Biochem. J. (1979) 177, 471-476 Printed in Great Britain

471

Purification and Properties of Glutathione Peroxidase from Human Placenta By YOGESH C. AWASTHI, DAT D. DAO, ANJANA K. LAL and SATISH K. SRIVASTAVA Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77550, U.S.A.

(Received 8 June 1978)

Glutathione peroxidase (glutathione-H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85 000 with a subunit size of about 22000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, f,-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta. Glutathione peroxidase (glutathione-H202 oxidoreductase; EC 1.11.1.9), which catalyses the reduction of H202 with the simultaneous oxidation of GSH, was first described in mammalian tissues by Mills (Mills, 1957; Mills &,Randall, 1958). The enzyme has since been implicated in the cellular defence mechanisms against oxidative stress (Cohen & Hochstein, 1963). Besides H202, the enzyme can also catalyse the reduction of various organic hydroperoxides (Little & O'Brien, 1968). Therefore it has been suggested that GSH peroxidase not only protects cellular components from H202 toxicity, but also prevents the autoxidation of structural lipids by cleaving lipid hydroperoxides, resulting in the disruption of the autocatalytic chain of lipid peroxidation (Little & O'Brien, 1968; Christopherson, 1969). This enzyme has been purified from bovine (Flohe et al., 1971, 1972) and sheep (Oh et al., 1974) erythrocytes, rat liver (Prohaska et al., 1977) and human erythrocytes (Awasthi et al., 1975). We have demonstrated earlier that the human erythrocyte GSH peroxidase has two immunologically similar forms, isoenzymes A and B, and that both the forms are selenoproteins (Awasthi et al., 1975). The rat liver enzyme has also been shown to exist in two forms (Lawrence & Burk, 1976). However, only one of these enzymes (isoenzyme I) is a selenoprotein, whereas the other isoenzyme (II),

Abbreviation used: GSH, glutathione. Vol. 177

which acts only on cumene hydroperoxide, is not a selenoprotein (Prohaska et al., 1977). We have now purified GSH peroxidase from human placenta and studied its kinetic and structural properties. Like the rat liver enzyme, selenium is essential for the activity of the human enzyme, because the release of selenium from the enzyme by cyanide results in complete inactivation. However, unlike rat liver, human placenta was found to have only one species of GSH peroxidase having similar affinities toward H202 and organic hydroperoxides. Materials and Methods Human placentae were collected soon after parturition, stored at 4°C and processed within 48h. Sephadex G-200 and G-150, GSH, NADPH, glutathione reductase (yeast, type III), 2,3-diaminonaphthalene and ECTEOLA-cellulose were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. DEAE-cellulose (DE-52) and CM-cellulose (CM-52) were obtained from Whatman Co., Springfield Mill, Maidstone, Kent, U.K. Ampholines were purchased from LKB-Produkter, Bromma, Sweden. Cumene hydroperoxide and t-butyl hydroperoxide were purchased from Koch-Light Laboratories, Colnbrook, Bucks., U.K. The enzyme assays with t-butyl hydroperoxide and

472

Y. C. AWASTHI, D. D. DAO, A. K. LAL AND S. K. SRIVASTAVA

cumene hydroperoxide were carried out by the procedure of Awasthi et al. (1975). The final concentration of cumene hydroperoxide and t-butyl hydroperoxide in the assay system was 0.1 mm. The exact concentrations of t-butyl hydroperoxide and cumene hydroperoxide in the commercial preparations were determined by iodometric titration (Kokatnur & Jelling, 1941). The enzyme assay with H202 as substrate was performed by the method of Beutler (1971). Polyacrylamide-disc-gel electrophoresis was carried out by the method of Davis (1964) and thin-layer isoelectrofocusing on Sephadex G-75 bed was performed by the method of Srivastava et al. (1976). Protein was determined by the method of Lowry et al. (1951). The presence off,-mercaptoethanol (1.4mM) did not interfere with the protein assay. Selenium was determined fluorimetrically by the method similar to that described by Watkinson (1966). The fluorescence of the 2,3-diaminonaphthalene-selenium complex (piazselenol) in cyclohexane solution was determined in a Turner fluorimeter, model 111, by using no. 7-60 primary filter and no. 58 as secondary filter, corresponding to excitation and emission wavelengths of 360 and 525 nm respec-

tively. Results

Purification of GSHfperoxidase Unless otherwise specified, all steps were performed at 4°C and the enzyme activity was determined by using t-butyl hydroperoxide. Placentae were cleaned of amniotic membrane and connective tissues and washed thoroughly with cold deionized distilled water to remove blood and blood clots. After cutting into small pieces, the placentae (3000g) were homogenized in SmM-potassium phosphate buffer, pH 7.4 (10 litres), containing 1.4mM-fl-mercaptoethanol. The homogenate was centrifuged for 30 min at lOOOOg and the clear supernatant was subjected to (NH4)2SO4 fractionation. Solid (NH4)2SO was slowly added to the supernatant to give 45 % saturation. The mixture was stirred gently overnight and centrifuged for 30min at 1OOO0g. The supernatant, virtually free of GSH peroxidase activity, was discarded and the pellet was resuspended in 5 mmpotassium phosphate buffer (1400ml), pH7.2, containing 1.4mM-fl-mercaptoethanol and was dialysed against the same buffer for 72h (4 x 15 vol.). The dialysed enzyme was centrifuged for 30 min at lOOOOg and the supernatant was passed through a CM-cellulose (CM-52) column (2.5 cmx40cm), preequilibrated with 5mM-potassium phosphate buffer, pH7.2, containing 1.4mM-,6-mercaptoethanol, at a rate of 30ml/h. The enzyme did not bind to the column, and the eluate was concentrated to 500 ml in an Amicon ultrafiltration cell by using a PM-10 mem-

brane and was subjected to DEAE-cellulose (DE-52) chromatography. DEAE-cellulose (DE-52) chromatography The pooled enzyme fraction after concentration was passed through a DEAE-cellulose (DE-52) column (2.5cmx4Ocm) at a rate of 30ml/h. The column was pre-equilibrated with 5mM-potassium phosphate buffer, pH 6.8, containing 1 .4mM-flmercaptoethanol. Almost all of the enzyme was adsorbed on the column. After washing with the equilibrating buffer, the enzyme was eluted with a 1.2-litre linear gradient of 0-lOOmM-NaCl in the application buffer. The enzyme was eluted as a single peak between 30mM- and 50mM-NaCl. The pooled fractions from the DEAE-cellulose column were concentrated to 75 ml in an Amicon ultrafiltration cell by using a PM-10 membrane, and subjected to Sephadex G-200 gel filtration.

Sephadex G-200 gelffiltration The Sephadex G-200 column (5cm x 100cm) was equilibrated with 5mM-potassium phosphate buffer, pH 7.2, containing l00mM-(NH4)2SO4 and 1.4mMfl-mercaptoethanol. The pooled DEAE-cellulose fraction after concentration to 75 ml was passed through the column by using upward flow at a rate of 60 ml/h. The enzyme was eluted as a major broad peak (Fig. 1) having more than 90 % of the total enzyme activity.

ECTEOLA-cellulose column chromatography The peak fractions of the Sephadex G-200 column were pooled and dialysed against 5mM-potassium

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GSHperoxidase The experimental details are given in the text: the enzyme activity was monitored by using t-butyl hydroperoxide (o) and H202 ().

1979

473

GLUTATHIONE PEROXIDASE FROM HUMAN PLACENTA phosphate buffer, pH6.8, containing 1.4mM-flmercaptoethanol (4x20vol.). The dialysed enzyme was passed through a column (2.5cmx40cm) of ECTEOLA-cellulose at a flow rate of 30ml/h. The column was washed with 200ml of 20mM-NaCi in 5mM-potassium phosphate buffer (pH6.8)/1.4mMfJ-mercaptoethanol, and the enzyme was eluted with a linear gradient (800ml) of 20-100mM-NaCI. The enzyme was eluted as a sharp peak between 30mMand 42mM-NaCl.

Sephadex G-150 gelfiltration The enzyme from the ECTEOLA-cellulose step was concentrated to 20ml in an Amicon ultrafiltration cell and passed through a Sephadex G-150 column (2.5 cm x 100cm) at a rate of 30 mI/h by using upward flow. The enzyme separated as a single peak when assayed with H202, t-butyl hydroperoxide or cumene hydroperoxide as substrates. In a separate experiment, the enzyme from the 10000g (30min) supernatant of the whole homogenate of placenta was subjected to gel filtration through the same column under identical conditions. The gel-filtration profile of the crude enzyme shown in Fig. 2 was exactly identical with that of the partially purified enzyme obtained after ECTEOLA-cellulose column chromatography. Preparative polyacrylamide-disc-gel electrophoresis The Sephadex G-150-gel-filtration step yielded GSH peroxidase in a highly purified state. A minor contaminating band was, however, observed on polyacrylamide-disc-gel electrophoresis. The mobility of the contaminating band was less anodal than that of GSH peroxidase, and attempts to separate this protein by using an affinity column of GSH bound to activated Sepharose beads (Awasthi et al., 1975)

were unsuccessful. Therefore the enzyme was subjected to preparative polyacrylamide-disc-gel electrophoresis as described previously (Awasthi et al., 1975). This step resulted in a homogeneous preparation of GSH peroxidase. The results of the purification are presented in Table 1. The final enzyme preparation moved on polyacrylamide-disc-gel electrophoresis as a single protein band (Fig. 3a). The enzyme was stable under a reduced atmosphere at 4°C for more than 4 months.

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100

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300

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Volume of eluate (ml)

Fig. 2. Sephadex G-150 gel filtrationi of crude GSH peroxidase from human placenta Placenta (2.5g) free of connective tissue, amniotic membrane and blood clots was homogenized in 25ml of 10mM-potassium phosphate buffer, pH7.2, with 1 .4mM-fl-mercaptoethanol. The homogenate was centrifuged at 10000g, and the clear supernatant (25ml) was applied to a Sephadex G-150 column (2.5cmxl00cm) with an upward flow of 30ml/h. Fractions (4ml) were collected and the enzyme activity was monitored by using H202 (0), t-butyl hydroperoxide (CI) and cumene hydroperoxide (e) as substrates.

Table. 1. Pufrification of GSH peroxidasefrom human placentta One unit of enzyme brings about oxidation of 1 umol of GSH in 1 min at 37°C. Specific Purificaactivity Activity (units) (units/mg Protein tion Yield of protein) (per ml) (total) (mg/ml) (fold) 0.42 5060 6.57 0.06 Supernatant 100 4.11 6580 27.5 (NH4)2SO4 fraction* 0.15 2.5 130 4.70 5640 7.56 111 CM-cellulose CM-52 0.62 10 14.2 5250 1.69 DEAE-cellulose DE-52 8.39 140 104 6.19 3400 0.62 166 9.98 Sephadex G-200 67 13.2 2190 0.15 87.8 1460 ECTEOLA-cellulose 43 27.3 2020 0.13 202.0 3370 40 Sephadex G-lSOt * During prolonged dialysis of the (NH4)2SO4 fraction with 1.4mM-fl-mercaptoethanol (72h, see the text) about 30% increase in the enzyme activity was observed. t Batches of enzyme protein containing 100 units were subjected to preparative polyacrylamide-disc-gel electrophoresis. The calculated overall yield of homogeneous enzyme thus obtained was 20 %. The specific activity of the final homogeneous preparation, however, was about 100 units/mg owing to the inactivation of the enzyme during gel electrophoresis. Vol. 177

474

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Fig. 3. Polyacrylamide-gel-disc electrophoresis (a) and ureal sodium dodecyl sulphate/polyacrylamide-gel-disc electrophoresis (b) of human placental GSH peroxidase (a) The electrophoresis was carried out on a 7.5 % gel by using a double buffer system, 0.052M-Tris/glycine, pH8.9, in the upper chamber and O.1 M-Tris/HCl, pH8.2, in the lower chamber: 20,ug of protein was applied to the gel. Protein was stained with Coomassie Blue R. (b) The enzyme and the standard protein samples (30ug) were incubated at 370C for 5h with 8 M-urea containing 1 Y. sodium dodecyl sulphate and l0OmM-/J-mercaptoethanol in a N2 atmosphere and dialysed in a N2 atmosphere overnight at 4°C against 50mM-Tris/borate buffer, pH7.4, containing 1 % sodium dodecyl sulphate, l0OmM-16-mercaptoethanol and 12mM-EDTA. Gels (7.5%) containing 0.1% sodium dodecyl sulphate and l0mM-/3-mercaptoethanol were used. The electrophoresis was carried out in 50mM-Tris/borate buffer, pH7.4, containing 0.1% sodium dodecyl sulphate and lOmM-/J-mercaptoethanol, and 12mM-EDTA. (i) GSH peroxidase; (ii) standards (from top to bottom): bovine serum albumin, bovine hepatic catalase, aldolase and cytochrome c.

Sephadex G-200 or G-150 gel filtration of human placental GSH peroxidase yields only one species of the enzyme (Figs. 1 and 2). The enzyme activity in the fractions obtained by Sephadex G-200 gel filtration was determined by using H202 and t-butyl hydroperoxide. The gel-filtration profile (Fig. 1) showed the presence of only one enzymeactivity peak, having no selective preference for any of these substrates. To rule out the possibility that other isoenzyme of GSH peroxidase might have been eliminated in the purification steps before gel filtration, Sephadex G-150 gel filtration of the supernatant (10000g; 30min) from the whole homogenate of human placenta was performed. The enzyme activity of the fractions from Sephadex G-150 column was monitored by using H202, t-butyl hydroperoxide and cumene hydroperoxide as substrates. As shown in Fig. 2, only one peak of enzyme activity for all the three substrates was obtained, indicating that human placenta has only one species of GSH peroxidase.

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Besides fi-mercaptoethanol, dithiothreitol and GSH also stabilize the enzyme activity. In the absence of either of these reducing agents, the enzyme activity was rapidly lost. When the enzyme was dialysed for 24h against 10mM-potassium phosphate buffer, pH 7.0, without fi-mercaptoethanol, GSH and dithiothreitol, more than 30% of the enzyme activity was lost. However, most of the lost activity could be regenerated when the enzyme was dialysed against the same buffer containing 1.4 mM-fi-mercaptoethanol. This indicates that a reducing environment is crucial for the activity of the enzyme. The enzyme was extremely unstable below pH5.0, because on dialysis against citrate/phosphate buffer (10mM-citrate), pH5.0, the enzyme activity was irreversibly lost. Owing to this property of the enzyme, column isoelectrofocusing could not be used as one of the steps in the enzyme purification, because the isoelectric pH of the enzyme was found to be 4.8.

5

10

20

30

10-4 x Molecular weight 4. Fig. Mfolecular-weight cletermination of GSH peroxidase by Sephadex G-200 gelfiltration In a total volume of 7ml, the enzyme and standard protein samples [Blue Dextran 2000, aldolase (1), bovine serum albumin (2), ovalbumin (3), chymotrypsinogen A (4) and cytochrome c (5), 5mg/ml each] were passed through a Sephadex G-200 (5 cm x 100cm) column, equilibrated with 10mM-potassium phosphate, pH 7.0, containing 0.1 M-(NH4)2SO4, by using an upward flow rate of 60ml/h. Fractions (5 ml) were collected and protein was monitored by measuring the A280. The enzyme was assayed using t-butyl hydroperoxide as substrate. The molecular weights wvere estimated by plotting K,* (Laurent & Killander,

1964) against log(molecular weight).

1979

GLUTATHIONE PEROXIDASE FROM HUMAN PLACENTA

Properties of GSHperoxidase The molecular weight of the purified enzyme, as determined by Sephadex G-200 gel filtration, was 85500 (Fig. 4). Urea/sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis dissociated the enzyme into subunits of mol.wts. about 22000 (Fig. 3b), indicating that human placental GSH peroxidase is a tetramer. The enzyme was found to have about 4g-atoms of selenium/mol of protein. Since long exposure below pH5 inactivates the enzyme, isoelectrofocusing of GSH peroxidase was performed on a thin-layer bed of Sephadex G-75. The enzyme focused as a single sharp peak with p14.8. Inhibition by cyanide Purified GSH peroxidase incubated in a nonreducing environment with 33mM-KCN was completely inhibited in 60min at 25°C (Table 2). However, if the enzyme was preincubated with GSH, f6mercaptoethanol or dithiothreitol, the inhibition by cyanide was not observed. When the homogeneous enzyme was dialysed overnight against 10mMpotassium phosphate buffer, pH7.0, to remove f6mercaptoethanol (used as a stabilizing agent for storage) and then incubated with 33mM-KCN for 1 h, it was completely inactivated. On the other hand, if the dialysed enzyme was preincubated for 10min with GSH, ,B-mercaptoethanol or dithiothreitol and then incubated with KCN, most of the enzyme activity was retained (Table 2). Preincubation with oxidizing agents such as H202, t-butyl hydroperoxide or cumene hydroperoxide, on the other hand, also resulted in complete inhibition of GSH peroxidase Table 2. Inhibition of GSH peroxidase by cyanide Homogeneous GSH peroxiclase was dialysed overnight against lOmM-potass.,ium phosphate buffer, pH7.0. The dialysed enzymite (90pl) was incubated reagent for with lOpl of lOmM-preincub, ad at 25°C. Then 366mM-KCN was theI samples were further incuba phosphate the control, lO,ul of lOmi buffer, pH7.0, was added iniplace of cyanide. GSH peroxidase was determined aLs described earlier. Percentage of activity .

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.

.

1ation lOmin Oitd) addedfo Me-potassium

Preincubation reagent None GSH t-Butyl hydroperoxide Cumene hydroperoxide H202

fi-Mercaptoethanol Dithiothreitol Sodium arsenite Vol. 177

Control 80 80 73 83 100 100 68

+ Cyanide 0 80 0

4 ioo 97 3.6

475

by cyanide. Preincubation of the enzyme with these oxidizing agents alone also resulted in some loss of the enzyme activity (Table 2), which was restored by providing the enzyme with a reducing environment. Inhibition by cyanide, however, was irreversible and resulted in the release of selenium.

Release of selenium The release of selenium by cyanide was studied by incubating the homogeneous enzyme with cyanide in the non-reducing environment and removing the selenium by filtration with a PM-10 membrane in an Amicon ultrafiltration cell. For this, 70,ug of purified GSH peroxidase was preincubated with 1.OmM-tbutyl hydroperoxide for 10min; subsequently it was incubated with 33 mM-KCN for 1 h at 25°C, and the reaction mixture was concentrated to about 0.2 ml in the small Amicon ultrafiltration cell by using a PM-10 membrane. Then 10mM-potassium phosphate buffer, pH 7.2 (2.5 ml), was added to the concentrate and the resulting mixture was again concentrated by ultrafiltration to 0.2ml. The procedure was repeated three times to remove most of the diffusible material, and the concentrated protein was collected in a total volume of 1.5ml by repeatedly washing the ultrafiltration cell. In the control experiment, performed simultaneously, the enzyme that had not been incubated with cyanide was subjected to similar incubation and filtration procedures. Selenium content in the protein fractions was determined as described in the Materials and Methods section. The cyanide-treated enzyme lost more than 80 % of its selenium. The control sample, however, lost less than 10% of its selenium, indicating that cyanide released selenium from human placental GSH peroxidase. Discussion GSH peroxidase purified from human placenta is a selenoprotein having 4g-atoms of selenium/mol. The enzyme is a tetramer with a subunit size of 22000 daltons. Sephadex gel filtration reveals only one species of enzyme that utilizes H202, t-butyl hydroperoxide and cumene hydroperoxide. The enzyme similar to rat liver GSH peroxidase II (Lawrence & Burk, 1976), which acts only on cumene

hydroperoxide, was not present in human placenta. The placental enzyme is inhibited by cyanide. However, in the presence of f,-mercaptoethanol, dithiothreitol or GSH, the enzyme is not inhibited by cyanide. Preincubation of the enzyme with H202 or t-butyl hydroperoxide, on the other hand, results in the inhibition of the activity by cyanide, indicating that the oxidized state of the enzyme is essential for the inhibition by cyanide. Similar results have been reported for the selenium-containing GSH peroxidase I of rat liver (Prohaska et al., 1977). During the

476

Y. C. AWASTHI, D. D. DAO, A. K. LAL AND S. K. SRIVASTAVA

inhibition by cyanide, selenium is released from the enzyme as a low-molecular-weight fragment, indicating that selenium is essential for the enzyme activity. Thus GSH peroxidase from human placenta appears to be similar to rat liver GSH peroxidase I in its inhibition and release of selenium by cyanide. The properties of the human placental enzyme appear to be similar to those of GSH peroxidase of human erythrocytes (Awasthi et al., 1975). However, in human placenta, GSH peroxidase B of human erythrocytes, which is immunologically similar to GSH peroxidase A, was not detected. This work was supported in part by National Institutes of Health grants EY01677 and 1 T32GM07204.

References Awasthi, Y. C., Beutler, E. & Srivastava, S. K. (1975) J. Biol. Chem. 250, 5144-5149 Beutler, E. (1971) Red Cell Metabolism, p. 66, Grune and Stratton, New York Christopherson, B. 0. (1969) Biochim. Biophys. Acta 176, 463470

Cohen, G. & Hochstein, P. (1963) Biochemistry 2, 14201428 Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404 427 Flohe, L., Bernhard, E. & Werdel, A. (1971) HoppeSeyler's Z. Physiol. Chem. 352, 151-158 Flohe, L., Loschen, G., Gunzler, W. A. & Eichle, E. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 987-999 Kokatnur, V. R. & Jelling, M. (1941) J. Am. Chem. Soc. 63, 1432-1433 Laurent, T. C. & Killander, J. (1964) J. Chromatogr. 14, 317-330 Lawrence, R. A. & Burk, R. F. (1976) Biochem. Biophys. Res. Commun. 71, 952-958 Little, C. & O'Brien, P. J. (1968) Biochem. Biophys. Res. Commun. 31, 145-150 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Mills, G. C. (1957) J. Biol. Chem. 229,189-197 Mills, G. C. & Randall, H. P. (1958) J. Biol. Chem. 232, 589-598 Oh, S. H., Ganther, H. E. & Hoekstra, W. G. (1974) Biochemistry 13, 1825-1829 Prohaska, J. R., Hoekstra, W. G. & Ganther, H. E. (1977) Biochem. Biophys. Res. Commun. 74, 64-71 Srivastava, S. K., Wiktorowicz, J. E. & Awasthi, Y. C. (1976) Proc. Natl. Acad. Sci. U.S.A. 78, 2833-2837 Watkinson, J. H. (1966) Anal. Chem. 38, 92-97

Purification and properties of glutathione peroxidase from human placenta.

Biochem. J. (1979) 177, 471-476 Printed in Great Britain 471 Purification and Properties of Glutathione Peroxidase from Human Placenta By YOGESH C...
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