Affinity chromatographic purification of the pregnancy zone protein

Journal o f Immunological Methods, 23 (1978) 117--125 © Elsevier/North-H~)lland Biomedical Press

117

A F F I N I T Y C H R O M A T O G R A P H I C P U R I F I C A T I O N OF THE P R E G N A N C Y ZONE P R O T E I N

J. FOLKERSEN 1, B. TEISNER 1, S. AHRONS 2, and S.-E. SVEHAG 1

1 Institute o f Medical Microbiology, Odense University, Odense and 2 Department o f Obstetrics and Gynecology, Odense University, Odense, Denmark (Received 19 January 1978, accepted 24 March 1978)

An immunospecific affinity chromatographic method for purification of human pregnancy zone protein (PZP) directly from serum is described. Highly purified goatanti-human PZP-immunoglobulin was applied as a ligand. Recovery of PZP varied from 56--75%. The impurities constituted maximally 5--10% of the total protein in the eluate. The purification factor was approximately 100.

INTRODUCTION

The first immunochemical study of serum from pregnant w o m e n was made by Thornes (1958) who established the existende of at least two pregnancy associated proteins. The designation 'pregnancy zone' protein was introduced by Smithies (1959) in his investigation of serum from pregnant w o m e n by starch gel electrophoresis. Pregnancy zone protein (PZP) has been studied more in detail by Stimson and Eubank-Scott (1972), and Von Schoultz and Stigbrand (1974). Its isoelectric point was estimated as 4.7, and the sedimentation coefficient was reported to be 12.2 S, b u t there is some uncertainty a b o u t its molecular weight which Stimson and Eubank-Scott (1972) reported as 506,000 while Von Schoultz and Stigbrand (1974) determined it as 359,000. PZP occurs in low concentrations in serum of normal men and nonpregnant w o m e n (Damber et al., 1976), but increased serum concentrations can be induced by intake of oestrogens (De Alvarez and Afonso, 1967; Beckman et al., 1971; H o m e et al., 1973 and Stimson, 1974). The use of PZP as a marker of certain types of breast cancer has been suggested by Stimson (1975a,b). The biological activity of PZP is unknown, although reports on 'immunosuppressive activities' in vitro of purified PZP-preparations have been published (Stimson, 1976; Von Schoultz et al., 1973). 'The immunological paradox of pregnancy' (Billingham, 1965; Burstein and Blumenthal, 1969; Hoffmann et al., 1974) has attracted much interest in recent years, and PZP has been suggested as one of the immunosuppressive factors operating in pregnancy. The aim of this study was to develop a purification m e t h o d which renders

118 it possible to produce large quantities of PZP for investigations of its immunological and biochemical properties. MATERIALS AND METHODS

Starting materials and preparations As starting material for the affinity chromatographic process, serum from pregnant w o m e n in the third trimester was employed. The same starting material was employed for the preparation of partially purified PZP-preparations for the immunization of animals. The partial PZP purification procedure was a combination of precipitation with aetacridine lactate and exclusion chromatography as earlier described (Folkersen et al., 1978a). Anti-PZP-immunoglobulin was purified from goat-anti-PZP hyperimmune sera. The immunization procedure has been described earlier (Folkersen et al., 1978a). Partially purified goat IgG was employed as ligand in preliminary affinity chromatography experiments. The partial purification was performed by recycling on Sephadex G-200 (Pharmacia, Uppsala, Sweden) in a K 5 0 / 1 0 0 column (Pharmacia, Uppsala, Sweden). Fractions containing IgG were pooled and concentrated by evaporation. Highly-purified monospecific goat-anti-PZP immunoglobulins were employed as ligands in the final affinity chromatographic experiments. This is the purification procedure described earlier (Folkersen et al., 1978a).

Affinity chromatography equipment The column used in all the affinity chromatographic procedures was a K 26/40 (Pharmacia, Uppsala, Sweden) with an inner diameter of 26 mm. F i f t e e n millilitres of packed cyanogen bromide-activated Sepharose 4B reached an approximate height of 30 mm in this column. A flow rate of 80 ml/h was used in all the procedures. Washing and elution processes were monitored at 280 nm by an ultraviolet absorption monitor, UA-5 (ISCO, Lincoln, NE). Fractions of 40 ml or 20 ml were collected on a fraction collector and dialysed against 0.15 M NaC1.

Selection of desorption conditions The choice of the final elution medium was decided by experiments in which PZP-containing samples were exposed to increasing concentrations of the main constituents. The highest ionic strength and the lowest pH, which gave no signs of denaturation, were chosen as optimal conditions. Changes in the height and appearance of the precipitate after rocket immunoelectrophoresis of the exposed samples were regarded as signs of partial denaturation. Complete disappearance of the precipitate was regarded as total loss of antigenicity. An unexposed PZP-containing sample constit u t e d the standard. All samples were exposed to the potentially denaturating constituents for 4 h and the treatment was terminated by dialysis against 0.15 M NaC1.

119

Ligand-coupling procedure The immunoglobulin preparation was dialysed overnight against a 0.1 M NaHCO3 solution containing 0.5 M NaC1 (pH 8.3). The dialysed antibody preparations were mixed with the swelled and cyanogen bromide-activated gel in an 'end-over-end' mixer for 2 h at room temperature. Blocking of excess activated groups was carried out with 1 M ethanolamine--HC1, pH 8. The gel was washed 4 times alternatively with an acetate buffer (pH 4), and borate buffer (pH 8), according to the manufacturer's prescription. After an additional wash with 0.07 M phosphate buffer (pH 7), the gel was suspended in a 0.4% solution of dextran blue 2 0 0 0 (Pharmacia, Uppsala, Sweden) corresponding to 5 times the swelled volume of the gel. The mixture was placed in an end-over-end mixer overnight at room temperature. The major part of the excess dextran blue was removed by 5 washings on a glass filter with 0.1 M barbital acetate--HC1 buffer (pH 8.5). After packing the column, the gel was washed for 24 h with the last-mentioned buffer (flow 80 ml/h). Antibodies o f very high affinity for PZP were blocked by perfusing the column with PZP containing serum, eluting with 5 M MgC12 and finally washing the column with 0.15 M NaC1 solution as in normal runs. The gel was then ready for use.

Affinity chromatography procedure The complete flow programme is given in Table 1. The gel was perfused with serum at a flow rate of 15 ml/h. All washing products were collected in TABLE 1 PROCESS AND FLOW PROGRAMME FOR ONE-STEP AFFINITY R A P H I C P U R I F I C A T I O N O F PZP D I R E C T L Y F R O M S E R U M Solution

Volume mt

Row mr/hour

Durofion hours

Procedure

0,15 M NoEL

80

8O

I

Prewosh

Sem~m

30

15

2

Perfusion wifh serum

0

0

3

Coupling

0,1 M Borbitotocelot HErbuffer pH 8,5

z,O0

80

5

1st woshing procedure

I M NoEl

320

80

4

2nd woshing procedure

3,0 Motor MgCL2 0,6 Molar NoEl pH 4,8

160

80

2

Elufion of PZP

0,15 I'4 NoEl

240

80

3

Wosh

CHROMATOG-

120 fractions of 40 ml, while the eluate was collected in fractions of 20 ml. The flow rate was 80 ml/h during these procedures. All fractions were dialysed against 0.15 M NaC1 for 24 h and concentrated by evaporation. The content of PZP and total protein was estimated. Selected fractions were tested in crossed immunoelectrophoresis against rabbit-anti-whole-pregnancy serum.

Electrophoretic methods Estimation o f PZP concentration. Quantitative estimations of PZP were carried out by rocket immunoelectrophoresis as described earlier (Folkersen et al., 1978a). The PZP-content was expressed in arbitrary PZP-units per ml (=APU/ml). Estimation o f specific antibody activity in antisera and antibody containing samples. Quantitative estimation of the antibody activity in anti-PZPimmunoglobulin containing samples was carried out by use of the antigen consumption-electroimmunoassay (ACE-assay) described earlier (Folkersen et al., 1978a). The activity was expressed in antigen consumption units (ACU), one ACU being defined as the amount of immunoglobulin that diminished the PZP content of a standard preparation with 1 APU under the test conditions. Purity test on fractions from the affinity chromatography column. Samples from the washing and elution steps were tested in crossed immunoelectrophoresis against rabbit-anti-whole-human antiserum (Behringwerke, Marburg) and rabbit-anti-whole-pregnancy serum (produced at our institute) dissolved in the second dimension gel. The first dimension was run at 10 V/ cm for 90 min and the second dimension at 1-~ V/cm for 18 h. After identification of the contaminating proteins in the eluate, by means of crossed immunoelectrophoresis and monospecific antisera, quantitative estimations were carried o u t with rocket immunoelectrophoresis using the same monospecific antisera to human plasma proteins (Behringwerke AG, Marburg). Protein-estimations Estimation of total protein content of test samples was made by photometric measurement of the absorption at 260 and 280 nm using bovine serum albumin as standard. Composition o f buffers and salt solutions used in the affinity chromatographic procedure (1) Barbital acetate--HC1 buffer (BAH-buffer), pH 8 . 5 : 2 0 . 6 h sodium barbital, 13.6 g sodium acetate and 15 ml I M HC1 were mixed with distilled water to a final volume of 1 liter (first washing fluid). (2) NaCl washing solutions: a) 1 M NaC1 (second washing fluid); b) 0.15 M NaC1 (inter-cyclus washing fluid). (3) MgC12-containing elution liquid: The MgC12 solution was used at a concentration of 5 M (corresponding to a molarity of about 3.0) containing 1 M NaC1 (corresponding to a molarity of 0.6).

121 RESULTS

Selection of desorption conditions The stability of PZP was studied by exposing it to a variety of possible constituents of the elution liquid. The use of different elution conditions gave the following results: (1) Exposure of PZP to pH ranging from 4.4-11.0 for 4 h had no denaturating effect, but pH values outside this range caused partial or complete denaturation. (2) Exposure of PZP to 3 M KSCN at pH 8 resulted in partial denaturation after 1 h and after 4 h only a trace of the immuno-precipitate was visible. (3) PZP was found to resist exposure to a 5 M solution of MgCl:, pH 4.4 for 4 h. (4) On exposure to a 5.0 M MgC12, 1.0 M NaC1 solution, pH 4.4 for 4 h, a slight change in the appearance of the precipitate was seen. However, when the pH was adjusted to 4.8 with 1 M NaOH, no sign of denaturation was observed within the same exposure time. (5) In a 6.0 M solution of urea, PZP was stable for 4 h, but denaturation occurred with higher concentrations. According to these results, low pH and KSCN were considered undesirable elution conditions. Desorption of PZP was achieved by repeated elution with 6.0 molal urea, but recovery proved to be too low. Both the MgC12containing liquids mentioned above had high desorption effect, but 5 M MgC12 containing 1.0 M NaC1, pH 4.8 was chosen as it gave the sharpest elution profile. BAH-Wash

I IM NaCl-Wash IMqCiEEluti°nl

0.150

100

E

JS0

E C)

-,~

0.100

60

E

40

o 0.05o

cL

N (2.

t~ <

20

,:A ....,IL i

5

10

i

15

20

25

3O

Fraction number Fig. 1. Ultraviolet profile for affinity chromatographic purification of PZP. The shaded areas indicate the concentration of PZP in the eluted fractions, expressed in APU/ml. The duration of the various washing and elution procedures is shown at the top of the figure.

122

Affinity chromatographic purification of PZP Figure 1 illustrates an affinity chromatographic run in which the column was not completely saturated by the PZP present in the serum sample. A combined washing procedure was employed consisting of 5 h wash with BAH-buffer (400 ml) and 4 h wash with a 1 M NaC1 solution (320 ml). The major part of the non-specifically bound proteins was removed in the BAH-wash (Fig. 2) and no PZP was detected in this washing liquid (Fig. 1). During the following wash with 1 M NaC1 solution, a slight desorption of PZP occurred (Fig. 1) simultaneously with the removal of contaminating proteins. This combined washing procedure was found optimal giving the highest recovery and purity of PZP u p o n subsequent elution with 5 M MgC12/ 1 M NaC1 solution. Exclusion of the NaC1 washing step required a prolonged BAH-wash and resulted in decreased PZP recovery, while the purity of PZP in the eluate was unchanged. The desorbed material was analysed by crossed immunoelectrophoresis using rabbit anti-human pregnancy serum and rabbit anti-whole human serum. The rabbit anti-human pregnancy serum produced in our laboratory was used routinely for identifying and quantifying PZP since its anti-PZP activity was greater than that of the commercial serum. Only one contaminating protein was visible in crossed immunoelectrophoresis (Fig. 3) and this protein was identified as a2-macroglobulin. However, careful examination of the concentrated (5×) elution product by rocket immunoelectrophoresis in addition revealed small amounts of albumin. Only these two contaminating proteins were detected by the two different antisera used.

Fig. 2. Crossed immunoelectrophoresis, using the BAH washing material as antigen and r a b b i t a n t i - w h o l e p r e g n a n c y s e r u m d i l u t e d 1 : 100 in the s e c o n d d i m e n s i o n gel.

123

/

.J

Fig. 3. Crossed i m m u n o e l e c t r o p h o r e s i s of the PZP-containing eluate as antigen and rabbit anti-wl~ole pregnancy serum diluted 1 : 100 in the second d i m e n s i o n gel. N o t e the precipitation line i m m e d i a t e l y above the baseline, which is due to c o n t a m i n a t i o n with ~-2-macroglobulin.

Quantitative rocket immunoelectrophoresis showed that ~2-macroglobulin and albumin constituted maximally 5--10% of the total protein in the eluate. The flow programme illustrated in Table 1 gave a PZP recovery of 56--75% (range from 16 runs) and a PZP purity in the eluate of 90--96%. Applying partially purified PZP to the chromatography column did not increase the purity or the recovery. By comparison with a serum pool obtained from pregnant women in the third trimester the purification factor was found to be approximately 100. DISCUSSION

Selection of desorption conditions Visible changes in the appearance and height of the precipitates after rocket immunoelectrophoresis of the exposed PZP-containing serum samples were regarded as signs of partial denaturation of the protein. This was supported by the results obtained when the same samples were analysed by crossed immunoelectrophoresis which showed that these changes in the height and morphology of the rockets were associated with the appearance of molecular populations of altered mobility, probably representing split products. Under the elution conditions described by Straube et al. (1974) exposure of ~t PZP containing sample to 3 M KSCN (pH 8) produced partial denaturation of the glycoprotein after 1 hour and complete denaturation after 4 h. Thus, in our hands 3 M KSCN could not be used for desorption of PZP.

124 Importance of ligand purity and the use of dextran blue In the initial experiments, partially purified goat-anti-PZP IgG preparations were employed for coupling to the cyanogen bromide activated gel. However, these gel preparations revealed an unacceptably high degree of nonspecific binding of 'nonsense' proteins to the gel ligand matrix in relation to the amount of specifically bound PZP. The following procedures were introduced in order to reduce this non-specific adsorption: (1) immunospecific purification of the ligand, (2) reduction of the time of ligand coupling, and (3) coupling of dextran blue to the gel-ligand matrix. Purification of the ligand by the immunosorbent technique previously described (Folkersen et al., 1978a) increased the specific activity of the goat-anti-PZP immunoglobulin preparation by a factor of 50--64, which in turn improved the affinity chromatographic method in two ways: (a) by reducing the amount of proteins in the ligand preparation which had a nonspecific affinity for certain plasma proteins; (b) by increasing the total binding capacity for PZP per millilitre gel-ligand preparation, which also reduced the relative amount of contaminating proteins in the PZP eluate. The non-specific binding of proteins to the gel matrix alone can be reduced by shortening the time of ligand coupling (Heinzel et al., 1976). This non-specific affinity was considered to be due to the formation of active urethane groups on the gel surface. Heinzel et al. (1976) concluded that 'the amount of non-specific adsorption is a function of both the cyanogen bromide concentration during activation and the time between activation and blocking with ethanolamine'. On these considerations we decided to reduce the time of ligand coupling from 24 h at 4°C to 2 h at room temperature. Heinzel et al. (1976) have also discussed the necessity of masking the remaining urethane groups by coupling with dextran blue. This dextran possesses non-specific adsorptive capacity, which, however, can be eliminated by introducing a 0.1 M BAH-buffer in the first washing step. The 1.0 M NaC1 solution used in the second washing step proved to be more effective for desorption of unspecifically bound proteins. The slight desorptive effect on PZP and the potentially denaturating effect of this washing fluid prevented its more extensive use. A marked increase of purity in the eluted PZP was achieved by the modifications of the affinity chromatographic procedure described above. The affinity chromatographic procedure can be modified to a fully automatic multi-programmed system. This system is described in detail in a subsequent communication (Folkersen et al., 1978b). ACKNOWLEDGEMENT This work was supported by the Danish Medical Research Council (Project No. 512-5685 and 512-7208). We thank Mrs. Jette Brandt for skilful technical assistance.

125 REFERENCES Beckman, L., Bo yon Schoultz, T. Stigbrand, 1971, Acta Obstet. Gynec. Scand. 50,369. Billingham, R.E., 1964, New Engl. J. Med. 270,667. Burstein, R.H., H.T. Blumenthal, 1969, Am. J. Obstet. Gynec. 104,691. Damber, M.G., B. yon Schoultz, T. Stigbrand, K. KarlstrSm, 1976, Clin. Chim. Acta 66, 85. De Alvarez, R.R., J.S. Afonso, 1967, Penn. Med. J. 70, 43. Folkersen, J., B. Teisner, P. Svendsen, S.-E. Svehag, 1978a, J. Immunol. Methods 23,127. Folkersen, J., B. Teisner, J. Westergaard, S.-E. Svehag, 1978b, J. Immunol. Methods 23, 137. Heinzel, W., I. Rahim-Laridjani, H. Grimmiger, 1976, J. Immunol. Methods 2,337. Hoffmann, R., H. Kyank, W. Straube, B. Klausch, 1974, Zbl. Gyn~k. 96, 1365. Home, C.H.W., A.L.C. McLay, H.B. Tavadia, I. Carmichael, A.C. Mallinson, A.A.C. Yeunk Laiwah, M.A. Thomas and R.N.H. Mac Sween 1973, Clin. Exp. Immunol. 13,603. Smithies, O., 1957, Adv. Protein Chem. 14, 65. Stimson, W.H., 1974, J. Endocrin. 61, 30. Stimson, W.H., 1975a, Lancet i, 777. Stimson, W.H., 1975b, Behring Inst. Mitt. 57, 42. Stimson, W.H., 1976, Clin. Exp. Immunol. 25, 199. Stimson, W.H., L. Eubank-Scott, 1972, FEBS Lett. 23,298. Straube, W., R. Hofmann, B. Klausch, G. Kiicken and J. Brock, 1974, Arch. Gyn~k. 217, 415. Thornes, R.D., 1958, Thesis, Dublin University. Von Schoultz, B., T. Stigbrand, A. T~rnvik, 1973. FEBS Lett. 38, 23. Von Schoultz, B., T. Stigbrand, 1974, Biochim. Biophys. Acta 359,303.