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A simple procedure for the purification of eosinophil peroxidase from normal human blood
Journal of Immunological Methods, 91 (1986) 283-288 Elsevier
283
JIM 04011
A simple procedure for the purification of eosinophil peroxidase from normal human blood Renzo Menegazzi *, Giuliano Zabucchi and Pierluigi Patriarca Istituto di Patologia Generale, Universith di Trieste, via A. Fleming 22, 34127, Trieste, ltaly (Received 3 March 1986, accepted 2 April 1986)
A simple procedure to purify human eosinophil peroxidase (EPO) is described. The method uses pure anucleated granule-rich eosinophil fragments (cytosomes) as a suitable starting material from which EPO can be quickly isolated. The enzyme obtained by this procedure has both the biochemical and the spectral properties of EPO and shows a reasonable degree of purity, as judged by its rz value. This procedure, besides its simplicity and reproducibility, offers at least two other advantages over the methods currently used for EPO purification, (1) the possibility of isolating EPO from small amounts of normal human blood and (2) a very high recovery of the enzyme activity. Key words: Eosinophil peroxidase
Introduction
The eosinophil granulocytes contain a large amounts of peroxidase, commonly known as eosinophil peroxidase (EPO), which is immunologically (Salmon et al., 1970) and genetically (Crarner et al., 1982) distinct from myeloperoxidase (MPO), the peroxidase of the neutrophils. The biological importance of EPO is related to a number of functions exhibited by this enzyme. EPO has been shown, for example, to have both bactericidal (Jong et al., 1980) and cytocidal (Jong and Klebanoff, 1980; Jong et al., 1981) activity, to inactivate leukotriene B4 (Henderson et al., 1982b), to potentiate macrophage killing against bacteria (Ramsey et al., 1982), protozoa (Locksley et al., 1982; Nogueira et al., 1982) and tumor cells (Nathan and Klebanoff, 1982), and to stimulate the release of histamine from mast cells (Henderson et al., 1982a). All these studies have been performed using guinea pig or horse EPO, but, to * To whom correspondence should be addressed.
our knowledge, data on the biological role of human EPO are very limited (Buys et al., 1984). Indeed, EPO has already been isolated from human blood, but the techniques commonly used to purify this enzyme are based on methods employing either blood from highly eosinophilic subjects (Migler and DeChatelet, 1978; Wever et al., 1980; Carlson et al., 1985) or multistep procedures starting from very large amounts of normal human blood (Wever et al., 1981; Olsen and Little, 1983; Bolsher et al., 1984). The rarity of the hypereosinophilic syndrome is by itself an evident limitation to the employment of eosinophilic blood as starting material for EPO isolation. On the other hand, the procedures which use buffy-coats for EPO extraction and purification are disadvantageous since time is required to harvest the buffycoats and, more important, the buffy-coat extract is an extremely heterogeneous material which contains other cationic proteins from white blood cells, including MPO which, in some catalytic assays, can interfere with EPO. In this paper we describe a simple procedure to
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
284
purify EPO by a single gel filtration step using pure anucleated granule-rich eosinophil fragments (cytosomes) (Zabucchi et al., 1985) as starting material.
Materials and methods
Isolation of eosinophilfragments (cytosomes) Cytosomes were obtained from normal human blood as previously described (Zabucchi et al., 1985). Briefly, whole blood was treated with 5 m g / m l saponin (Merck, Darmstadt, F.R.G.) dissolved in Krebs-Ringer phosphate buffer (KRP) containing 1% bovine serum albumin (BSA) (Miles, Elkhart, IN). After centrifugation, the pellet was resuspended in K R P containing 2% BSA and layered on Percoll (Pharmacia, Uppsala, Sweden) (density: 1.068-1.072). After 15 rain centrifugation, the band formed at the interface was treated with 70/~g/ml DNAase, diluted in KRP containing 2% BSA and centrifuged once again on Percoll. The pellet formed contained virtually 100% pure cytosomes.
Extraction of cytosomes Cytosomes from about 4 liters of blood were suspended in 25 ml of 0.1 M sodium acetate buffer p H 4.7 containing 0.1 M Na2SO 4 and 0.075% cetyltrimethylammonium bromide (CTAB) (Eastman Kodak, Rochester, NY, U.S.A.) and incubated with continuous stirring for 90 min at 4 ° C. The mixture was then centrifuged for 20 min at 20 000 × g and the supernatant collected while the pellet was extracted once again with the same buffer. The supernatants were then combined and processed for EPO purification.
formed using 1.5% purified agarose (Istituto Behring, L'Aquila, Italy) dissolved in 0.2 M acetate buffer pH 4.5 which was also used as electrode buffer. Slabs were stained for both protein, with Coomassie brilliant blue, and peroxidase activity, using 3-3'-diaminobenzidine (Serva, Heidelberg, F.R.G.) as substrate, as previously described (Patriarca et al., 1977).
MPO purification MPO was purified according to the method described by Wever et al. (1981). The rz of the enzyme preparation was 0.8.
Determination of peroxidase activity Peroxidase activity was measured by the guaiacol method described by Chance and Mahley (1955) and modified by Romeo et al. (1973).
Protein determination Protein was determined by the method described by Lowry et al. (1951).
Spectral analysis Spectral analyses were obtained Perkin-Elmer 576 spectrophotometer.
using
a
Results
Extraction of peroxidase activity from cytosomes Table I shows the high efficiency of the extraction procedure used to solubilize peroxidase activity from cytosomes. In three experiments the peroxidase activity contained in the cytosome suspension was almost completely recovered in the acetate soluble extract.
Gel filtration chromatography The cytosome extract was concentrated by ultra filtration in an Amicon chamber (Amicon, U.K.) equipped with a PM-10 Diaflo membrane (Amicon, U.K.); the extract was then chromatographed by an Ultrogel AcA-44 gel filtration column (LKB, Sweden) equilibrated with 0.025 M acetate buffer pH 4.7 containing 0.1 M NaC1 and 0.02% CTAB.
Agarose gel electrophoresis Analytical agarose gel electrophoresis was per-
TABLE I RECOVERY OF PEROXIDASE ACTIVITY IN THE CYTOSOME ACETATE EXTRACT Experiment
I II llI
GU a in cytosome suspension 825.6 848.0 793.4
GU in acetate extract 842.1 797.1 777.5
% Recovery in acetate extract 102 94 98
a GU: guaiacolunits =/xmol of oxidizedguaiacol/min.
285
oo! GU,
"1.5
ml
0.06
0.3
100'
OD 413nm
280nm
OD
0.02
\
0.1 " 0.7
0.7"
o
,/ .......
70
0.5'
go
- -
\
0.3
-J
0
110
90
280nm
B 0.5
0,15
"0.06
.......
~
~k
0.3
l i t " ~\
0.05
0.02
0
o.~ 0
/
.................
/
. . . . . . 60 70 80
"7-----,"
90
k .~ ' -
"o~
.
100 fraction
110 number
Fig. 1. A: Elution profile of cytosome extract fractionated by Ultrogel AcA-44. Each fraction was analyzed for both protein (OD at 280 nm ) and EPO (OD at 413 nm . . . . . . ) absorption. B: Peroxidase activity eluted from Ultrogel AcA44. Peroxidase activity was measured by the guaiacol oxidation assay. (GU: guaiacol units vmol of oxidized guaiacol/min.) Arrow indicates peroxidase activity contributed by MPO. =
Gel filtration The extract was concentrated to a few ml in an Amicon ultrafiltration chamber equipped with a PM-10 membrane and then applied to a column (1.5 cm x 90 cm) of Ultrogel AcA-44 equilibrated with 0.025 M acetate buffer pH 4.7 containing 0.1 M NaCI and 0.02% CTAB. Fractions of about 1.5 ml were eluted with the same buffer. We exploited a distinctive feature of EPO, that is its characteristic absorption peak in the Soret region at 413 nm (Wever et al., 1981), to check spectrophotometrically the presence of the enzyme in each fraction. Fig. 1A shows a typical elution profile. It is evident that only one clear-cut absorbance peak at 413 nm was detectable in the elution profile and that this peak was practically superimposable on one of the two major protein peaks eluted from the column (peak B). Peak A, as judged by an immunoprecipitation assay using the Ouchterlony
0.3
0.06 1
0.02
0.1 I
0 I
I
I
370
430
490
i
550
610 Z
I nm
Fig. 2. Spectral analysis of EPO. A: Absorption spectrum of native EPO (110 G U / m l , rz 0.9) in 0.025 M acetate buffer pH 4.7 containing 0.1 M NaC1 and 0.02% CTAB. B: Difference spectrum (reduced - oxidized) of EPO in 0.01 M acetate buffer pH 5.6 containing 0.5 M NaC1. C: Difference spectrum (reduced - oxidized) of purified MPO (97 G U / m l , rz 0.8) in 0.01 M acetate buffer pH 5.6 containing 0.5 M NaC1. Left ordinates: continuous lines; right ordinates: dotted lines. The addition of purified MPO (2% of the total peroxidase activity) modified the difference spectrum of purified EPO (arrow in B).
plate technique, was shown to contain BSA, one of the components of the medium used for cytosome isolation (Zabucchi et al., 1985).
Biochemical analysis Fig. 1B shows the peroxidase distribution pattern in the fractions eluted from the gel filtration column. The three fractions forming peak B contained 90% of the total peroxidase activity re-
286
Fig. 3. Agarose gel electrophoresis of the three pooled fractions containing EPO. The electrophoretic procedure is described in the materials and methods section. Lane A: stained for protein; lane B: stained for peroxidase activity. covered from the column, as measured by the guaiacol oxidation assay. A further characterization of this peroxidase activity was carried out on the basis of the selective inhibition of EPO by aminotriazole (Cramer et al., 1984). In all three fractions the peroxidase activity was more than 90% inhibited by aminotriazole, suggesting that the bulk of this activity was accounted for by EPO. Some peroxidase activity, amounting to less than 2% of the total peroxidase activity, was found in fractions 73-79 (arrow in Fig. 1B). This peroxidase activity was not inhibited by aminotriazole, suggesting that it was contributed by neutrophil myeloperoxidase which may have become bound to the eosinophil cytosomes during their preparation. In fact, immunoprecipitation of the proteins in these fractions with anti-MPO I g G was observed. Ouchterlony immunoprecipitation was negative when the three main fractions containing EPO were reacted with anti-MPO IgG, suggesting that EPO was not contaminated with MPO or, at
worst, that the contamination was less than 2%. Fig. 2A illustrates an absorbance spectrum showing absorption maxima at 413 and 640 nm and shoulders at 550 and 500 nm which are typical of the native form of EPO (Olsen and Little, 1983). Fig. 2B shows the difference spectrum of EPO which is characteristic of the purified enzyme (Wever et al., 1981). When purified MPO was added to the EPO at a concentration of 2% or less of the total peroxidase activity, it was detected as a shoulder at 637 nm (arrow in Fig. 2B) where the M P O difference spectrum exhibits a characteristic peak (Fig. 2C). This suggests that our EPO preparation was not significantly contaminated by MPO. The r~ value (ratio between absorption in the Soret region and at 280 nm), which is commonly used as a criterion of purity for heme peroxidases, was 0.9 in one fraction and 0.8 in the three pooled fractions.
Agarose gel electrophoresis Fig. 3 shows the electrophoretic pattern of the three pooled fractions stained for both protein (lane A) and peroxidase activity (lane B). Lane B showed one strong peroxidase-positive band, corresponding to EPO, and another weakly staining peroxidase-positive band, probably due to the presence, in our preparation, o~ aggregated forms of EPO previously reported by others (Olsen and Little, 1983). In lane A, in addition to the bands corresponding to EPO, another minor band, containing a contaminant more cationic than EPO itself, stained for protein.
Efficiency of the purification procedure Table II shows that the procedure employed permitted a 90% recovery in peak B of the EPO activity present in the original acetate extract. The
TABLE II EFFICIENCY OF EPO PURIFICATION PROCEDURE Purification step Cytosome extract Gel-filtration on Ultrogel AcA-44 (peak B)
Volume (ml) 35.0 a 4.5
Total peroxidase activity (GU) 560.7 504.9
a Derived from about 4000 ml of normal human blood.
Total protein (mg) 28.0 1.8
Specific activity (GU/mg protein) 20.0 280.0
% Recovery 100 90
287
total peroxidase recovery in all fractions was about 98%.
Discussion We describe here a simple procedure yielding EPO from pure anucleated granule-rich eosinophil fragments (cytosomes) prepared from normal human blood. EPO was almost completely extracted from the cytosomes according to the method of Bolsher et al. (1984); the soluble extract was concentrated by ultrafiltration and EPO was purified from the ultrafiltrate by a single gel filtration step. 90% of the original enzyme activity was recovered in a sharp peak formed by three fractions of 1.5 ml each. No contamination by another human leukocyte peroxidase (i.e., myeloperoxidase) was detectable. The rz value of at least one of the three fractions was 0.9, suggesting a reasonable degree of EPO purity. The enzyme has both the biochemical (selective inhibition by aminotriazole) and spectral characteristics of EPO. EPO is a highly cationic protein with a M r of about 71000 (Bolsher et al., 1984). It was unexpectedly eluted from the column later than BSA, an anionic protein with a closely similar M r of 68000. This anomalous behaviour of EPO was thought to be due to its remarkable tendency to stick to negatively charged surfaces (Olsen and Little, 1983) such as gel filtration media. Evidently, the presence in the elution buffer of 0.02% CTAB, a cationic detergent, was insufficient to completely eliminate the electrostatic interactions between the gel filtration media and EPO. The use of CTAB, instead of non ionic detergents such as Tween 80, was possibly due to the exclusive use of gel filtration instead of cationic exchange chromatography, which has been used in most of the methods previously described. The use of CTAB offers three advantages, (1) complete EPO extraction, (2) a critical miceUar concentration higher than that of Tween 80, which permits the concentration of the enzyme without reaching the critical miceUar concentration, and (3) ease of removal by dialysis. The procedure for the isolation of EPO described in this paper offers the following advantages over the methods currently employed: (1) the use of normal human blood instead of hyper-
eosinophilic blood; (2) the possibility of using small amounts of blood compared with the volumes used by other authors; (3) very high recovery of enzyme activity; and (4) simplicity and the reproducibility of the whole procedure.
Acknowledgements We gratefully acknowledge the Blood Banks of the Hospitals of Trieste, Udine and Pordenone for supplying blood. Supported by grants from the National Research Council of Italy (CNR - Progetto Finalizzato ControUo Malattie da Infezione, no. 84.02024.52) and from the Ministero Pubblica Istruzione (60% and 40%). We are indebted to Miss Valentina Ceschia for her skilful technical assistance
References Bolsher, B.G.J.M., H. Plat and R. Wever, 1984, Biochim. Biophys. Acta 784, 177. Buys, G., R. Wever and E.J. Ruitenberg, 1984, J. Immunol. 51, 601. Carlson, M.G.C., C.G.B. Peterson and P. Venge, 1985, J. Immunol. 134, 1875. Chance, B. and A.C. Mahley, 1955, in: Methods in Enzymology, Vol. 2, eds. S.P. Colowick and N.O. Kaplan (Academic Press, New York) p. 764. Cramer, R., M.R. Soranzo, P. Dri, G.D. Rottini, M. Bramezza, S. Cirielli and P. Patriarca, 1982, Br. J. Haematol. 51, 81. Cramer, R., M.R. Soranzo, P. Dri, R. Menegazzi, A. Pitotti, G. Zabucchi and P. Patriarca, 1984, J. Immunol. Methods 70, 119. Henderson, W.R., E.Y. Chi and S.J. Klebanoff, 1982a, J. Exp. Med. 152, 265. Henderson, W.R., A. JSrg and S.J. Klebanoff, 1982b, J. Immunol. 128, 2609. Jong, E.C. and S.J. Klebanoff, 1980, J. Immunol. 124, 1949. Jong, E.C., W.R. Henderson and S.J. Klebanoff, 1980, J. Immunol. 124, 1378. Jong, E.C., A.A.F. Mahmoud and S.J. Klebanoff, 1981, J. Immunol. 126, 468. Locksley, R.M., B.B. Wilson and S.J. Klebanoff, 1982, J. Clin. Invest. 69, 1099. Lowry, O.H., N.J. Rosebrough, A.L. Fair and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Migler, R. and L.R. DeChatelet, 1978, Biochem, Med. 19, 16. Nathan, C.F. and S.J. Klebanoff, 1982, J. Exp. Med. 155, 1291. Nogueira, N.M., S.J. Klebanoff and Z.A. Cohn, 1982, J. Immunol. 128, 1705.
288 Olsen, R.L. and C. Little, 1983, Biochem. J. 209, 781. Patriarca, P., P. Dri and M. Snidero, 1977, J. Lab. Clin. Med. 90, 289. Ramsey, P.G., T. Martin, E. Chi and S.J. Klebanoff, 1982, J. Immunol. 128, 415. Romeo, D., R. Cramer, T. Marzi, M.R. Soranzo, G. Zabucchi and F. Rossi, 1973, J. Reticuloendothel. Soc. 13, 399. Salmon, S.E., M.J. Cline, J. Schultz and R.I. Lehrer, 1970, N. Engl. J. Med. 282, 250.
Wever, R., M.N. Hamers, R.S. Weening and D. Roos, 1980, Eur. J. Biochem. 108, 491. Wever, R., H. Plat and M.N. Hamers, 1981, FEBS Lett. 123, 327. Zabucchi, G., B. Skerlavaj, R. Menegazzi, L. Talarico Bidoli and P. Patriarca, 1985, J. Immunol. Methods 85, 393.
283
JIM 04011
A simple procedure for the purification of eosinophil peroxidase from normal human blood Renzo Menegazzi *, Giuliano Zabucchi and Pierluigi Patriarca Istituto di Patologia Generale, Universith di Trieste, via A. Fleming 22, 34127, Trieste, ltaly (Received 3 March 1986, accepted 2 April 1986)
A simple procedure to purify human eosinophil peroxidase (EPO) is described. The method uses pure anucleated granule-rich eosinophil fragments (cytosomes) as a suitable starting material from which EPO can be quickly isolated. The enzyme obtained by this procedure has both the biochemical and the spectral properties of EPO and shows a reasonable degree of purity, as judged by its rz value. This procedure, besides its simplicity and reproducibility, offers at least two other advantages over the methods currently used for EPO purification, (1) the possibility of isolating EPO from small amounts of normal human blood and (2) a very high recovery of the enzyme activity. Key words: Eosinophil peroxidase
Introduction
The eosinophil granulocytes contain a large amounts of peroxidase, commonly known as eosinophil peroxidase (EPO), which is immunologically (Salmon et al., 1970) and genetically (Crarner et al., 1982) distinct from myeloperoxidase (MPO), the peroxidase of the neutrophils. The biological importance of EPO is related to a number of functions exhibited by this enzyme. EPO has been shown, for example, to have both bactericidal (Jong et al., 1980) and cytocidal (Jong and Klebanoff, 1980; Jong et al., 1981) activity, to inactivate leukotriene B4 (Henderson et al., 1982b), to potentiate macrophage killing against bacteria (Ramsey et al., 1982), protozoa (Locksley et al., 1982; Nogueira et al., 1982) and tumor cells (Nathan and Klebanoff, 1982), and to stimulate the release of histamine from mast cells (Henderson et al., 1982a). All these studies have been performed using guinea pig or horse EPO, but, to * To whom correspondence should be addressed.
our knowledge, data on the biological role of human EPO are very limited (Buys et al., 1984). Indeed, EPO has already been isolated from human blood, but the techniques commonly used to purify this enzyme are based on methods employing either blood from highly eosinophilic subjects (Migler and DeChatelet, 1978; Wever et al., 1980; Carlson et al., 1985) or multistep procedures starting from very large amounts of normal human blood (Wever et al., 1981; Olsen and Little, 1983; Bolsher et al., 1984). The rarity of the hypereosinophilic syndrome is by itself an evident limitation to the employment of eosinophilic blood as starting material for EPO isolation. On the other hand, the procedures which use buffy-coats for EPO extraction and purification are disadvantageous since time is required to harvest the buffycoats and, more important, the buffy-coat extract is an extremely heterogeneous material which contains other cationic proteins from white blood cells, including MPO which, in some catalytic assays, can interfere with EPO. In this paper we describe a simple procedure to
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
284
purify EPO by a single gel filtration step using pure anucleated granule-rich eosinophil fragments (cytosomes) (Zabucchi et al., 1985) as starting material.
Materials and methods
Isolation of eosinophilfragments (cytosomes) Cytosomes were obtained from normal human blood as previously described (Zabucchi et al., 1985). Briefly, whole blood was treated with 5 m g / m l saponin (Merck, Darmstadt, F.R.G.) dissolved in Krebs-Ringer phosphate buffer (KRP) containing 1% bovine serum albumin (BSA) (Miles, Elkhart, IN). After centrifugation, the pellet was resuspended in K R P containing 2% BSA and layered on Percoll (Pharmacia, Uppsala, Sweden) (density: 1.068-1.072). After 15 rain centrifugation, the band formed at the interface was treated with 70/~g/ml DNAase, diluted in KRP containing 2% BSA and centrifuged once again on Percoll. The pellet formed contained virtually 100% pure cytosomes.
Extraction of cytosomes Cytosomes from about 4 liters of blood were suspended in 25 ml of 0.1 M sodium acetate buffer p H 4.7 containing 0.1 M Na2SO 4 and 0.075% cetyltrimethylammonium bromide (CTAB) (Eastman Kodak, Rochester, NY, U.S.A.) and incubated with continuous stirring for 90 min at 4 ° C. The mixture was then centrifuged for 20 min at 20 000 × g and the supernatant collected while the pellet was extracted once again with the same buffer. The supernatants were then combined and processed for EPO purification.
formed using 1.5% purified agarose (Istituto Behring, L'Aquila, Italy) dissolved in 0.2 M acetate buffer pH 4.5 which was also used as electrode buffer. Slabs were stained for both protein, with Coomassie brilliant blue, and peroxidase activity, using 3-3'-diaminobenzidine (Serva, Heidelberg, F.R.G.) as substrate, as previously described (Patriarca et al., 1977).
MPO purification MPO was purified according to the method described by Wever et al. (1981). The rz of the enzyme preparation was 0.8.
Determination of peroxidase activity Peroxidase activity was measured by the guaiacol method described by Chance and Mahley (1955) and modified by Romeo et al. (1973).
Protein determination Protein was determined by the method described by Lowry et al. (1951).
Spectral analysis Spectral analyses were obtained Perkin-Elmer 576 spectrophotometer.
using
a
Results
Extraction of peroxidase activity from cytosomes Table I shows the high efficiency of the extraction procedure used to solubilize peroxidase activity from cytosomes. In three experiments the peroxidase activity contained in the cytosome suspension was almost completely recovered in the acetate soluble extract.
Gel filtration chromatography The cytosome extract was concentrated by ultra filtration in an Amicon chamber (Amicon, U.K.) equipped with a PM-10 Diaflo membrane (Amicon, U.K.); the extract was then chromatographed by an Ultrogel AcA-44 gel filtration column (LKB, Sweden) equilibrated with 0.025 M acetate buffer pH 4.7 containing 0.1 M NaC1 and 0.02% CTAB.
Agarose gel electrophoresis Analytical agarose gel electrophoresis was per-
TABLE I RECOVERY OF PEROXIDASE ACTIVITY IN THE CYTOSOME ACETATE EXTRACT Experiment
I II llI
GU a in cytosome suspension 825.6 848.0 793.4
GU in acetate extract 842.1 797.1 777.5
% Recovery in acetate extract 102 94 98
a GU: guaiacolunits =/xmol of oxidizedguaiacol/min.
285
oo! GU,
"1.5
ml
0.06
0.3
100'
OD 413nm
280nm
OD
0.02
\
0.1 " 0.7
0.7"
o
,/ .......
70
0.5'
go
- -
\
0.3
-J
0
110
90
280nm
B 0.5
0,15
"0.06
.......
~
~k
0.3
l i t " ~\
0.05
0.02
0
o.~ 0
/
.................
/
. . . . . . 60 70 80
"7-----,"
90
k .~ ' -
"o~
.
100 fraction
110 number
Fig. 1. A: Elution profile of cytosome extract fractionated by Ultrogel AcA-44. Each fraction was analyzed for both protein (OD at 280 nm ) and EPO (OD at 413 nm . . . . . . ) absorption. B: Peroxidase activity eluted from Ultrogel AcA44. Peroxidase activity was measured by the guaiacol oxidation assay. (GU: guaiacol units vmol of oxidized guaiacol/min.) Arrow indicates peroxidase activity contributed by MPO. =
Gel filtration The extract was concentrated to a few ml in an Amicon ultrafiltration chamber equipped with a PM-10 membrane and then applied to a column (1.5 cm x 90 cm) of Ultrogel AcA-44 equilibrated with 0.025 M acetate buffer pH 4.7 containing 0.1 M NaCI and 0.02% CTAB. Fractions of about 1.5 ml were eluted with the same buffer. We exploited a distinctive feature of EPO, that is its characteristic absorption peak in the Soret region at 413 nm (Wever et al., 1981), to check spectrophotometrically the presence of the enzyme in each fraction. Fig. 1A shows a typical elution profile. It is evident that only one clear-cut absorbance peak at 413 nm was detectable in the elution profile and that this peak was practically superimposable on one of the two major protein peaks eluted from the column (peak B). Peak A, as judged by an immunoprecipitation assay using the Ouchterlony
0.3
0.06 1
0.02
0.1 I
0 I
I
I
370
430
490
i
550
610 Z
I nm
Fig. 2. Spectral analysis of EPO. A: Absorption spectrum of native EPO (110 G U / m l , rz 0.9) in 0.025 M acetate buffer pH 4.7 containing 0.1 M NaC1 and 0.02% CTAB. B: Difference spectrum (reduced - oxidized) of EPO in 0.01 M acetate buffer pH 5.6 containing 0.5 M NaC1. C: Difference spectrum (reduced - oxidized) of purified MPO (97 G U / m l , rz 0.8) in 0.01 M acetate buffer pH 5.6 containing 0.5 M NaC1. Left ordinates: continuous lines; right ordinates: dotted lines. The addition of purified MPO (2% of the total peroxidase activity) modified the difference spectrum of purified EPO (arrow in B).
plate technique, was shown to contain BSA, one of the components of the medium used for cytosome isolation (Zabucchi et al., 1985).
Biochemical analysis Fig. 1B shows the peroxidase distribution pattern in the fractions eluted from the gel filtration column. The three fractions forming peak B contained 90% of the total peroxidase activity re-
286
Fig. 3. Agarose gel electrophoresis of the three pooled fractions containing EPO. The electrophoretic procedure is described in the materials and methods section. Lane A: stained for protein; lane B: stained for peroxidase activity. covered from the column, as measured by the guaiacol oxidation assay. A further characterization of this peroxidase activity was carried out on the basis of the selective inhibition of EPO by aminotriazole (Cramer et al., 1984). In all three fractions the peroxidase activity was more than 90% inhibited by aminotriazole, suggesting that the bulk of this activity was accounted for by EPO. Some peroxidase activity, amounting to less than 2% of the total peroxidase activity, was found in fractions 73-79 (arrow in Fig. 1B). This peroxidase activity was not inhibited by aminotriazole, suggesting that it was contributed by neutrophil myeloperoxidase which may have become bound to the eosinophil cytosomes during their preparation. In fact, immunoprecipitation of the proteins in these fractions with anti-MPO I g G was observed. Ouchterlony immunoprecipitation was negative when the three main fractions containing EPO were reacted with anti-MPO IgG, suggesting that EPO was not contaminated with MPO or, at
worst, that the contamination was less than 2%. Fig. 2A illustrates an absorbance spectrum showing absorption maxima at 413 and 640 nm and shoulders at 550 and 500 nm which are typical of the native form of EPO (Olsen and Little, 1983). Fig. 2B shows the difference spectrum of EPO which is characteristic of the purified enzyme (Wever et al., 1981). When purified MPO was added to the EPO at a concentration of 2% or less of the total peroxidase activity, it was detected as a shoulder at 637 nm (arrow in Fig. 2B) where the M P O difference spectrum exhibits a characteristic peak (Fig. 2C). This suggests that our EPO preparation was not significantly contaminated by MPO. The r~ value (ratio between absorption in the Soret region and at 280 nm), which is commonly used as a criterion of purity for heme peroxidases, was 0.9 in one fraction and 0.8 in the three pooled fractions.
Agarose gel electrophoresis Fig. 3 shows the electrophoretic pattern of the three pooled fractions stained for both protein (lane A) and peroxidase activity (lane B). Lane B showed one strong peroxidase-positive band, corresponding to EPO, and another weakly staining peroxidase-positive band, probably due to the presence, in our preparation, o~ aggregated forms of EPO previously reported by others (Olsen and Little, 1983). In lane A, in addition to the bands corresponding to EPO, another minor band, containing a contaminant more cationic than EPO itself, stained for protein.
Efficiency of the purification procedure Table II shows that the procedure employed permitted a 90% recovery in peak B of the EPO activity present in the original acetate extract. The
TABLE II EFFICIENCY OF EPO PURIFICATION PROCEDURE Purification step Cytosome extract Gel-filtration on Ultrogel AcA-44 (peak B)
Volume (ml) 35.0 a 4.5
Total peroxidase activity (GU) 560.7 504.9
a Derived from about 4000 ml of normal human blood.
Total protein (mg) 28.0 1.8
Specific activity (GU/mg protein) 20.0 280.0
% Recovery 100 90
287
total peroxidase recovery in all fractions was about 98%.
Discussion We describe here a simple procedure yielding EPO from pure anucleated granule-rich eosinophil fragments (cytosomes) prepared from normal human blood. EPO was almost completely extracted from the cytosomes according to the method of Bolsher et al. (1984); the soluble extract was concentrated by ultrafiltration and EPO was purified from the ultrafiltrate by a single gel filtration step. 90% of the original enzyme activity was recovered in a sharp peak formed by three fractions of 1.5 ml each. No contamination by another human leukocyte peroxidase (i.e., myeloperoxidase) was detectable. The rz value of at least one of the three fractions was 0.9, suggesting a reasonable degree of EPO purity. The enzyme has both the biochemical (selective inhibition by aminotriazole) and spectral characteristics of EPO. EPO is a highly cationic protein with a M r of about 71000 (Bolsher et al., 1984). It was unexpectedly eluted from the column later than BSA, an anionic protein with a closely similar M r of 68000. This anomalous behaviour of EPO was thought to be due to its remarkable tendency to stick to negatively charged surfaces (Olsen and Little, 1983) such as gel filtration media. Evidently, the presence in the elution buffer of 0.02% CTAB, a cationic detergent, was insufficient to completely eliminate the electrostatic interactions between the gel filtration media and EPO. The use of CTAB, instead of non ionic detergents such as Tween 80, was possibly due to the exclusive use of gel filtration instead of cationic exchange chromatography, which has been used in most of the methods previously described. The use of CTAB offers three advantages, (1) complete EPO extraction, (2) a critical miceUar concentration higher than that of Tween 80, which permits the concentration of the enzyme without reaching the critical miceUar concentration, and (3) ease of removal by dialysis. The procedure for the isolation of EPO described in this paper offers the following advantages over the methods currently employed: (1) the use of normal human blood instead of hyper-
eosinophilic blood; (2) the possibility of using small amounts of blood compared with the volumes used by other authors; (3) very high recovery of enzyme activity; and (4) simplicity and the reproducibility of the whole procedure.
Acknowledgements We gratefully acknowledge the Blood Banks of the Hospitals of Trieste, Udine and Pordenone for supplying blood. Supported by grants from the National Research Council of Italy (CNR - Progetto Finalizzato ControUo Malattie da Infezione, no. 84.02024.52) and from the Ministero Pubblica Istruzione (60% and 40%). We are indebted to Miss Valentina Ceschia for her skilful technical assistance
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