One-step purification and properties of catalase from leaves of Zantedeschia aethiopica

One-step purification and properties of catalase from leaves of Zantedeschia aethiopica

Biochimie 70 (1988) 1759-1763 (~) Soci6t6de Chimie biologique/Elsevier, Paris 1759 Research article One-step purification and properties of catalas...

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Biochimie 70 (1988) 1759-1763 (~) Soci6t6de Chimie biologique/Elsevier, Paris

1759

Research article

One-step purification and properties of catalase from leaves of Zantedeschia aethiopica Helena T R I N D A D E 1, Amin KARMALI* and Maria S. PAIS .1

L N E T I / DTIQ-Bioqufmica, Estrada das Palmeiras 2745 Queluz de Bai.,:.o; and IFalcudade de Ci#ncias de Lisboa, Departamento de Biologica Vegetal, Edificio C2, Campo Grande 1600, Lisbon, Portugal (Received 25-1-1988, acceptedafter revision 11-5-1988)

Summary - - Catalase (E.C 1.11.1.6) was purified from leaves of Zandedeschia aethiopica to apparent homogeneity by a one-step hydrophobic interaction chromatography on a phenyl Sepharose CL-4B column. The purified enzyme preparation was obtained with a final recovery of enzyme activity of about 61% and a specific activity of 146 U / m g protein. The purified enzyme ran as a single protein band when analyzed both by native PAGE and S D S - P A G E corresponding to an Mr of 220,000 Da, which consists of 4 subunits with identical Mr of 54,000 Da. The pI of purified enzyme was found to be 5.2 by isoelectric focusing on ultrathin polyacrylamide gels. The purified catalase has an optimum temperature of activity at 40oC, whereas it is stable between 0 ° and 50oC. As regards pH, the enzyme has an optimum activity at pH 7.0 and it is stable in the range pH 6 - 8 . The absorption spectrum of the purified enzyme exhibited 2 peaks at 280 nm and 405 nm. one-step purification / catalase / Zantedesehia aethiopica / hydrophobic interaction chromatography/ properties

Introduction Catalase (EC i.11.1.6) was first isolated by Chance from bovine liver [1]. Since then~ this enzyme has been found to occur in a number of mammalian tissues, plants, and microorganisms [2-5]. The enzyme from plants and animals is located in microbodies (i.e peroxisomes and glyoxysomes) and often serves as a marker for these cellular organelles [6, 7]. The regreening of Zantedeschia aethiopica fruiting spathe following the action of endogenous cytokinins is accompanied by peroxisome restructuring and proliferation, as well as by an increase in catalase and glycollate oxidase content of these cellular organelles [8, 9]. Therefore, the availability of a highly purified preparation of catalase from Z. aethiopica would be of interest to study the biogenesis and proliferation of the peroxisomes of this plant. Furthermore, this would permit detailed study of the effect of cytokinins on

*Correspondence and reprints.

biogenesis and proliferation of such peroxisomes [8, 91. Catalase has been purified from a number of sources to apparent homogeneity by means of a series of steps involving precipitation either with organic solvents or ammonium sulfate and conventional chromatography, which result in low yields of enzyme activity [10-15]. The present work reports a one-step purification of catalase from Z. aethiopica by hydrophobic interaction chromatography, and some of its physicochemical properties are presented. Materials and methods

Materials Polyvinylpolypyrrolidone (PVP), bovine serum albumin (BSA), ovalbumin, chymotrypsin, alcohol dehydrogenase (yeast), amyloglucosidase, glucose oxidase, catalase (bovine liver), peroxidase (horseradish), diaminobenzidine, bovine liver catalase,

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Triton X-100, and myoglobin were purchased from Sigma Chemical Company. Sephacryl S-300, phenylSepharose CL 4B, and Pharmalyte 3-10 were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Fresh leaves of Z. aethiopica were harvested from a 1-yr-old tree in the Lisbon botanical garden and used immediately for enzyme isolation. All other reagents used were analytical grade.

Methods Proteins were determined by the Coomassie blue binding method [16]. Catalase activity was assayed as described previously [17], with some minor modifications. The reaction mixture contained 20 mM hydrogen peroxide in 50 mM phosphate buffer pH 7.0. The enzyme reaction was followed at 240 nm and one enzyme unit is defined as 1/zmol of H202 oxidized per rain at 22°C. SDS-PAGE and native PAGE were carded out as mentioned previously [18, 19] and stained for protein with Coomassie blue. lsoelectric focusing on ultrathin (0.1 mm) polyacrylamide gels was performed as reported previously [20] and stained for protein with Coomassie blue [21]. Native PAGE and ultrathin isoelectric focusing gels were stained for activity with H202, peroxidase, and diaminobenzidine by means of a negative staining method reported previously [22]. Mr determination of native catalase was carded out on a column packed with Sephacryl S-300 previously equilibrated with 50 mM phosphate buffer pH 7.0, with several protein markers as described previously [23]. Absorption spectrum of the purified enzyme solution in 50 mM phosphate buffer pH 7.0 was recorded with Shimadzu U V / V i s recording spectrophotometer at room temperature.

Enzyme isolation Fresh leaves (100 g) were cut into small pieces and homogenized with 2 vol of 0.1 M phosphate buffer pH 7.0 containing polyvinylpolypyrrolidone (5% wt/vol) and 2 mM mercaptoethanol in a Waring blendor for 5 rain at 4°C. The homogenate was centrifuged at 20,000 g for 30 min, the sediment was discarded, and the clear supernate (200 ml) was used as the source of catalase. The enzyme extract (10 ml) was applied to phenyl-Sepharose CL 4B (50 g wet wt), which was previously equilibrated in 50 mM phosphate buffer pH 7.0 by means of the batch procedure described previously [24]. The suspension was stirred for 1 h at room temperature, packed on a column (2 x 30 cm), and washed with 50 mM phosphate buffer pH 7.0 until A280was less than 0.03. The enzyme was eluted with a linear gradient of Triton (0-0.5% vol/vol) in the same buffer system, and fractions containing catalase activity were pooled and treated with an equal volume of isoamyl alcohol for removal of Triton. The mixture was stirred vigorously and centrifuged at 3000 g for 5 min, and the lower phase containing catalase free of Triton was concentrated by pressure dialysis using a P-30

membrane at 4°C. Alternatively, column fractions containing catalase were pooled and precipitated with ammonium sulfate at 60% saturation and centrifuged, and the pellet was resuspended in a small volume (10 ml) of 50 mM phosphate buffer pH 7.0.

Results Enzyme isolation Catalase was purified to apparent homogeneity from Zantedeschia aethiopica by means of a onestep isolation scheme by hydrophobic interaction chromatography on a phenyl Sepharose CL-4B column (Fig. 1) with a final recovery of enzyme activity of about 61% and a specific activity of about 146 U / m g protein (Table I). The purified preparation of catalase ran as a single protein band when analyzed by native P A G E and S D S - P A G E , corresponding to Mr values of 220,000 and 54,000 daltons, respectively (Figs. 2, 3). The native gel was also stained for enzyme activity, which was found to be coincident with the protein band (Fig. 3B). So far as the subunit structure of this enzyme is concerned, the native molecule is apparently a tetramer containing 4 subunits with identical Mr of 54,000 Da. The Mr of native enzyme was also determined by gel filtration on Sephacryl S-300 having obtained a value of 215,000 daltons. The purified enzyme was also analyzed by isoelectric focusing on ultrathin polyacrylamide gel migrating with a pI value of 5.2 and the protein band was coincident with the activity stain (Fig. 4).

Effect of pH and temperature on enzyme activity The optimum pH of catalase activity was found to be 7.0, whereas stability studies revealed that the enzyme is most stable between 6 and 8. The optimum temperature for catalase activity was found to be 40oC but, on the other hand, stability studies showed that the enzyme is most stable between 0 o and 20oC. The absorption spectrum of purified catalase exhibited 2 peaks at 280 nm and 405 nm characteristic of a hemoprotein (Fig. 5). The absorption ratio (A40s/A280) of this purified enzyme was found to.be 1.085.

Discussion Catalase has been purified from a number of sources to apparent homogeneity by means of purification schemes that involve extraction with

Catalase purification and properties

1761

,4[

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.-.

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2C

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15

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0.4

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56

64

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Fraction No. (4ml)

Fig. 1. Hydrophobic chromatography on a phenyl Sepharose CL-4B column of homogenate containing eatalase from Z. aethiopica. The catalase bound to the column was eluted with a linear gradient of Triton (0-0.05%) in 50 mM phosphate buffer pH 7.0. Protein at Aza0in eluted fractions could not be read due to Triton interference.

Table l o Purification of catalase from leaves of Z. aethiopica by hydrophobic interaction chromatography. Purification steps

Total protein (mg)

Total actitivy (U)

1. Crude homogenate

609.8

995.1

2. Supernate after centrifugation 3. Phenyl Se pharose

63 4.15

Sp. act. Recovery (U / mg protein) (%)

1.63

100

Purification factor

1

970.14

15.39

97.4

9.44

608.58

146.64

61.1

89.96

H. Trindade et al.

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ed regarding the use of this technique for catalase purification. In the present case, catalase from Z. aethiopica was adsorbed to phenyl sepharose CL-4B in the absence of a high salt concentration, suggesting that this enzyme may be highly hydrophobic (Fig. 1). Although most of the enzymes reported in the literature have been adsorbed to hydrophobic matrices in the presence of high salt concentration, there are

A

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organic solvents, ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography, resulting in low yields of enzyme activity [10-15]. The isolation scheme presented in this work involves a simple and rapid purification of catalase by phenyl sepharose CL-4B chromatography (Fig. 1), as opposed to the multistep isolation procedures reported for catalase from spinach leaves and cucumber cotyledons [13-15]. The crude homogenate of Z. aethiopica previously centrifuged at 20,000 g was applied to the resin by a batch procedure, since it was observed that both the recovery of enzyme activity as well as the purification factor were much higher when this technique was used, as compared with the standard chromatographic procedure. Although hydrophobic interaction chromatography has been widely used in enzyme isolation schemes [25], no work has been publish-

_lSlkd _120kd

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Fig. 3. Native PAGE of purified catalase' sample (10/~g) using a 7.5% separating gel. A, Protein stain of the gel; B, Activity stain of the gel. On the right margins are represented molecular weight markers.

Catalase purification and properties

A

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some enzymes (e.g., uroporphyrinogen I synthetase) that can bind to such matrices ia the absence of high ionic strength, due to their high capacity to form hydrophobic interactions

B

pl

[26, 27].

~i

~•

As regards catalase concentration in leaves of

l ~ ~ ~:i ~i~!~

Z. aethiopica, this enzyme represents 1.1% of i

the total soluble leaf protein, which is higher than the values of 0 . 1 - 0 . 5 % reported for catalase from spinach leaves and cotydelons [13-151. The purified enzyme was apparently homogenous when analyzed either by native P A G E or by S D S - P A G E (Figs. 2, 3), suggesting that it is a tetramer consisting of 4 subunits with identical Mr of 54.000 Da. The subunit size (Mr) of catalase from Z. aethiopica is close to the values published for catalase from cucumber and pumpkin cotyledons [14, 15]. However, on the other hand, it is lower when compared to the value of 72.000 Da reported for catalase subunit from spinach leaves [13]. A single pretein band was also observed when the enzyme was analyzed by isoelectric focusing running with a pl value

iiii

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;~ ~:~

+ Fig. 4. Isoelectric focusing on ultrathin (0.1 mm) polyacrylamide gel of purified catalase sample (8/zg). A, Protein

9g

stain of the gel; II, Activity stain of the gel. On the left margin are represented pI markers.

+

1.2 w

1.0m

08_ C~

0.6-

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0.:~

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250

I

300

I

I

350

400

~(NM)

Fig. 5. Absorption spectrum of purified catalase in 50 mM phosphate buffer pH 7.0.

I

450

500

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H. Trindade et al.

of 5.2 (Fig. 4), as opposed to the value of 5.7 reported for maize catalase [28]. The optimum pH of catalase activity was found to be 7.0, which is identical to liver catalase [29]. As regards pH stability studies, catalase from Z. aethiopica was stable in the pH range of 6 - 8 , which is close to that of catalase from spinach leaves [29]. The optimum temperature of catalase activity was found to be 40oC, whereas stability studies revealed that the enzyme is most stable between 0 o and 20oC. Catalase isolated from spinach leaves was reported to be very stable in range 2 - 5 o C at pH 6.0 [29]. The absorption spectrum of the purified catalase showed 2 peaks at 280 nm and 405 nm, characteristic of a hemoprotein (A405/A280 = 1.085), which is similar to the spectra published for catalase from other plant sources [13-15]. However, besides these 2 peaks, catalase from spinach leaves also exhibited 3 minor absorption peaks at 510, 545, and 620 nm [30].

Acknowledgments We would like to thank Mr. Costa Ferreira and Mr. Jo~o Pedroso for their technical assistance during the preparation of the figures.

A

Keferences 1 Chance B. (1948) Nature 161,914-918 2 Grieshaber C.K. & Hoffman H.A. (1974) Anal. Biochem. 60, 537-544 3 Nies D. & Schlegel H.G. (1984) Biotech. Bioeng. 26, 737-741 4 Lucas K., Boland M.J. & Schubert K.R. (1983) Arch. Biochem. Biophys. 226, 190-197 5 Sakai Y., Tamura K. & Tany Y. (1987) Agric. Biol. Chem. 51, 2177-2184 6 Rainbird R.M. & Atkins C.A. (1981) Biochim. Biophys. Acta 659, 132-140 7 Baudhuin P., Beaufay H. & De Duve C. (1965) J. Cell Biol. 26, 219-243 8 Pais M.S.S. (1981) Ann. Sci. Nat. (Bot.) Paris 2-3, 109-114

9 Pais M.S.S. & Neves C. (1982) Plant Growth Reg. 1,233-242 10 Chance B. & Mahehly A.C. (1955) in: Methods in Enzymology (Colowick S.P. & Kaplan N.O., eds.), Academic Press Inc., New York, vol. II, pp. 775-779 11 Deisseroth A. & Dounce A.L. (1970) Physiol. Rev. 50, 319-375 12 Jacob G.S. & Orm-Johnson W.H. (1979) Biochemistry 18, 2967-2975 13 Gregory R.P.F. (1968) Biochim. Biophys. Acta 159, 429-439 14 Lamb J.E., Riezman H. & Becker W.M. (1978) Plant Physiol. 62, 754-760 15 Yamaguchi J. & Nishimura M. (1984) Plant Physiol. 74, 261-267 16 Bradford M.M. (1976)Anal. Biochem. 72, 248-254 17 Aebi H.E. (1983) in: Methods of Enzymatic Analysis (Bergmeyer H.U., Bergmeyer J. & Grabl M., eds.), Verlag Chemie GMbH, Weinheim, vol. III, pp. 273-285 18 Laemmli U.K. (1970) Nature (Lond.) 227, 680-685 19 Davies B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427 20 Righetti P.G. (1983) in: Laboratory Techniques in Biochemistry and Molecular Biology- Isoelectric Focusing (Work T.S. & Burdon R.H., eds.), Elsevier Biomedical Press, New York, pp. 173-198 21 Rhigetti P.G. & Drysdale J.W. (1974) J. Chromatogr. 98, 271-321 22 Gregory E.M. & Frido.vich I. (1974) Anal. Biochem~ 58, 57-62 23 Andrews P. (1965) Biochem. J. 96, 595-606 24 Osborne W.R.A. & Spencer N. (1973) Biochem. J. 133, 117-126 25 Sudhakaran V.K. & Shewale J.G. (1987) Biotech. Lett. 9, 539-542 26 Anderson P.M. & Desnick R.J. (1980)J. Biol. Chem. 255, 1993-1999 27 Lindahl L. & Vogel H. (1984) Anal. Biochem. 140, 394- 402 28 Scandalios J.G., Lui E.H. & Campeau M.A. (1972) Arch. Biochem. Biophys. 153, 695-705 29 Scott D. (1985) in: Enzymes of Food Processing (Reed G., ed.), Academic Press Inc., New York, pp. 247-254 30 Galton A.W. (1955) in: Methods in Enzymology (Colowick S. and Kaplan N., eds.), Academic Press Inc., New York, vol. II, pp. 789-791