine serum albumin, and thus similar in nature to the observations of Westphal.

The calculations resulting from the dinitrophenylation technique have two potential sources of error, neither affecting the results by more than 10 per cent. First of all, no correction has been made for the possibility that substitution may occur on sites other than the E-amino groups. In order to evaluate this possibility, T-17-BSA was examined for 0-tyrosyl substitution by the method of Herriot (24). A maximum of two steroid moieties was found to be combined in this manner. It is probable that the extent of 0-tyrosyl substitution in the other conjugates is just as low or even negligible, since they were prepared by the mixed anhydride procedure, a method which has been shown (25) to produce tyrosine peptides without concomitant 0 substitution.

Incomplete dinitrophenylation due to steric interference by steroid residues on neighboring amino groups may introduce another error in the estimation of the number of steroid residues by this technique. Since this possibility has not been examined before, it cannot be evaluat.ed, but it is probable that no more than a few lysine residues could be affected in this manner.

Despite the uncertainties mentioned above, there is little doubt that the conjugates contain between twenty-two and thirty-five steroid moieties per molecule of BSA. The immunochemical specificity observed with these conjugates provides further substantiation for a high steroid content.

The electrophoretic patterns indicate that T-17-BSA and C-21-BSA are remarkably homogeneous and therefore that the extent of substitution may fall within a very narrow range. The crystallizability of T-17-BSA sup- ports this contention. The relative mobilities of the conjugates and BSA demonstrate that the latter is more positively charged, as would be expected

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726 STEROID-PROTEIN CONJUGATES. I

if the steroid residues were linked by amide bonds to the t-amino groups of BSA.

The decreased solubility of the conjugates after acetone treatment may be due to a process of dehydration caused by the removal of hydrogen- bonded water molecules. In support of this conclusion is the observation that a sample of acetone-treated material doubled its weight upon exposure to air for several months. Drying in vacua at 78” over PZOS for 4 hours removed the absorbed moisture. As noted above, the acetone-treated materials were similar to the original conjugates in all other respects.

It is evident from Table III that many of the infrared absorption bands which characterize the simple related steroid derivatives in the fingerprint region are also present in the spectra of the steroid-protein conjugates themselves. Thus certain vibrational frequencies of the steroid molecules remain almost unchanged, even when these compounds are associated with bovine serum albumin. On the other hand, the absorption bands in the spectra of the steroids are considerably more intense and are better resolved than the corresponding bands in the spectra of the conjugates, as might be expected from a consideration of the relative dimensions of the molecules involved.

The preparation of antigenic steroid-protein molecules makes possible a rational approach to the development and study of antihormonal principles. The availability of these compounds may provide a means for the investi- gation of the mechanism of action and of transport of the steroid hormones, as well as a practical tool for counteracting their biological effects. With these objectives in mind, the synthesis and study of steroid hormone- protein conjugates other than those described here are in progress.

SUMMARY

In an attempt to produce substances which could elicit antibodies with antihormonal properties, derivatives of the steroids, testosterone and cortisone, were linked by amide bonds to bovine serum albumin. The preparation of two conjugates of testosterone, one linked through Ca and the other through C17, and one of cortisone joined through C&r, are de- scribed.

These conjugates have been analyzed and characterized by their ultra- violet and infrared spectra, electrophoretic behavior, and by dinitrophenyl- ation studies. By means of these techniques it has been estimated that at least 20 steroid residues have been covalently linked to each molecule of bovine serum albumin. These compounds have been found to be antigenic in rabbits. A description of these immunochemical experiments will be published shortly.

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ERLANGER, BOREK, BEISER, AND LIEBERMAN 727

The authors wish to express their gratitude to Dr. Helena De Roetth for some of the electrophoretic determinations and to Mr. James Finkelstein and Mr. Richard Epand for technical assistance. They also wish to thank Mr. L. M. Brancone and his associates at the Research Division, American Cyanamid Company, Pearl River, New York, for performing some of the elemental analyses reported in this paper.

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Beiser and Seymour LiebermanBernard F. Erlanger, Felix Borek, Sam M.

WITH CORTISONEALBUMIN WITH TESTOSTERONE AND

CONJUGATES OF BOVINE SERUM CHARACTERIZATION OF

PREPARATION AND STEROID-PROTEIN CONJUGATES: I.

1957, 228:713-728.J. Biol. Chem. 

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