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Specificity and stability of guinea pig anti-progesterone antibodies
Molrculw Immunoioq~. Vol. Prmted nn Great Britam
19. No. 2. pp. 267-279.
IY82
0l61-58Y0/82/020267-l3SO3.00/0 0 1982 Pergamon Press Ltd
SPECIFICITY AND STABILITY OF GUINEA ANTI-PROGESTERONE ANTIBODIES G. B. HARDING,*
R. J. DOYLEt
and
PIG
U. WESTPHAL*
*Department of Biochemistry, and TDepartment of Microbiology and Immunology. University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. (First receiced
8 April
1981; accepted
in recisedform
12 June 1981)
Abstract-Antibodies against progesterone were induced in guinea pigs of both sexes by injection of progesterone-6[&hemisuccinate conjugated to bovine serum albumin (BSA) in a ratio of 16 moles of steroid per mole of protein. The concentration of antibody binding sites for progesterone of the animals studied ranged from 5 to 20@4. The expected heterogeneity of binding affinity for progesterone was observed with two major populations apparently predominating. One bound progesterone with an average affinity greater than 2 x IO9 M-i and the other showed an average affinity 56 x IO6 M-‘. The antibodies were found to be stable to extremes of pH and temperature in serum as well as in solutions of ammonium sulfate precipitates. The antibodies were not stable, however, in a more highly purified form. Attempts to obtain active preparations in high yield by purification beyond the ammonium sulfate step were unsuccessful. Competition studies and direct analysis with radiolabeled steroids showed the highaffinity population to be relatively specific for progesterone binding, whereas other steroids were bound according to the polarity rule indicating that the binding forces are predominantly hydrophobic,
INTRODUCTION
site. It was hoped that such an evaluation of an artificially-induced steroid-binding protein, synthesized in the same species that produces the pregnancy-associated progesterone-binding globulin (Blanford et al., 1978), would further characterize the common chemical nature of steroid binding sites.
Recent interest in high-affinity steroid-binding proteins has focused upon the nature and structure of the steroid binding site. This interest has been generated in the hope of discovering the chemical requirements for efficient binding of steroid hormones to serum proteins and target tissue receptors. It is thought that a clear understanding of the nature of the binding chemistry will reveal the mechanism and function of the protein-steroid complex, especially as related to the target tissue receptors. Various techniques have been used to compare the relative affinities of closely related steroids (Blanford et al., 1978; Lea, 1973; Westphal et al., 1977; Le Gaillard & Dautrevaux, 1977; Mickelson & Westphal, 1979, 1980) and thus probe the nature of the steroid binding site. Erlanger et al. (1957) produced the first antibodies against steroid hormones. Interest in antibodies against steroids has been directed primarily toward the determination of specificity and use in radioimmunoassays (Gross et al., 1968; Grover & Odell, 1977; Niswender et al., 1975; Peron & Caldwell, 1970). It was the purpose of the present studies to obtain in male and female nonpregnant guinea pigs, where progesterone binding is undetectable by conventional analysis (Diamond et al., 1969) an antibody of high specificity and high affinity for progesterone and to employ the techniques cited earlier to study its binding
MATERIALS
Reagents
AND
METHODS
and supplies
Progesterone-6/J-hemisuccinate covalently bound to e-amino groups of lysyl residues of bovine serum albumin (BSA) in a molar ratio of 16:l (BSA-prog,,) was purchased from Steraloids Inc., Wilton, NH. Incorporation of 16 moles of progesterone-6/I-hemisuccinate per mole BSA was confirmed by the procedure of Erlanger et al. (1957, 1959). [ 1,2-3H] Progesterone, 55.7 Ci/mmole, [1,2-3H]testosterone, 59 Ci/mmole, [1,2-‘HIdeoxycorticosterone, 46.8 Ci/mmole, and [1,2-3H]corticosterone, 40 Ci/mmole, were purchased from New England Nuclear Corp., Boston, Massachusetts. Purity of radiolabeled steroids was verified by thin-layer chromatography on commercial silica gel plates at the time of purchase and every 3-6 months afterwards. Solvent systems were chosen from Randerath (1966) on the basis of an R, of ap267
G. R. HARDING.
268
R. J. DOYLE
proximately 0.5 for the steroid under consideration. Tritium-labeled compound R.5020, was obtained from New England Nuclear Corp., Boston, Massachusetts, and nonlabeled R5020 from the Estrogen Receptor Screening Laboratory of the University of Louisville. Nonlabeled steroids were taken from our collection for which acknowledgments have been made in previous publications. Water used throughout these studies was doubly deionized and glass distilled. All chemicals were reagent grade or highest purity available. Adult Hartley albino guinea pigs of both sexes were purchased from Kentucky Cavies, Fern Creek, Kentucky, and housed in individual cages. Five-milliliter preimmune blood samples were drawn from each animal. Animals were immunized by injection of 0.1 or 0.4 mg BSA-prog,, dissolved in 0.4-0.5 ml of sterile saline and emulsified with an equal volume of Freund’s complete adjuvant. Injections were made intradermally into multiple sites of the foot pads and/or neck region. The first three boosters containing the same quantity of immunogen in Freund’s incomplete adjuvant-saline were given in multiple intradermal sites of the back at weekly intervals. Subsequent boosters were given in the same manner at monthly or 6-week intervals, dependent upon the duration of dermal necrosis. Five milliliter samples of blood were drawn on a bi-weekly schedule by heart puncture under Metafane anesthesia. Fluid volume and electrolyte were maintained by an intraperitoneal injection of 5 ml of lactated Ringer solution (Abbott Laboratories, North Chicago, Illinois) immediately before blood withdrawal. The blood was allowed to clot at 4C overnight and the serum stored at -85°C in individually labeled and dated vials.
Quantitation
qf binding
parameters
Equilibrium dialysis was performed on diluted serum or partially purified antibodies as previously described (Mickelson & Westphal, 1980). Buffer used was 50 mM phosphate, pH 7.4, containing mM EDTA and 0.02% (w/v) sodium azide, unless otherwise specified. Scatchard analysis data were evaluated by computer. semiquantitative or Rapid qualitative analyses of progesterone binding to antibodies were performed on temperature-regulated
and U. WESTPHAL
0.6 x 1Ocm ‘micro’ G-50 Sephadex columns (Pharmacia Fine Chemicals, Piscataway, New Jersey) at 2°C. Binding analyses were performed by filtering 0.1 ml of a dilute proteinp[3H]progesterone complex in 50 mM phosphate, pH 7.4, over the column at 2’C. Column effluent was collected directly into scintillation vials for counting. The void volume (25 drops) was collected in a single vial and 5 drop fractions thereafter. For comparathe bound steroid was tive evaluations, assumed to encompass the fractions between the void volume and the fraction of lowest counts per minute at the leading edge of the free steroid peak, generally drops 2650. Gel rlectrophoresis
and irnnlunoelectrophoresis
Gel electrophoresis was performed using only 7% acrylamide separator gels similar to the procedure of Ornstein (1964). Immunoelectrophoresis was run on Pfizer immunophoresis films (Pfizer Diagnostics Division, New York, New York) with a barbitalacetate buffer (0.06 M barbital, 0.05 M acetate, pH 8.6) in the electrode chambers. Current was applied for 1.25 hr at 100 V and the appropriate antiserum (10 ,ul) applied to the troughs. The films were incubated for 24-48 hr in a humid chamber, cleared of unprecipitated protein in 0.2 M saline, dried and stained for 60 set with Coomassie blue.
Pur$cution
of antibodies
Serum drawn from individual guinea pigs was evaluated for titer and affinity by multiple equilibrium dialysis. Samples that contained predominantly antibodies of high affinity for progesterone were pooled and submitted to ammonium sulfate fractionation as described by Herbert et al. (1973). Further purification of the gamma globulin fraction was attempted by a minor modification of the DEAE-cellulose method described by Garvey ef al. (1977). The sulfate free solution of gamma globulin was equilibrated to 17.5 mM phosphate, pH 6.3, and then with a saturating level of [3H]progesterone. Quantitation of recovery of antibody binding capacity from DEAE-cellulose columns was performed using the ammonium sulfate fraction from 3 ml of pooled antiserum saturated with [3H]progesterone on a 1 x 30cm DEAE-cellulose column. A linear gradient con-
Anti-progesterone
Antibodies in Guinea Pig
sisting of lOOmI each of starting buffer (17.5 mM phosphate, pH 6.3) and limit buffer (starting buffer made 1 M in NaCI) was applied. Affinity chromatography was performed according to the method of Cheng et al. (1976) for PBG, using deoxycorticosterone-21-hemisuccinate (10 E*moles per milliliter of gel) as ligand. Ammonium sulfate fractions, whole immune serum or preimmune serum was applied to the affinity gel at 4°C (0.5 ml~co~~lrnn) and the nonbinding protein eluted with 50mM phosphate, pH 7.4. The columns were incubated 1 hr at ambient temperatl~r~ with perturbant buffer and eluted under the same conditions. Perturbants employed were low pH (3.0, 4.5) buffer, a saturated solution of progesterone in loo/, ethanol--phosphate buffer, pH 7.4, 4 M urea, alanine-acetate buffer, pH 4.5, combined with electrophoresis or 1 M NH_+OH (Grenot & Cuilleron, 1977). Prechromatography controls and the pooled fractions of each peak were analysed for binding on the micro G-50 Sephadex columns. St&es
on antibody stffbjlity
InJluence of pH on antibody in serum and after partial purijication. The influence of pH
on the binding affinity of antibodies in serum and after partial purification was evaluated by performing dialysis experiments at 4°C and pH 2, 4, 6, 7.4, 8, 10 and 12. Similarly, the in~uence of pH on the stability of the binding proteins was evaluated by incubating samples for 20 hr at 4°C and the pH values given above. The solutions were adjusted to pH 7.4, diluted and subjected to multiple equilibrium dialysis at pH 7.4. Influence of temperature on antibody in serum and after partial pur$cation. The influence of
temperature on binding and protein stability was evaluated by performing equilibrium dialyses at 4, 15, 25, 40 and 6c?“C or by preincubating dialysis solutions at these temperatures, then readjusting them to 4°C to evaluate the renewable binding. in~uence
~~~ee~jng and thanking on partially
~z~r~~e~~ntibo~~~. An ammonium sulfate fraction was clarified by centrifugation for 40min at 17,OOOgand diluted to 10nM binding sites with 50 mM phosphate buffer, pH 7.4, and the same buffer containing 0.1 M NaCl, or 1 mM P-mercaptoethanol. Controls were refrigerated at 4°C and the test solutions frozen overnight
269
at -85°C. The protein solutions were brought to room temperature and the absorbances recorded at 280 and 350nm. Separate equilibrium dialysis series were executed for each protein solution and evaluated by the method of Scatchard as described. Similarly, protein was diluted to 10nM binding sites with 50mM phosphate, pH 7.4, and incubated with [3H]progesterone at 10 and 1OOnM concentration for 1.5 hr at 20°C. Controls were stored at 4°C and experimen~l samples frozen at -85°C overnight. Duplicate multiple equilibriLlm dialyses were run for both concentrations of progesterone. Binding was calculated as per cent of the controls. f$luettce of remocat of steroid from partially pur$ed antibody by Sephadex G-50 Jiltration or Amberlite XAD-2. A bed of Sephadex G-50
(1 x 28 cm) in a jacketed column was equilibrated with alanine-acetate buffer, pH 4.5, or 50 mM phosphate buffer, pH 7.4, at 40” or 20°C. Protein for these experiments was from a single ammol~ium sulfate fraction, 3 ml of which had been further purified on DEAE-cellulose with a saturation level of C3H]progesterone as described. The proteins were incubated for 1 hr using various combinations of pH and temperature and analysed for binding or conversely stripped on the Sephadex G-50 column at various combinations of pH and temperature, then analysed for binding. Binding was analysed by bringing the proteins to a final dilution of 1:45 or I:50 relative to the concentration in the ammonium sulfate fraction. The diluted protein was incubated with 2-fold excess of [3H]progesterone and analysed for binding on micro Sephadex G-50 columns. The binding of the protein solutions subjected to various treatments (temperature, pH and stripping) was calculated and presented as a percentage of the control protein solution. In one experiment [3H]-progesterone bound to DEAE-fractionated protein was removed by treatment with Amberlite XAD-2 (Rohm & Haas, Cincinnati, OH) in 0.05 M phosphate, 0.1% gelatin, in a similar way to that described by Chan & Slaunwhite (1977) and evaluated as described earlier. Variability in the evaluations, by micro co’lumn filtration of the various treatments discussed earlier, suggested that a careful quantitation be made of recovery from fractionation and steroid removal experiments. Each of the following antibody samples was adjusted to 1:500 dilution. relative to serum concentration,
270
G. B. HARDING.
R. J. DOYLE
with 50mM phosphate, pH 7.4, and a multiple equilibrium dialysis run against progesterone at 4°C: (1) antiserum; (2) 3 times precipitated gamma-globulin; (3) DEAE-cellulose chromatography pool 1 (see Fig. 1); (4) DEAE-cellulose chromatography pool 1 stripped of progesterone by filtration on a Sephadex G-50 column (1 x 30 cm) at 30°C pH 7.4; (5) DEAE-cellulose chromatography pool 1 stripped of progesterone by incubation for 4 hr at 20°C with XAD-2 in the presence of 0.1% gelatin. RESULTS
PuriJication
of antibody
Attempts at further purification of the ammonium sulfate-precipitated antibodies by 3.5 .
n
I
and U. WESTPHAL
DEAE-cellulose chromatography (Fig. 1) with traces or saturation levels of progesterone resulted in severe losses of binding activity. Duplicate experiments yielded 88% recovery of protein and of [3H]progesterone but only about 10% of the original serum binding activity. Improvement in antibody purity was only minimal, as demonstrated by gel and immunoelectrophoresis (Fig. 2). In every affinity chromatography experiment, pool 1 was essentially devoid of binding activity (Fig. 3) indicating binding of the antiprogesterone antibodies to the affinity resin. There was, in contrast, minimal loss when similar samples were filtered over Sepharose 4B columns or activated Sepharose free of the DOC-hemisuccinate. Recovery of active binding protein (peak 2) from the columns has not been accomplished in a reproducible manner. Specific precipitation of the progesterone binding antibodies was accomplished by incubation of ammonium sulfate fraction with an equivalence of BSA-prog,, at 4°C overnight. The precipitate was pelleted by centrifugation, washed with cold buffer and dissociated with an equivalence or IO-fold excess of C3H]progesterone. The binding activity was evaluated by dialysis without attempting to separate antibody and antigen. Calculation of the molarity of bound steroid showed approximately 10% of the expected concentration with considerable improvement in protein purity as shown by gel and immunoelectrophoresis (Fig. 4). Studies on antibody
,
75 Fraction
Number
Fig. 1. DEAE-cellulose chromatography of gamma-globulin fraction which was saturated with [3H]progesterone. Three milliliters of reconstituted ammonium sulfate fraction which has been equilibrated with 17.5 mM phosphate, pH 6.3, and clarified by spinning 2 times at 17,OOOg, 4°C. for 40min. was incubated with an equivalent of [3H]progesterone and run at 4°C on a 1 x 30cm DEAE-cellulose column. A linear gradient was applied consisting of 100 ml each of 17.5 mM phosphate, pH 6.3, and 17.5 mM phosphate, pH 6.3, containing 250mM sodium chloride. Absorbance at 280nm -0, cpm [3H]progesterone per lOO$ q ---- q, pmhos conductivity O---O. Recovery of [‘H]progesterone and protein were evaluated (see Table 1).
stability
The effect of pH on binding and stability of binding was investigated by equilibrium dialysis of serum at a final dilution of 1: 500 and of ammonium sulfate fractions at a comparable concentration of binding sites. Table 1 presents the results of these experiments. Only at the extreme alkaline pH did complete and irreversible inactivation occur. At pH 2, approximately half the total sites are inactivated but the affinity of the neutralized protein is not different from control values. The influence of temperature on diluted serum and ammonium sulfate fractions may be seen in Table 2. Only at 6OC was there any detectable irreversible inactivation of binding sites. There was a strong temperature effect on the binding affinity for progesterone which was completely reversible when the temperature was returned to 4°C. A similar decrease of K,
Anti-progesterone
Antibodies
in Guinea
Pig
271
A 0
100
50
Effluent
,
80
draps
Fig. 3. Evaluation of the recovery of progesterone binding activity from a DOC-affinity column by filtration on Sephadex G-50 micro-columns at Z’C, pH 7.4. Ammonium sulfate fraction was filtered on a DOC affinity column and the eluted pools evaluated for binding by adjusting the eluate to 1:lOO dilution (relative to the antiserum) with 50mM phosphate. pH 7.4. and incubation with [‘HIprogesterone for 30 min at 2OC, 5 min at 0-C. and filtering the mixture on Sephadex G-50 micro-columns at 2 C.I . ammonium sulfate fraction control: n. pool I eluate; pool 2 eluate. Inset: Affinity chromatogram: 3x and?--!. ammonium sulfate precipitated gamma-globulin fraction of 2ml of anti-serum were run on a DOC-hemisuccinateSepharose column as described.
Fig. 2. Immunoelectrophoresis and gel electrophoresis of protein samples from ammonium sulfate precipitation and DEAE-cellulose purification. (A) Immunoelectrophoresis. (1) Whole immune serum pool, (2) ammonium sulfate supernate 1, (3) ammonium sulfate supernate 2, (4) ammonium sulfate supernate 3, (5) 3 x -precipitated gammaglobulin, (6) DEAE-cellulose chromatography pool I. (7) preimmune serum. After electrophoresis IO nl of anti-guinea pig IgG (RPI) was placed in troughs a, c, e and g; and 10~1 of anti-whole guinea pig serum (Mann) in troughs b. d and f. (B) Disc gel electrophoresis. From left to right: (1) whole immune serum pool, (2) ammonium sulfate supernate I, (3) ammonium sulfate supernate 2. (4) ammonium sulfate supernate 3, (5) 3 x -precipitated gamma-globulin. (6) DEAE-cellulose chromatography pool I.
with increasing temperature was observed with the DEAE-cellulose-purified antibodies. Serum and concentrated solutions of redis-
solved ammonium sulfate precipitates were routinely stored at -85°C without significant loss of activity. Dilute solutions (10 nM sites), when frozen, were observed to consistently lose activity and to become visibly turbid upon thawing (Table 3 and Fig. 5). Ten-fold excess of progesterone, 0.1 M NaCl, or P-mercaptoethano1 did not prevent the aggregation or loss of binding activity. The removal of small mol. wt compounds by filtration on Sephadex G-50 columns had a considerable influence upon the stability of the binding capacity as shown in Table 4. Perturbation alone did not affect the site, whereas filtration on Sephadex G-50 always produced significant losses in activity. This observation is
212
G. B. HARDING,
R. .I. DOYLE
and U. WESTPHAL
protein that was simply filtered over the Sephadex G-50 (1 Y 30 cm) column at 40°C pH 7.4. In the first instance 91% of the binding was retained, whereas, only 38% of the binding of the stripped protein was retained. The single exception was a stripping run made with buffer containing O.l’/” gelatin which appears to have afforded significant protection. A more quantitative assessment of instability was shown by Scatchard analysis of multiple equilibrium dialyses (4°C pH 7.4) performed on protein at various stages of purification and removal of small molecular weight molecules. Table 5 shows that there is significant loss of activity by dialysis against low ionic strength buffer, chromatography on DEAE-cellulose or stripping on Sephadex G-50. Affinity chromatography of binding protein and elution by free ligand is considered to be a highly protective method of isolating such proteins. As shown earlier, almost quantitative removal of binding protein has been observed in every experiment performed. Various perturbants have been successful in eluting UV absorbing material in a pattern similar to that observed by Cheng et ul. (1976) for PBG. However, active protein has not been recovered in significant quantity.
Binding bodies
Fig. 4. Immunoelectrophoresis and disc gel electrophoresis of an immunospecific precipitate and its supernate. (A) Immunoelectrophoresis. (I) Antiserum pool, (2) 3 x precipitated gamma-globulin, (3) DEAE-cellulose chromatography pool I, (4) supernate of immunospecific precipitation, (5) immunospecific precipitate dissolved with 100-fold excess progesterone. After electrophoresis 10 ~1 of anti-whole guinea pig serum (Mann) was placed in troughs a, c and e, and 10~1 of anti-guinea pig IgG (RPI) in troughs b, d and f. The protein load was equal to IpI of serum except for the immunospecific precipitate which was equal to 4~1 of serum. (B) Disc gel electrophoresis. From left to right: (1) whole immune serum pool, (2) 3 x -precipitated gamma-globulin, (3) DEAE-cellulose chromatography pool 1, (4) supernate of immunospecific precipitation, (5) immunospecific precipitate dissolved with IOO-fold excess progesterone.
particularly evident in the comparison of three times precipitated gamma-globulin which was incubated 1 hr at 40°C pH 4.5, and the same
specijicity
of’ partiully
purified
anti-
The steroid binding specificity of ammonium and of DEAE-cellulosesulfate fractions purified antibodies was determined in equilibrium dialyses by either direct interaction of the protein with tritium-labeled steroid or by competitive inhibition of [3H]progesteroneeantibody interaction as applied by Mickelson & Westphal (1980) to guinea pig CBG (see illustration, Fig. 6). In a few cases both methods have been applied to assess the accuracy of the competitive inhibition method. When possible, duplicate determinations have been performed. Table 6 shows the results of duplicate experiwith DEAE-cellulose-purified antiments bodies. These experiments were performed at the same protein concentration with several weeks intervening time and are indicative of the range of variabilities encountered. Table 7 shows the results obtained with the ammonium sulfate fraction of pooled serum compared with guinea pig PBG affinity constants for the same steroids (Blanford et al., 1978).
Anti-progesterone
Table
I. Effect of pH
Antibodies
on progesterone binding gamma-globulin
2 4 6 7.4 8 10 12 Stability globulin
Sites (nM)
(X%, 0.4 0.9 1.8 ND” 1.9 0.9 0.02
in serum
3 x -precipitated
Serum (1:500)
PH
in Guinea
19.6 30.8 30.6
and
3 x-precipitated
gamma-globulin Sites (nM)
0.6 1.3 2.2 2.5 2.2 1.4 0.02
30.2 31.0 7.8
2.Y 2.5 2.6 ND 2.5 2.5 0.5
273
(&“)
of progesterone binding antibody in serum to pH perturbation for 20 hr at 4°C
2 --+ 7.4 4-+ 7.4 6-7.4 1.4 8-+ 7.4 lO-+ 7.4 1247.4
Pig
15.3 26.2 25.7
14.2 31.9 36.1 35.9 36.1 34.2 2.4
and 3 x -precipitated 2.2 1.5 1.8 2.5 1.8 1.7 0.6
25.3 25.5 4.6
gamma17.4 33.5 33.3 35.9 31.4 30.4 2.1
‘Not determined. ‘Proteins were incubated 20 hr at the pH indicated, titrated to pH 7.4, adjusted to 1:500 and a multiple equilibrium dialysis vs progesterone run for 48 hr at 4°C.
DISCUSSION
These studies have demonstrated that antibodies against the steroid hormone, progesterone, can be induced in guinea pigs when the hormone is covalently attached to a suitable carrier protein. The response is relatively rapid with a dose of 0.4mg per injection, which established both maximum affinity and titer at Table 2. Effect of temperature
on progesterone binding tated gamnla-globulin
Serum (1:500)” Temperature (‘C) 4 25 45 60 Stability globulin 4 2544 45-+4 6044
(XF& 2.1 0.7 0.2 0.06
of progesterone to temperature 2.1 2.8 2.4 1.4 (2.6)
about 8 weeks of immunization. Animals given 0.1 mg per injection under the same regimen as the higher dose group responded more slowly and were more variable in titer of binding protein during the course of several months of immunization. The conventional ammonium sulfate isolation of the gamma-globulin fraction was successfully applied to the antisera. Removal of in serum
3 x -precipitated Sites (nM) 30.7 28.7 27.1 17.9
binding antibody in serum perturbation for 24 hrh 30.7 26.9 28.8 23.0 (17.6)
(X%)
and 3 x -precipi-
gamma-globulin Sites Wf)
2.2 0.8 0.3 0.07 and 3 x -precipitated 2.2 2.5 2.3 (5::)
33.5 31.2 27.6 18.9 gamma 33.5 32.4 31.2 27.0 (22)
“Average of two experiments done on separate days. ‘Dialyses versus progesterone were prepared and incubated at the indicated temperature for 24 hr and then for 48 hr at 4’C. ‘These curves exhibited a significant increase in low affinity binding. Linear regression comparison showed that -59% of the original concentration of sites remained with a K, identical to controls,
274
Table
G. B. HARDING,
R. J. DOYLE
3. Absorbance of 10 nA4 solutions of 3 x -precipitated gamma-globulin before and after freezing _. A **onIll
10 nM control protein 1OnM protein in 1 mM P-mercaptoethanol 10 n&J protein frozen - 85°C 10 nM protein in 1 mM /?-mercaptoethanol frozen - 85’C See Fig. 5 for binding
A 350 Llill
0.013 0.019
0 0.006
0.120 0.130
0.072 0.080
data.
sulfate by dialysis against water or solutions of low ionic strength resulted in significant Iosses in binding capacity. The inability to recover significant quantities of binding activity from ion exchange chromatographies and affinity chromatographies suggested possible lability of the purified anti-progesterone antibodies. Veri-
a 10
5
Progesterone
bound,
nmoles
Fig. 5. Stability of progesterone binding to freezing in dilute solutions. Solutions of 3 x -precipitated gamma-globulin fraction were made (10 nM binding sites) in 50 mM phosphate, pH 7.4, and plus 1 mA4 ~-mercaptoethanol. Controls were refrigerated overnight and experimental samples frozen at - 85°C. -0, control in 50 mA4 PO,; O--O, control in 50 mM PO., f P-mercaptoethanol; -85°C (inset a-+); 50 mM 50mM PO, frozen PO1 + fl-mercaptoethanol frozen - 85°C (inset V---f). Similar results were observed in an experiment with DEAE-purified antibody using 0.1 M NaCi in 50 mM PO+
and U. WESTPHAL
fication of the loss of binding activity by quantitation of duplicate DEAE-cellulose chromatographies made it practical to use the ammonium sulfate fraction for the remainder of these studies. Analytical disc gel electrophoresis and immunoelectrophoresis demonstrated the absence of known steroid-binding proteins, such as PBG and CBG. The studies on stability arc noteworthy in two aspects. The ability of the protein to withstand the extremes of heat and pH perturbation, in whole serum and ammonium sulfate fractions, without severe losses in binding activity are similar to observations with the progesterone-binding globulin (PBG) of the pregnant guinea pig (Harding et al., 1974; MacLaughlin et al., 1972; Milgrom et al., 1973). No other known steroid-binding protein has the ability to retain binding activity under such severe conditions. In contrast, when the antibody was submitted to purification beyond the ammonium sulfate fractionation, losses in binding activity were observed. Routine dissociation of other antigen-antibody complexes with low pH buffers and various perturbant ions have given the impression of relatively high stability of all immunoglobulins (Dandliker et al., 1967; Garvey et al., 1977; Grenot & Cuilleron, 1977). In the present studies, however, the simple removal of steroid and/or other small ions or molecules by Sephadex G-50 filtration of ammonium sulfate fractions or DEAE-cellulose-purified antibodies irreversibly destroyed binding activity. Part of the observed instability of the isolated protein is related to aggregation or polymerization, as indicated by the increased absorbance and particle formation with concomitant loss of binding activity. The loss of binding affinity when the antibodies are removed from the protective milieu of the serum proteins and deprived of their stabilizing ligand is reminiscent of the lability of unprotected human CBG (Westphal, 1971, pp. 315.-324). The aggregation or polymerization with concomitant loss of binding activity are similar to observations made by Chader & Westphal on rabbit CBG (1968@ and rat CBG (1968a) which polymerized when steroid was removed from the purified proteins. It is dissimilar in that polymerization and inactivation of antibody is not reversed or inhibited by the presence of an equivalent or lo-fold excess amount of ligand as had been found for CBG (Chader & Westphal, 1968a, b; Chader er al., 1972).
Anti-progesterone
Table
4. Stability
Antibodies
in Guinea
of an ammonium sulfate fraction fied antibody
Pig
275
and DEAE-cellulose-puri-
Binding: 9,; control” Run 1 Control (3 x-precipitated antibody) 3 x -precipitated antibody incubated 1 hr, pH 4.5, 4O’C 3 x -precipitated antibody incubated 1hr. pH 4.5, 40-C, and stripped at pH 4.5, 40-C, on Sephadex G-50 3 x-precipitated antibody incubated 1 hr, pH 4.5, 4o’C, and stripped at pii 7.4,4O”C, on Sephadex G-50 3 x-precipitated antibody stripped at pH 7.4. 4o”C, on Sephadex G-50 DEAE-cellulose-purified antibody stripped at pH 7,4,4O”C, on Sephadex C-50 DEAE-cellulose-puri~ed antibody stripped at pH 7.4, 2o’C, on Sephadex G-50 DEAE-cellulose-purified antibody 0.1% gelatin stripped with XAD-2
Run 2
Run 3
100
100
100
19
85
109
34
60
ND
47
11
46
ND
29
19
51
44
38
9
13
ND
11
I7
23
ND
20
ND
76
ND
“Analysed for binding at pH 7.4 on a Sephadex hND = not determined.
The reasons for the observed instability are not clear. Other investigations with haptenspecific antibodies produced in guinea pigs have not reported comparable lability of the isolated proteins (Benacerraf & Gell, 1959; Edelman et al., 1961; Ovary et al., 1963; Quijada et al., 1974). Grenot & Cuilleron (1977), however, have observed the loss of binding acTable
5, Recovery of binding activity during isolation anti-progesterone from guinea pig serum Preparation (kW
Serum 3x pptd* 3x pptd’ DEAE-pool 1 DEAF-pool 1 XAD + gelatin DEALpool 1 Sephadex G-50 stripped
15 7
1
Preparation (wW
of
2
12 .-
4.2 2.5 3.2
4.6 0.4 2.7
1.7
0.8
“Binding site concentration corrected to undiluted serum. “Prior to dialysis against 17.5 mM phosphate, pH 6.3 (DEAE-starting buffer); determined in a separate equilibrium dialysis. ‘After dialysis against DEAE-starting buffer.
G-50 micro column
Average
91
at 2°C.
tivity in rabbit anti-dihydrotestosterone antibodies during the process of dialyzing out the steroid used to elute the antibodies from affinity columns. Other elution methods successfully employed by them, that is, electrophoretic elution in pH 4.5 alanine-acetate buffer and elution with 1 A4 NH40H solution, were not successful in eluting active protein from the DOCSepharose gels. Similar loss of activity upon purification was observed in ovine antiestrogens by Chard (personal communication); see also Chard (1978). The primary objective of the current study was to probe the structure and nature of the progesterone binding site of anti-progesterone antibodies. The instability of the protein and the inability to isolate the specific progesterone binding populations have prevented the exploration of this objective. However, the influence ofcertain polar and nonpolar groups as well as spatial or steric effects have been examined in a manner similar to that employed for PBG by Blanford et af. (1978). Because the technique of fluorescence quenching used by these authors could not be utilized, only a small number of different steroids have been studied. Table 7
276
G. B. HARDING,
R. J. DOYLE
and U. WESTPHAL
Substitutions R-OH
CNo.30 6p* Ila 170, 208, 21
R=CH,
CNo. 2a,
( R5020)
and 8.
6a
- 17a and 21 with 9, IO -ene
Side chain - removed testosterone = 17 - OH Ethynodrel = 17 - ethynl f I9 - CH, removed Fig. 6. Modifications of progesterone by substitution, reduction or removal of certain groups have been tested for binding by direct measurement of the radiolabeled compound in equilibrium dialysis or competitive displacement of [‘HIprogesterone in dialysis experiments. The structure of the progesterone molecule, the numbering of the carbons, and the modifications studied are shown.
shows that the antibodies produced in the guinea pig have certain similarities to cavian PBG with respect to their binding specificities. Introduction or removal of any group on the progesterone molecule resulted in a depression of the association constant in the limited survey performed in the present studies. Introduction of a single hydroxyl group decreased the association constants of the steroid with both proteins. In the case of PBG, the depression of affinity for these steroids is about an order of magnitude. In the antibodies the depression ranges from 80- to 165-fold, with the exception of the hapten 6/?-hydroxyprogesterone (6/3-hy-
droxy-4-pregnen-3,20-one), which shows only a 2- or 3-fold reduction. It is also of interest that the 1I/?-hydroxyl has less effect than those at carbons 1ICC, 208 or 21. The result with [3H]corticosterone appears to be a combination of the inhibition observed for Cl1 and C21 hydroxyls. The effect of the third hydroxyl introduced in the cortisol molecule is marked; the reduction in affinity is more than four orders of magnitude. The above results indicate that the antibody binding of steroids is governed by the polarity rule (Westphal, 1971, pp. 216236) which means that binding is primarily a hydrophobic interaction.
Table 6. Affinity constants, K,s, of anti-progesterone antibodies tation with ammonium sulfate and subsequent chromatography Run Steroid [-‘HIProgesterone Progesterone (competition)
I
(x IO%V) 21 23 0.021 7.4
purified by precipion DEAE-cellulose
Run 2
(x ,o%w- 1) Average 23 11 ND” 4.0
22 17
Desoxycorticosterone 6/?-Hydroxy-4-prcgnene3,20-dione Cortisol
0.0016
0.~05
5cc-Pregnanedione 5P-Pregnanedione
ND 2.7
2.0 4.0
3.4
3a-Hydroxy-5/?-pregnan-20-one
0.030
0.027
0.029
Testosterone
0.08 1
0.13
0.11
*ND = not determined.
5.1 0.001
Anti-progesterone
Antibodies in Guinea Pig
Table 7. Affinity constants (K,s) of anti-progesterone
277
and PBG
Anti-progesterone
(x m%-
Steroid [3H]Progesterone Progesterone (competition) Introduction of OH [aH]Deoxycorticosterone (DOC) DOC (competition) 6~-H~droxy-4-pre~nene-3,2O-diorl~ 1la-Hydroxy-4-pregnene-3,20-dione 1Ifi-Hydroxy-4-pregnene-3,2O-dione 4-Pregnen-20b-ol-3-ene [3H]Corticosterone Cortisol Reduction of 4-ene Sa-Pregnanedione 5~-Pr~gnanedione Reduction of 3 keto and 4-ene 3z+Hydroxy-S&pregnan-20-one Introduction of methyl group 2x-Methyl-progesterone 6a-Methyl-progesterone Removal of side chain E3H]Testosterone Testosterone (competition) Other [3H]R5020 Testosterone acetate BSA-prog, s
38 25 (n = 2)”
1.4 0.19 14 0.18 0.40 0.31 0.17 0.0024 17 8.8
‘f
PBG*
(x ~~~~~-1) I8 8.6 3.2 2.8 1.8 4.2 1.4 0.022 21 3.3
0.073
0.44
(n = 0.5) (n = 0.8)
0.60 0.51
2.3 -
(n = 0.57)
1.1 0.058
2.9 -
(n = 0.7)
0.20 0.46 4.8
0.85 9.2 -
“n = Concentration of sites detected relative to progesterone control. *Ammonium sulfate fraction of anti-serum and affinity purified guinea pig PBG: Scatchard analysis of equilibrium dialyses. PBG values from Blanford cf al. (1978).
Reduction of the Cl-5 double bond does not produce a large reduction in binding for either PBG or antibodies. The effect of the ‘bent’ conformation of the S&reduced compound is similar for both proteins. Reduction of the 3-0~0 group of 5~-pregnanedione, however, has a much greater effect on the binding affinity in the antibodies (-loo-fold) than in PBG (&fold). This suggests a greater importance of hydrophobi~ity in the antibody binding site. The greater sensitivity to alteration of the progesterone molecule may be largely due to a tighter spatial arrangement as shown by the similarity in binding to the two proteins of the pregnanediones and 6/I-hydroxyprogesterone, and the dissimilarity of binding in the case of testosterone acetate. As Blanford et cd. (1978) have indicated, testosterone acetate is structurally similar to progesterone except for the insertion of the ester oxygen between the acetyl group and (217. This oxygen produces a very slight increase in axial length of the molecule which decreases the binding to PBG to about half the progesterone value. However, in the antibodies studied, the reduction of K, is two orders of magnitude.
The differences between the association constants and concentrations of binding sites observed when the determinations were made by competition with [3H]progesterone and those obtained directly by equilibrium dialysis with the radiolabeled steroids deserve comment. Zimmering er al. (1967) have observed a similar phenomenon in the study of the binding of ‘heterologous’ steroid haptens to anti-testosterone succinate antibodies. As in the present study, they assume the sites for the ‘homologous’ steroid to be the total sites available. They do not consider the association constant an equilibrium constant since saturation by the ‘heteroiogous’ hapten occurs at a concentration lower than the total available sites. They do not comment on the observation that their Sips’ heterogeneity index rises above unity in some cases. It is our belief that all three phenomena are a result of the heterogeneous population of antibodies present. It is just as reasonable to believe’ that additional sites may be available to some ‘heterologous’ haptens that are not available to, or are of low affinity for, the immunizing hapten as it is to believe that sites available to the immunizing hapten
G. B. HARDING,
278
R. J. DOYLE
might not be available to some ‘heterologous’ haptens. In fact, this is what the differences in the concentration of sites measured by competition and by direct determination in the current studies suggest. The values of K, determined by direct measurement are more nearly true average equilibrium constants. The K, determined by competition can only be an accurate value for the population of antibodies from which it displaces progesterone. This concept of the binding of the ‘heterologous’ haptens is in harmony with the fact that the association constants for both testosterone and DOC determined by direct measurement are higher than those determined by competition, while the binding sites are 0.6 and 2.0 respectively, compared to progesterone. The higher-affinity sites for the heterologous hapten are undetectable in the competition experiments because they are inaccessible to progesterone. On the other hand, the K, value for progesterone determined by competition with radiolabeled progesterone is the same as that obtained directly by equilibrium dialysis because the two values are measured with the same population of antibodies. Acknowledgemenrs~The authors wish to thank Drs R. L. McGeachin and G. Sonnenfeld for their critique of this paper, and Mrs Ellen Ford for preparation of the manuscript. REFERENCES Benacerraf B. & Cell P. G. H. (1959) Studies on hypersensitivity I. Delayed and Arthus-type skin reactivity to protein conjugates in guinea pigs. Immunology 2, 53363. Blanford A. T., Wittman W.. Stroupe S. D. & Westphal U. (1978) Steroid-protein interactions--XXXVIII. Influence of steroid structure on affinity to the progesterone-binding globulin. J. Steroid Biochem. 9, 187-201. Chader G. J., Rust N., Burton R. M. & Westphal U. (1972) Steroid protein interactions-XXVI. Studies on the polymeric nature of the corticosteroid-binding globulin of the rabbit. J. biol. Chem. 247, 6581-6588. Chader G. J. 8~ Westphal U. (1968~) Steroid-protein interactions-XVIII. Isolation and observations on the polymeric nature of the corticosteroid-binding globulin of the rat. Biochemistry 7, 4272-4282. Chader G. J. & Westphal U. (1968b) Steroid-protein interactions-XVI. Isolation and characterization of the corticosteroid-binding globulin of the rabbit. J. biol. Chem. 243, 928-939.
Chan D. W. & Slaunwhite W. R., Jr. (1977) The chemistry of human transcortin: the effects of pH. urea, salt, and temperature on the binding of cortisol and progesterone. Archs Biochem.
Biophys.
182, 437.-442.
Chard T. (1978) An introduction to radioimmunoassay and related techniques. In Laboratory Techniques in Biochemistry and kolecular Biology (Edited by Work T. S. & Work E.). Vol. 6. North-Holland. Amsterdam. Cheng S-L., Stroupe S. D. & Westphal U. (1976) Steroidprotein interactions. Purification of progesterone-bind-
and U. WESTPHAL
ing globuhn
by affinity chromatography.
them. Sot. Left.
Frdn
Eur. Bio-
64, 38@384.
Dandliker W. B.. Alson R., de Saussure V. A., Kiersaenbaum F. & Levison S. A. (1967) The effect of chaotropic ions on the dissociation of antigen antibody complexes. Biochemisfry 6, 1460~1467. Diamond M., Rust N. 6i Westphal U. (1969) High-afhnity binding of progesterone, testosterone and cortisol in normal and androgen-treated guinea pigs during various reproductive stages: relationship to masculinization. Endocrino/og_v 84, I 143-l 15 1. Edelman G. M.. Benacerraf B.. Ovary Z. & Poulik M. D. (1961) Structural differences among antibodies of different specificities. Proc. ntrtn. Acud. Sci. I’.S.A. 47, 1751-1758.
Erlanger B. F., Borek F., Beiser S. M. & Lieberman S. (1957) Steroid-protein conjugates+ I. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J. hiol. Churn. 228, 7 13m727. Erlanger B. F., Borek, F.. Beiser S. M. & Lieberman S. (1959) Steroid protein conjugates II. Preparation and characterization of conjugates of bovine serum albumin with progesterone, deoxycorticosterone and estrone. J. hiol. Chrm. 234, 109@-1094. Garvey J. S., Cremer N. E. & Susdorf D. H. (1977) Mvrhods in Immunologic. W. A. Benjamin, London, Grenot C. & Cuilleron C-Y. (1977) Isolation of antibodies to steroid hormones by affinity chromatography on antigen-linked Sepharose: an efficient electrophoretic elution procedure. Biochrm. biophJ,.s. Res. Commun. 79, 274 279. Gross S. J.. Campbell D. H. & Weetall H. H. (1968) Production of antisera to steroids coupled to proteins directly through the phenolic ring. Immunocl~emisrr~~ 5, 55 -65.
Grover P. K. & Ode11 W. D. (1977) Specificity of antisera to sex steroids I. The effect of substitution and stereochemistry. J. Steroid Biochem. 8, 121 126. Harding G. B.. Burton R. M., Stroupe S. D. & Westphal. U. (1974) Steroid-protein interactions -XXVIII. The isoelectric point and pH stability of the progesteronebinding globulin. Life Sci. 14, 240552412. Herbert G-A., Pelham P. L. & Pittman B. (1973) Determination of the optimal ammonium sulfate concentration for the fractionation of rabbit. sheep. horse and goat antisera. Appl. Microbial. 25, 26-36. Kute T. E. (1975) Characterization of steroid binding sites in human a,-acid glycoprotein and cavian progesterone binding globulin. Ph.D. dissertation, University of Louisville. Lea 0. A. (1973) On the steroid specificity of the pregnant guinea pig progesterone-binding globulin. Biochim. hiophys. Acta 322, 68-74. Le Gaillard F. & Dautrevaux M. (1977) Affinity labeling of human transcortin. Biochim. hiophys. Acfa 495, 312.-323. MacLaughlin D. T.. Harding G. B. & Westphal U. (1972) Steroid-protein interactions XXV. Binding of progesterone and cortisol in pregnancy sera; progesteronebinding globulin and uterine cytosol receptor in the pregnant guinea pig. Am. J. Anut. 135. 1799186. Mickelson K. E. & Westphal U. (1979) Purification and characterization of the corticosteroid-binding globulin of pregnant guinea pig serum. Biochrmisrry 18;?685P2690. Mickelson K. E. & Westphal U. (1980) Steroid-protein interactions. Influence of steroid structure and temperature on the binding of steroids to guinea pig corticosteroid-binding globulin. Biochemistry 19, 585-590. Milgrom E., Allouch P., Atger M. & Baulieu E. E. (1973) Progesterone-binding plasma protein of pregnant guinea pig. Purification and characterization. J. biol. Chew. 248, 11061114. Niswender G. D.. Akbar A. M. & Nett T. M. (1975) Use of specific antibodies for quantification of steroid hor-
Anti-progesterone mones. In Methods in Enzpmloyy (Edited by O’Malley B. W. & Hardman J. G.), Vol. 36, pp. 16-34. Academic Press, New York. Ornstein L. (1964) Disc electrophoresis-----I. Background and theory. Ann. N.Y. Acad. Sci. 121. 321-349. Ovary Z., Benacerraf B. & Bloch K. (1963) Properties of guinea pig 7s antibodies-II. Identification of antibodies in passive cutaneous and systemic anaphylaxis. J. exp. Mud. 117, 951-964. Peron F. G. & Caldwell B. V., Eds (1970) Immunologic Merhods in Sferoid ~~~f~r~~ifzf~r~~ff. Appleton-CenturyCrofts, ?lew York. Quijada L.. Kim Y. T., Siskind G. W. & Ovary Z. (1974)
Antibodies
in Guinea
Pig
279
Immunogenicity of lowly and highly derivatized dinitrophenyl-bovine y-globulin in different strains of guinea pigs. J. Immun. 113, 129&1301. Randerath K. (1966) Thin Layer C~z~~~~f~gruph~. Verlag Chemie~Academic Press, New York. Westphal U. (1971) Steroid-Prorein Interactions. Springer, New York. Westphal U., Stroupe S. D., Kute T. & Cheng S.-L. (1977) Steroid interactions with progesterone-binding globulin. J. Steroid Biochm. 8. 367.-374. Zimmering P. E.. Lieberman S. & Erlanger B. F. (1967) Binding of steroids to steroid-specific antibodies. Biochemistr? 6, 154-164.
19. No. 2. pp. 267-279.
IY82
0l61-58Y0/82/020267-l3SO3.00/0 0 1982 Pergamon Press Ltd
SPECIFICITY AND STABILITY OF GUINEA ANTI-PROGESTERONE ANTIBODIES G. B. HARDING,*
R. J. DOYLEt
and
PIG
U. WESTPHAL*
*Department of Biochemistry, and TDepartment of Microbiology and Immunology. University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. (First receiced
8 April
1981; accepted
in recisedform
12 June 1981)
Abstract-Antibodies against progesterone were induced in guinea pigs of both sexes by injection of progesterone-6[&hemisuccinate conjugated to bovine serum albumin (BSA) in a ratio of 16 moles of steroid per mole of protein. The concentration of antibody binding sites for progesterone of the animals studied ranged from 5 to 20@4. The expected heterogeneity of binding affinity for progesterone was observed with two major populations apparently predominating. One bound progesterone with an average affinity greater than 2 x IO9 M-i and the other showed an average affinity 56 x IO6 M-‘. The antibodies were found to be stable to extremes of pH and temperature in serum as well as in solutions of ammonium sulfate precipitates. The antibodies were not stable, however, in a more highly purified form. Attempts to obtain active preparations in high yield by purification beyond the ammonium sulfate step were unsuccessful. Competition studies and direct analysis with radiolabeled steroids showed the highaffinity population to be relatively specific for progesterone binding, whereas other steroids were bound according to the polarity rule indicating that the binding forces are predominantly hydrophobic,
INTRODUCTION
site. It was hoped that such an evaluation of an artificially-induced steroid-binding protein, synthesized in the same species that produces the pregnancy-associated progesterone-binding globulin (Blanford et al., 1978), would further characterize the common chemical nature of steroid binding sites.
Recent interest in high-affinity steroid-binding proteins has focused upon the nature and structure of the steroid binding site. This interest has been generated in the hope of discovering the chemical requirements for efficient binding of steroid hormones to serum proteins and target tissue receptors. It is thought that a clear understanding of the nature of the binding chemistry will reveal the mechanism and function of the protein-steroid complex, especially as related to the target tissue receptors. Various techniques have been used to compare the relative affinities of closely related steroids (Blanford et al., 1978; Lea, 1973; Westphal et al., 1977; Le Gaillard & Dautrevaux, 1977; Mickelson & Westphal, 1979, 1980) and thus probe the nature of the steroid binding site. Erlanger et al. (1957) produced the first antibodies against steroid hormones. Interest in antibodies against steroids has been directed primarily toward the determination of specificity and use in radioimmunoassays (Gross et al., 1968; Grover & Odell, 1977; Niswender et al., 1975; Peron & Caldwell, 1970). It was the purpose of the present studies to obtain in male and female nonpregnant guinea pigs, where progesterone binding is undetectable by conventional analysis (Diamond et al., 1969) an antibody of high specificity and high affinity for progesterone and to employ the techniques cited earlier to study its binding
MATERIALS
Reagents
AND
METHODS
and supplies
Progesterone-6/J-hemisuccinate covalently bound to e-amino groups of lysyl residues of bovine serum albumin (BSA) in a molar ratio of 16:l (BSA-prog,,) was purchased from Steraloids Inc., Wilton, NH. Incorporation of 16 moles of progesterone-6/I-hemisuccinate per mole BSA was confirmed by the procedure of Erlanger et al. (1957, 1959). [ 1,2-3H] Progesterone, 55.7 Ci/mmole, [1,2-3H]testosterone, 59 Ci/mmole, [1,2-‘HIdeoxycorticosterone, 46.8 Ci/mmole, and [1,2-3H]corticosterone, 40 Ci/mmole, were purchased from New England Nuclear Corp., Boston, Massachusetts. Purity of radiolabeled steroids was verified by thin-layer chromatography on commercial silica gel plates at the time of purchase and every 3-6 months afterwards. Solvent systems were chosen from Randerath (1966) on the basis of an R, of ap267
G. R. HARDING.
268
R. J. DOYLE
proximately 0.5 for the steroid under consideration. Tritium-labeled compound R.5020, was obtained from New England Nuclear Corp., Boston, Massachusetts, and nonlabeled R5020 from the Estrogen Receptor Screening Laboratory of the University of Louisville. Nonlabeled steroids were taken from our collection for which acknowledgments have been made in previous publications. Water used throughout these studies was doubly deionized and glass distilled. All chemicals were reagent grade or highest purity available. Adult Hartley albino guinea pigs of both sexes were purchased from Kentucky Cavies, Fern Creek, Kentucky, and housed in individual cages. Five-milliliter preimmune blood samples were drawn from each animal. Animals were immunized by injection of 0.1 or 0.4 mg BSA-prog,, dissolved in 0.4-0.5 ml of sterile saline and emulsified with an equal volume of Freund’s complete adjuvant. Injections were made intradermally into multiple sites of the foot pads and/or neck region. The first three boosters containing the same quantity of immunogen in Freund’s incomplete adjuvant-saline were given in multiple intradermal sites of the back at weekly intervals. Subsequent boosters were given in the same manner at monthly or 6-week intervals, dependent upon the duration of dermal necrosis. Five milliliter samples of blood were drawn on a bi-weekly schedule by heart puncture under Metafane anesthesia. Fluid volume and electrolyte were maintained by an intraperitoneal injection of 5 ml of lactated Ringer solution (Abbott Laboratories, North Chicago, Illinois) immediately before blood withdrawal. The blood was allowed to clot at 4C overnight and the serum stored at -85°C in individually labeled and dated vials.
Quantitation
qf binding
parameters
Equilibrium dialysis was performed on diluted serum or partially purified antibodies as previously described (Mickelson & Westphal, 1980). Buffer used was 50 mM phosphate, pH 7.4, containing mM EDTA and 0.02% (w/v) sodium azide, unless otherwise specified. Scatchard analysis data were evaluated by computer. semiquantitative or Rapid qualitative analyses of progesterone binding to antibodies were performed on temperature-regulated
and U. WESTPHAL
0.6 x 1Ocm ‘micro’ G-50 Sephadex columns (Pharmacia Fine Chemicals, Piscataway, New Jersey) at 2°C. Binding analyses were performed by filtering 0.1 ml of a dilute proteinp[3H]progesterone complex in 50 mM phosphate, pH 7.4, over the column at 2’C. Column effluent was collected directly into scintillation vials for counting. The void volume (25 drops) was collected in a single vial and 5 drop fractions thereafter. For comparathe bound steroid was tive evaluations, assumed to encompass the fractions between the void volume and the fraction of lowest counts per minute at the leading edge of the free steroid peak, generally drops 2650. Gel rlectrophoresis
and irnnlunoelectrophoresis
Gel electrophoresis was performed using only 7% acrylamide separator gels similar to the procedure of Ornstein (1964). Immunoelectrophoresis was run on Pfizer immunophoresis films (Pfizer Diagnostics Division, New York, New York) with a barbitalacetate buffer (0.06 M barbital, 0.05 M acetate, pH 8.6) in the electrode chambers. Current was applied for 1.25 hr at 100 V and the appropriate antiserum (10 ,ul) applied to the troughs. The films were incubated for 24-48 hr in a humid chamber, cleared of unprecipitated protein in 0.2 M saline, dried and stained for 60 set with Coomassie blue.
Pur$cution
of antibodies
Serum drawn from individual guinea pigs was evaluated for titer and affinity by multiple equilibrium dialysis. Samples that contained predominantly antibodies of high affinity for progesterone were pooled and submitted to ammonium sulfate fractionation as described by Herbert et al. (1973). Further purification of the gamma globulin fraction was attempted by a minor modification of the DEAE-cellulose method described by Garvey ef al. (1977). The sulfate free solution of gamma globulin was equilibrated to 17.5 mM phosphate, pH 6.3, and then with a saturating level of [3H]progesterone. Quantitation of recovery of antibody binding capacity from DEAE-cellulose columns was performed using the ammonium sulfate fraction from 3 ml of pooled antiserum saturated with [3H]progesterone on a 1 x 30cm DEAE-cellulose column. A linear gradient con-
Anti-progesterone
Antibodies in Guinea Pig
sisting of lOOmI each of starting buffer (17.5 mM phosphate, pH 6.3) and limit buffer (starting buffer made 1 M in NaCI) was applied. Affinity chromatography was performed according to the method of Cheng et al. (1976) for PBG, using deoxycorticosterone-21-hemisuccinate (10 E*moles per milliliter of gel) as ligand. Ammonium sulfate fractions, whole immune serum or preimmune serum was applied to the affinity gel at 4°C (0.5 ml~co~~lrnn) and the nonbinding protein eluted with 50mM phosphate, pH 7.4. The columns were incubated 1 hr at ambient temperatl~r~ with perturbant buffer and eluted under the same conditions. Perturbants employed were low pH (3.0, 4.5) buffer, a saturated solution of progesterone in loo/, ethanol--phosphate buffer, pH 7.4, 4 M urea, alanine-acetate buffer, pH 4.5, combined with electrophoresis or 1 M NH_+OH (Grenot & Cuilleron, 1977). Prechromatography controls and the pooled fractions of each peak were analysed for binding on the micro G-50 Sephadex columns. St&es
on antibody stffbjlity
InJluence of pH on antibody in serum and after partial purijication. The influence of pH
on the binding affinity of antibodies in serum and after partial purification was evaluated by performing dialysis experiments at 4°C and pH 2, 4, 6, 7.4, 8, 10 and 12. Similarly, the in~uence of pH on the stability of the binding proteins was evaluated by incubating samples for 20 hr at 4°C and the pH values given above. The solutions were adjusted to pH 7.4, diluted and subjected to multiple equilibrium dialysis at pH 7.4. Influence of temperature on antibody in serum and after partial pur$cation. The influence of
temperature on binding and protein stability was evaluated by performing equilibrium dialyses at 4, 15, 25, 40 and 6c?“C or by preincubating dialysis solutions at these temperatures, then readjusting them to 4°C to evaluate the renewable binding. in~uence
~~~ee~jng and thanking on partially
~z~r~~e~~ntibo~~~. An ammonium sulfate fraction was clarified by centrifugation for 40min at 17,OOOgand diluted to 10nM binding sites with 50 mM phosphate buffer, pH 7.4, and the same buffer containing 0.1 M NaCl, or 1 mM P-mercaptoethanol. Controls were refrigerated at 4°C and the test solutions frozen overnight
269
at -85°C. The protein solutions were brought to room temperature and the absorbances recorded at 280 and 350nm. Separate equilibrium dialysis series were executed for each protein solution and evaluated by the method of Scatchard as described. Similarly, protein was diluted to 10nM binding sites with 50mM phosphate, pH 7.4, and incubated with [3H]progesterone at 10 and 1OOnM concentration for 1.5 hr at 20°C. Controls were stored at 4°C and experimen~l samples frozen at -85°C overnight. Duplicate multiple equilibriLlm dialyses were run for both concentrations of progesterone. Binding was calculated as per cent of the controls. f$luettce of remocat of steroid from partially pur$ed antibody by Sephadex G-50 Jiltration or Amberlite XAD-2. A bed of Sephadex G-50
(1 x 28 cm) in a jacketed column was equilibrated with alanine-acetate buffer, pH 4.5, or 50 mM phosphate buffer, pH 7.4, at 40” or 20°C. Protein for these experiments was from a single ammol~ium sulfate fraction, 3 ml of which had been further purified on DEAE-cellulose with a saturation level of C3H]progesterone as described. The proteins were incubated for 1 hr using various combinations of pH and temperature and analysed for binding or conversely stripped on the Sephadex G-50 column at various combinations of pH and temperature, then analysed for binding. Binding was analysed by bringing the proteins to a final dilution of 1:45 or I:50 relative to the concentration in the ammonium sulfate fraction. The diluted protein was incubated with 2-fold excess of [3H]progesterone and analysed for binding on micro Sephadex G-50 columns. The binding of the protein solutions subjected to various treatments (temperature, pH and stripping) was calculated and presented as a percentage of the control protein solution. In one experiment [3H]-progesterone bound to DEAE-fractionated protein was removed by treatment with Amberlite XAD-2 (Rohm & Haas, Cincinnati, OH) in 0.05 M phosphate, 0.1% gelatin, in a similar way to that described by Chan & Slaunwhite (1977) and evaluated as described earlier. Variability in the evaluations, by micro co’lumn filtration of the various treatments discussed earlier, suggested that a careful quantitation be made of recovery from fractionation and steroid removal experiments. Each of the following antibody samples was adjusted to 1:500 dilution. relative to serum concentration,
270
G. B. HARDING.
R. J. DOYLE
with 50mM phosphate, pH 7.4, and a multiple equilibrium dialysis run against progesterone at 4°C: (1) antiserum; (2) 3 times precipitated gamma-globulin; (3) DEAE-cellulose chromatography pool 1 (see Fig. 1); (4) DEAE-cellulose chromatography pool 1 stripped of progesterone by filtration on a Sephadex G-50 column (1 x 30 cm) at 30°C pH 7.4; (5) DEAE-cellulose chromatography pool 1 stripped of progesterone by incubation for 4 hr at 20°C with XAD-2 in the presence of 0.1% gelatin. RESULTS
PuriJication
of antibody
Attempts at further purification of the ammonium sulfate-precipitated antibodies by 3.5 .
n
I
and U. WESTPHAL
DEAE-cellulose chromatography (Fig. 1) with traces or saturation levels of progesterone resulted in severe losses of binding activity. Duplicate experiments yielded 88% recovery of protein and of [3H]progesterone but only about 10% of the original serum binding activity. Improvement in antibody purity was only minimal, as demonstrated by gel and immunoelectrophoresis (Fig. 2). In every affinity chromatography experiment, pool 1 was essentially devoid of binding activity (Fig. 3) indicating binding of the antiprogesterone antibodies to the affinity resin. There was, in contrast, minimal loss when similar samples were filtered over Sepharose 4B columns or activated Sepharose free of the DOC-hemisuccinate. Recovery of active binding protein (peak 2) from the columns has not been accomplished in a reproducible manner. Specific precipitation of the progesterone binding antibodies was accomplished by incubation of ammonium sulfate fraction with an equivalence of BSA-prog,, at 4°C overnight. The precipitate was pelleted by centrifugation, washed with cold buffer and dissociated with an equivalence or IO-fold excess of C3H]progesterone. The binding activity was evaluated by dialysis without attempting to separate antibody and antigen. Calculation of the molarity of bound steroid showed approximately 10% of the expected concentration with considerable improvement in protein purity as shown by gel and immunoelectrophoresis (Fig. 4). Studies on antibody
,
75 Fraction
Number
Fig. 1. DEAE-cellulose chromatography of gamma-globulin fraction which was saturated with [3H]progesterone. Three milliliters of reconstituted ammonium sulfate fraction which has been equilibrated with 17.5 mM phosphate, pH 6.3, and clarified by spinning 2 times at 17,OOOg, 4°C. for 40min. was incubated with an equivalent of [3H]progesterone and run at 4°C on a 1 x 30cm DEAE-cellulose column. A linear gradient was applied consisting of 100 ml each of 17.5 mM phosphate, pH 6.3, and 17.5 mM phosphate, pH 6.3, containing 250mM sodium chloride. Absorbance at 280nm -0, cpm [3H]progesterone per lOO$ q ---- q, pmhos conductivity O---O. Recovery of [‘H]progesterone and protein were evaluated (see Table 1).
stability
The effect of pH on binding and stability of binding was investigated by equilibrium dialysis of serum at a final dilution of 1: 500 and of ammonium sulfate fractions at a comparable concentration of binding sites. Table 1 presents the results of these experiments. Only at the extreme alkaline pH did complete and irreversible inactivation occur. At pH 2, approximately half the total sites are inactivated but the affinity of the neutralized protein is not different from control values. The influence of temperature on diluted serum and ammonium sulfate fractions may be seen in Table 2. Only at 6OC was there any detectable irreversible inactivation of binding sites. There was a strong temperature effect on the binding affinity for progesterone which was completely reversible when the temperature was returned to 4°C. A similar decrease of K,
Anti-progesterone
Antibodies
in Guinea
Pig
271
A 0
100
50
Effluent
,
80
draps
Fig. 3. Evaluation of the recovery of progesterone binding activity from a DOC-affinity column by filtration on Sephadex G-50 micro-columns at Z’C, pH 7.4. Ammonium sulfate fraction was filtered on a DOC affinity column and the eluted pools evaluated for binding by adjusting the eluate to 1:lOO dilution (relative to the antiserum) with 50mM phosphate. pH 7.4. and incubation with [‘HIprogesterone for 30 min at 2OC, 5 min at 0-C. and filtering the mixture on Sephadex G-50 micro-columns at 2 C.I . ammonium sulfate fraction control: n. pool I eluate; pool 2 eluate. Inset: Affinity chromatogram: 3x and?--!. ammonium sulfate precipitated gamma-globulin fraction of 2ml of anti-serum were run on a DOC-hemisuccinateSepharose column as described.
Fig. 2. Immunoelectrophoresis and gel electrophoresis of protein samples from ammonium sulfate precipitation and DEAE-cellulose purification. (A) Immunoelectrophoresis. (1) Whole immune serum pool, (2) ammonium sulfate supernate 1, (3) ammonium sulfate supernate 2, (4) ammonium sulfate supernate 3, (5) 3 x -precipitated gammaglobulin, (6) DEAE-cellulose chromatography pool I. (7) preimmune serum. After electrophoresis IO nl of anti-guinea pig IgG (RPI) was placed in troughs a, c, e and g; and 10~1 of anti-whole guinea pig serum (Mann) in troughs b. d and f. (B) Disc gel electrophoresis. From left to right: (1) whole immune serum pool, (2) ammonium sulfate supernate I, (3) ammonium sulfate supernate 2. (4) ammonium sulfate supernate 3, (5) 3 x -precipitated gamma-globulin. (6) DEAE-cellulose chromatography pool I.
with increasing temperature was observed with the DEAE-cellulose-purified antibodies. Serum and concentrated solutions of redis-
solved ammonium sulfate precipitates were routinely stored at -85°C without significant loss of activity. Dilute solutions (10 nM sites), when frozen, were observed to consistently lose activity and to become visibly turbid upon thawing (Table 3 and Fig. 5). Ten-fold excess of progesterone, 0.1 M NaCl, or P-mercaptoethano1 did not prevent the aggregation or loss of binding activity. The removal of small mol. wt compounds by filtration on Sephadex G-50 columns had a considerable influence upon the stability of the binding capacity as shown in Table 4. Perturbation alone did not affect the site, whereas filtration on Sephadex G-50 always produced significant losses in activity. This observation is
212
G. B. HARDING,
R. .I. DOYLE
and U. WESTPHAL
protein that was simply filtered over the Sephadex G-50 (1 Y 30 cm) column at 40°C pH 7.4. In the first instance 91% of the binding was retained, whereas, only 38% of the binding of the stripped protein was retained. The single exception was a stripping run made with buffer containing O.l’/” gelatin which appears to have afforded significant protection. A more quantitative assessment of instability was shown by Scatchard analysis of multiple equilibrium dialyses (4°C pH 7.4) performed on protein at various stages of purification and removal of small molecular weight molecules. Table 5 shows that there is significant loss of activity by dialysis against low ionic strength buffer, chromatography on DEAE-cellulose or stripping on Sephadex G-50. Affinity chromatography of binding protein and elution by free ligand is considered to be a highly protective method of isolating such proteins. As shown earlier, almost quantitative removal of binding protein has been observed in every experiment performed. Various perturbants have been successful in eluting UV absorbing material in a pattern similar to that observed by Cheng et ul. (1976) for PBG. However, active protein has not been recovered in significant quantity.
Binding bodies
Fig. 4. Immunoelectrophoresis and disc gel electrophoresis of an immunospecific precipitate and its supernate. (A) Immunoelectrophoresis. (I) Antiserum pool, (2) 3 x precipitated gamma-globulin, (3) DEAE-cellulose chromatography pool I, (4) supernate of immunospecific precipitation, (5) immunospecific precipitate dissolved with 100-fold excess progesterone. After electrophoresis 10 ~1 of anti-whole guinea pig serum (Mann) was placed in troughs a, c and e, and 10~1 of anti-guinea pig IgG (RPI) in troughs b, d and f. The protein load was equal to IpI of serum except for the immunospecific precipitate which was equal to 4~1 of serum. (B) Disc gel electrophoresis. From left to right: (1) whole immune serum pool, (2) 3 x -precipitated gamma-globulin, (3) DEAE-cellulose chromatography pool 1, (4) supernate of immunospecific precipitation, (5) immunospecific precipitate dissolved with IOO-fold excess progesterone.
particularly evident in the comparison of three times precipitated gamma-globulin which was incubated 1 hr at 40°C pH 4.5, and the same
specijicity
of’ partiully
purified
anti-
The steroid binding specificity of ammonium and of DEAE-cellulosesulfate fractions purified antibodies was determined in equilibrium dialyses by either direct interaction of the protein with tritium-labeled steroid or by competitive inhibition of [3H]progesteroneeantibody interaction as applied by Mickelson & Westphal (1980) to guinea pig CBG (see illustration, Fig. 6). In a few cases both methods have been applied to assess the accuracy of the competitive inhibition method. When possible, duplicate determinations have been performed. Table 6 shows the results of duplicate experiwith DEAE-cellulose-purified antiments bodies. These experiments were performed at the same protein concentration with several weeks intervening time and are indicative of the range of variabilities encountered. Table 7 shows the results obtained with the ammonium sulfate fraction of pooled serum compared with guinea pig PBG affinity constants for the same steroids (Blanford et al., 1978).
Anti-progesterone
Table
I. Effect of pH
Antibodies
on progesterone binding gamma-globulin
2 4 6 7.4 8 10 12 Stability globulin
Sites (nM)
(X%, 0.4 0.9 1.8 ND” 1.9 0.9 0.02
in serum
3 x -precipitated
Serum (1:500)
PH
in Guinea
19.6 30.8 30.6
and
3 x-precipitated
gamma-globulin Sites (nM)
0.6 1.3 2.2 2.5 2.2 1.4 0.02
30.2 31.0 7.8
2.Y 2.5 2.6 ND 2.5 2.5 0.5
273
(&“)
of progesterone binding antibody in serum to pH perturbation for 20 hr at 4°C
2 --+ 7.4 4-+ 7.4 6-7.4 1.4 8-+ 7.4 lO-+ 7.4 1247.4
Pig
15.3 26.2 25.7
14.2 31.9 36.1 35.9 36.1 34.2 2.4
and 3 x -precipitated 2.2 1.5 1.8 2.5 1.8 1.7 0.6
25.3 25.5 4.6
gamma17.4 33.5 33.3 35.9 31.4 30.4 2.1
‘Not determined. ‘Proteins were incubated 20 hr at the pH indicated, titrated to pH 7.4, adjusted to 1:500 and a multiple equilibrium dialysis vs progesterone run for 48 hr at 4°C.
DISCUSSION
These studies have demonstrated that antibodies against the steroid hormone, progesterone, can be induced in guinea pigs when the hormone is covalently attached to a suitable carrier protein. The response is relatively rapid with a dose of 0.4mg per injection, which established both maximum affinity and titer at Table 2. Effect of temperature
on progesterone binding tated gamnla-globulin
Serum (1:500)” Temperature (‘C) 4 25 45 60 Stability globulin 4 2544 45-+4 6044
(XF& 2.1 0.7 0.2 0.06
of progesterone to temperature 2.1 2.8 2.4 1.4 (2.6)
about 8 weeks of immunization. Animals given 0.1 mg per injection under the same regimen as the higher dose group responded more slowly and were more variable in titer of binding protein during the course of several months of immunization. The conventional ammonium sulfate isolation of the gamma-globulin fraction was successfully applied to the antisera. Removal of in serum
3 x -precipitated Sites (nM) 30.7 28.7 27.1 17.9
binding antibody in serum perturbation for 24 hrh 30.7 26.9 28.8 23.0 (17.6)
(X%)
and 3 x -precipi-
gamma-globulin Sites Wf)
2.2 0.8 0.3 0.07 and 3 x -precipitated 2.2 2.5 2.3 (5::)
33.5 31.2 27.6 18.9 gamma 33.5 32.4 31.2 27.0 (22)
“Average of two experiments done on separate days. ‘Dialyses versus progesterone were prepared and incubated at the indicated temperature for 24 hr and then for 48 hr at 4’C. ‘These curves exhibited a significant increase in low affinity binding. Linear regression comparison showed that -59% of the original concentration of sites remained with a K, identical to controls,
274
Table
G. B. HARDING,
R. J. DOYLE
3. Absorbance of 10 nA4 solutions of 3 x -precipitated gamma-globulin before and after freezing _. A **onIll
10 nM control protein 1OnM protein in 1 mM P-mercaptoethanol 10 n&J protein frozen - 85°C 10 nM protein in 1 mM /?-mercaptoethanol frozen - 85’C See Fig. 5 for binding
A 350 Llill
0.013 0.019
0 0.006
0.120 0.130
0.072 0.080
data.
sulfate by dialysis against water or solutions of low ionic strength resulted in significant Iosses in binding capacity. The inability to recover significant quantities of binding activity from ion exchange chromatographies and affinity chromatographies suggested possible lability of the purified anti-progesterone antibodies. Veri-
a 10
5
Progesterone
bound,
nmoles
Fig. 5. Stability of progesterone binding to freezing in dilute solutions. Solutions of 3 x -precipitated gamma-globulin fraction were made (10 nM binding sites) in 50 mM phosphate, pH 7.4, and plus 1 mA4 ~-mercaptoethanol. Controls were refrigerated overnight and experimental samples frozen at - 85°C. -0, control in 50 mA4 PO,; O--O, control in 50 mM PO., f P-mercaptoethanol; -85°C (inset a-+); 50 mM 50mM PO, frozen PO1 + fl-mercaptoethanol frozen - 85°C (inset V---f). Similar results were observed in an experiment with DEAE-purified antibody using 0.1 M NaCi in 50 mM PO+
and U. WESTPHAL
fication of the loss of binding activity by quantitation of duplicate DEAE-cellulose chromatographies made it practical to use the ammonium sulfate fraction for the remainder of these studies. Analytical disc gel electrophoresis and immunoelectrophoresis demonstrated the absence of known steroid-binding proteins, such as PBG and CBG. The studies on stability arc noteworthy in two aspects. The ability of the protein to withstand the extremes of heat and pH perturbation, in whole serum and ammonium sulfate fractions, without severe losses in binding activity are similar to observations with the progesterone-binding globulin (PBG) of the pregnant guinea pig (Harding et al., 1974; MacLaughlin et al., 1972; Milgrom et al., 1973). No other known steroid-binding protein has the ability to retain binding activity under such severe conditions. In contrast, when the antibody was submitted to purification beyond the ammonium sulfate fractionation, losses in binding activity were observed. Routine dissociation of other antigen-antibody complexes with low pH buffers and various perturbant ions have given the impression of relatively high stability of all immunoglobulins (Dandliker et al., 1967; Garvey et al., 1977; Grenot & Cuilleron, 1977). In the present studies, however, the simple removal of steroid and/or other small ions or molecules by Sephadex G-50 filtration of ammonium sulfate fractions or DEAE-cellulose-purified antibodies irreversibly destroyed binding activity. Part of the observed instability of the isolated protein is related to aggregation or polymerization, as indicated by the increased absorbance and particle formation with concomitant loss of binding activity. The loss of binding affinity when the antibodies are removed from the protective milieu of the serum proteins and deprived of their stabilizing ligand is reminiscent of the lability of unprotected human CBG (Westphal, 1971, pp. 315.-324). The aggregation or polymerization with concomitant loss of binding activity are similar to observations made by Chader & Westphal on rabbit CBG (1968@ and rat CBG (1968a) which polymerized when steroid was removed from the purified proteins. It is dissimilar in that polymerization and inactivation of antibody is not reversed or inhibited by the presence of an equivalent or lo-fold excess amount of ligand as had been found for CBG (Chader & Westphal, 1968a, b; Chader er al., 1972).
Anti-progesterone
Table
4. Stability
Antibodies
in Guinea
of an ammonium sulfate fraction fied antibody
Pig
275
and DEAE-cellulose-puri-
Binding: 9,; control” Run 1 Control (3 x-precipitated antibody) 3 x -precipitated antibody incubated 1 hr, pH 4.5, 4O’C 3 x -precipitated antibody incubated 1hr. pH 4.5, 40-C, and stripped at pH 4.5, 40-C, on Sephadex G-50 3 x-precipitated antibody incubated 1 hr, pH 4.5, 4o’C, and stripped at pii 7.4,4O”C, on Sephadex G-50 3 x-precipitated antibody stripped at pH 7.4. 4o”C, on Sephadex G-50 DEAE-cellulose-purified antibody stripped at pH 7,4,4O”C, on Sephadex C-50 DEAE-cellulose-puri~ed antibody stripped at pH 7.4, 2o’C, on Sephadex G-50 DEAE-cellulose-purified antibody 0.1% gelatin stripped with XAD-2
Run 2
Run 3
100
100
100
19
85
109
34
60
ND
47
11
46
ND
29
19
51
44
38
9
13
ND
11
I7
23
ND
20
ND
76
ND
“Analysed for binding at pH 7.4 on a Sephadex hND = not determined.
The reasons for the observed instability are not clear. Other investigations with haptenspecific antibodies produced in guinea pigs have not reported comparable lability of the isolated proteins (Benacerraf & Gell, 1959; Edelman et al., 1961; Ovary et al., 1963; Quijada et al., 1974). Grenot & Cuilleron (1977), however, have observed the loss of binding acTable
5, Recovery of binding activity during isolation anti-progesterone from guinea pig serum Preparation (kW
Serum 3x pptd* 3x pptd’ DEAE-pool 1 DEAF-pool 1 XAD + gelatin DEALpool 1 Sephadex G-50 stripped
15 7
1
Preparation (wW
of
2
12 .-
4.2 2.5 3.2
4.6 0.4 2.7
1.7
0.8
“Binding site concentration corrected to undiluted serum. “Prior to dialysis against 17.5 mM phosphate, pH 6.3 (DEAE-starting buffer); determined in a separate equilibrium dialysis. ‘After dialysis against DEAE-starting buffer.
G-50 micro column
Average
91
at 2°C.
tivity in rabbit anti-dihydrotestosterone antibodies during the process of dialyzing out the steroid used to elute the antibodies from affinity columns. Other elution methods successfully employed by them, that is, electrophoretic elution in pH 4.5 alanine-acetate buffer and elution with 1 A4 NH40H solution, were not successful in eluting active protein from the DOCSepharose gels. Similar loss of activity upon purification was observed in ovine antiestrogens by Chard (personal communication); see also Chard (1978). The primary objective of the current study was to probe the structure and nature of the progesterone binding site of anti-progesterone antibodies. The instability of the protein and the inability to isolate the specific progesterone binding populations have prevented the exploration of this objective. However, the influence ofcertain polar and nonpolar groups as well as spatial or steric effects have been examined in a manner similar to that employed for PBG by Blanford et af. (1978). Because the technique of fluorescence quenching used by these authors could not be utilized, only a small number of different steroids have been studied. Table 7
276
G. B. HARDING,
R. J. DOYLE
and U. WESTPHAL
Substitutions R-OH
CNo.30 6p* Ila 170, 208, 21
R=CH,
CNo. 2a,
( R5020)
and 8.
6a
- 17a and 21 with 9, IO -ene
Side chain - removed testosterone = 17 - OH Ethynodrel = 17 - ethynl f I9 - CH, removed Fig. 6. Modifications of progesterone by substitution, reduction or removal of certain groups have been tested for binding by direct measurement of the radiolabeled compound in equilibrium dialysis or competitive displacement of [‘HIprogesterone in dialysis experiments. The structure of the progesterone molecule, the numbering of the carbons, and the modifications studied are shown.
shows that the antibodies produced in the guinea pig have certain similarities to cavian PBG with respect to their binding specificities. Introduction or removal of any group on the progesterone molecule resulted in a depression of the association constant in the limited survey performed in the present studies. Introduction of a single hydroxyl group decreased the association constants of the steroid with both proteins. In the case of PBG, the depression of affinity for these steroids is about an order of magnitude. In the antibodies the depression ranges from 80- to 165-fold, with the exception of the hapten 6/?-hydroxyprogesterone (6/3-hy-
droxy-4-pregnen-3,20-one), which shows only a 2- or 3-fold reduction. It is also of interest that the 1I/?-hydroxyl has less effect than those at carbons 1ICC, 208 or 21. The result with [3H]corticosterone appears to be a combination of the inhibition observed for Cl1 and C21 hydroxyls. The effect of the third hydroxyl introduced in the cortisol molecule is marked; the reduction in affinity is more than four orders of magnitude. The above results indicate that the antibody binding of steroids is governed by the polarity rule (Westphal, 1971, pp. 216236) which means that binding is primarily a hydrophobic interaction.
Table 6. Affinity constants, K,s, of anti-progesterone antibodies tation with ammonium sulfate and subsequent chromatography Run Steroid [-‘HIProgesterone Progesterone (competition)
I
(x IO%V) 21 23 0.021 7.4
purified by precipion DEAE-cellulose
Run 2
(x ,o%w- 1) Average 23 11 ND” 4.0
22 17
Desoxycorticosterone 6/?-Hydroxy-4-prcgnene3,20-dione Cortisol
0.0016
0.~05
5cc-Pregnanedione 5P-Pregnanedione
ND 2.7
2.0 4.0
3.4
3a-Hydroxy-5/?-pregnan-20-one
0.030
0.027
0.029
Testosterone
0.08 1
0.13
0.11
*ND = not determined.
5.1 0.001
Anti-progesterone
Antibodies in Guinea Pig
Table 7. Affinity constants (K,s) of anti-progesterone
277
and PBG
Anti-progesterone
(x m%-
Steroid [3H]Progesterone Progesterone (competition) Introduction of OH [aH]Deoxycorticosterone (DOC) DOC (competition) 6~-H~droxy-4-pre~nene-3,2O-diorl~ 1la-Hydroxy-4-pregnene-3,20-dione 1Ifi-Hydroxy-4-pregnene-3,2O-dione 4-Pregnen-20b-ol-3-ene [3H]Corticosterone Cortisol Reduction of 4-ene Sa-Pregnanedione 5~-Pr~gnanedione Reduction of 3 keto and 4-ene 3z+Hydroxy-S&pregnan-20-one Introduction of methyl group 2x-Methyl-progesterone 6a-Methyl-progesterone Removal of side chain E3H]Testosterone Testosterone (competition) Other [3H]R5020 Testosterone acetate BSA-prog, s
38 25 (n = 2)”
1.4 0.19 14 0.18 0.40 0.31 0.17 0.0024 17 8.8
‘f
PBG*
(x ~~~~~-1) I8 8.6 3.2 2.8 1.8 4.2 1.4 0.022 21 3.3
0.073
0.44
(n = 0.5) (n = 0.8)
0.60 0.51
2.3 -
(n = 0.57)
1.1 0.058
2.9 -
(n = 0.7)
0.20 0.46 4.8
0.85 9.2 -
“n = Concentration of sites detected relative to progesterone control. *Ammonium sulfate fraction of anti-serum and affinity purified guinea pig PBG: Scatchard analysis of equilibrium dialyses. PBG values from Blanford cf al. (1978).
Reduction of the Cl-5 double bond does not produce a large reduction in binding for either PBG or antibodies. The effect of the ‘bent’ conformation of the S&reduced compound is similar for both proteins. Reduction of the 3-0~0 group of 5~-pregnanedione, however, has a much greater effect on the binding affinity in the antibodies (-loo-fold) than in PBG (&fold). This suggests a greater importance of hydrophobi~ity in the antibody binding site. The greater sensitivity to alteration of the progesterone molecule may be largely due to a tighter spatial arrangement as shown by the similarity in binding to the two proteins of the pregnanediones and 6/I-hydroxyprogesterone, and the dissimilarity of binding in the case of testosterone acetate. As Blanford et cd. (1978) have indicated, testosterone acetate is structurally similar to progesterone except for the insertion of the ester oxygen between the acetyl group and (217. This oxygen produces a very slight increase in axial length of the molecule which decreases the binding to PBG to about half the progesterone value. However, in the antibodies studied, the reduction of K, is two orders of magnitude.
The differences between the association constants and concentrations of binding sites observed when the determinations were made by competition with [3H]progesterone and those obtained directly by equilibrium dialysis with the radiolabeled steroids deserve comment. Zimmering er al. (1967) have observed a similar phenomenon in the study of the binding of ‘heterologous’ steroid haptens to anti-testosterone succinate antibodies. As in the present study, they assume the sites for the ‘homologous’ steroid to be the total sites available. They do not consider the association constant an equilibrium constant since saturation by the ‘heteroiogous’ hapten occurs at a concentration lower than the total available sites. They do not comment on the observation that their Sips’ heterogeneity index rises above unity in some cases. It is our belief that all three phenomena are a result of the heterogeneous population of antibodies present. It is just as reasonable to believe’ that additional sites may be available to some ‘heterologous’ haptens that are not available to, or are of low affinity for, the immunizing hapten as it is to believe that sites available to the immunizing hapten
G. B. HARDING,
278
R. J. DOYLE
might not be available to some ‘heterologous’ haptens. In fact, this is what the differences in the concentration of sites measured by competition and by direct determination in the current studies suggest. The values of K, determined by direct measurement are more nearly true average equilibrium constants. The K, determined by competition can only be an accurate value for the population of antibodies from which it displaces progesterone. This concept of the binding of the ‘heterologous’ haptens is in harmony with the fact that the association constants for both testosterone and DOC determined by direct measurement are higher than those determined by competition, while the binding sites are 0.6 and 2.0 respectively, compared to progesterone. The higher-affinity sites for the heterologous hapten are undetectable in the competition experiments because they are inaccessible to progesterone. On the other hand, the K, value for progesterone determined by competition with radiolabeled progesterone is the same as that obtained directly by equilibrium dialysis because the two values are measured with the same population of antibodies. Acknowledgemenrs~The authors wish to thank Drs R. L. McGeachin and G. Sonnenfeld for their critique of this paper, and Mrs Ellen Ford for preparation of the manuscript. REFERENCES Benacerraf B. & Cell P. G. H. (1959) Studies on hypersensitivity I. Delayed and Arthus-type skin reactivity to protein conjugates in guinea pigs. Immunology 2, 53363. Blanford A. T., Wittman W.. Stroupe S. D. & Westphal U. (1978) Steroid-protein interactions--XXXVIII. Influence of steroid structure on affinity to the progesterone-binding globulin. J. Steroid Biochem. 9, 187-201. Chader G. J., Rust N., Burton R. M. & Westphal U. (1972) Steroid protein interactions-XXVI. Studies on the polymeric nature of the corticosteroid-binding globulin of the rabbit. J. biol. Chem. 247, 6581-6588. Chader G. J. 8~ Westphal U. (1968~) Steroid-protein interactions-XVIII. Isolation and observations on the polymeric nature of the corticosteroid-binding globulin of the rat. Biochemistry 7, 4272-4282. Chader G. J. & Westphal U. (1968b) Steroid-protein interactions-XVI. Isolation and characterization of the corticosteroid-binding globulin of the rabbit. J. biol. Chem. 243, 928-939.
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