Purification and some properties of l -ascorbic acid-specific peroxidase in Euglena gracilis z

ARCHIVESOF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 1, April 15, pp. 121-127, 1980

Purification

and Some Properties of L-Ascorbic Acid-Specific Peroxidase in Euglena gracilis z

SHIGERU SHIGEOKA, YOSHIHISA Department

~fAgricultum1

Chemistry,

NAKANO,

UnPuersity

Received

AND

SHOZABURO KITAOKA

qf Osaka Pr
November

9, 1979

L-Ascorbic acid-specific peroxidase was purified approximately 280-fold from a cell extract ofEugle)&a gracilis. L-Ascorbic acid was virtually the only natural electron donor; the activity with glutathione was less than 1.1% of that with L-ascorbic acid. Cytochrome c, NADH, NADPH, palmitic acid, and triose reductone did not donate electrons to this enzyme. Among artificial electron donors D-araboascorbic acid and pyrogallol had considerable activities but guaiacol, iodide, and reductic acid showed only weak activities. The EugleTLa peroxidase showed an oxidase activity only with triose reductone. The enzyme had a molecular weight of about 76,000 as estimated from gel filtration data. The K,,, values of the enzyme for L-ascorbic acid and hydrogen peroxide were 0.41 and 0.056 mM, respectively, while those for pyrogallol and hydrogen peroxide were 8.6 and 0.22 mM, respectively. These results indicate that the Etiglena peroxidase belongs to a new type of peroxidase to be designated as L-ascorbic acid peroxidase. The inhibition of the enzyme by cyanide and azide as well as absorption spectra of the enzyme alone and in complex with hydrogen peroxide showed that it is a hemoprotein. Organic hydroperoxides were also reduced by the ElLglelza peroxidase. Its physiological function to decompose lipid peroxides as well as hydrogen peroxide generated in cells for the protection of cellular integrity was discussed.

Eugiena gracilis contains in cytosol a new peroxidase which specifically requires L-ascorbic acid as the natural electron donor. This enzyme solely disposes of hydrogen peroxide in the Euglena cells which lacks catalase (1). Although L-ascorbic acid is an electron donor, among many other compounds, for various nonspecific peroxidases (2) none has been known to which only Lascorbic acid, but not other substrates, can donate electrons under physiological conditions. The present paper reports the purification and properties of this peroxidase and discusses its possible physiological functions other than disposing of hydrogen peroxide. MATERIALS

AND METHODS

Cell culture. E. gmcilis, strain z, maintained at 26°C under illumination (2000 lx), was cultured in the Karen-Hutner medium (3) at 26°C for 6 days. Preparation c$ crude enzyme. Cells (54.2 g, wet basis) were washed with 25 mM Verona1 buffer (pH 6.8) containing 30% (w/v) sucrose, and disrupted in 116 ml 121

of the same buffer by sonication (10 kc) for total 50 min with nine intervals of 5 min each. The supernatant obtained by centrifugation of the sonicate at 20,OOOg for 15 min was used as a crude enzyme. The typical preparation showed an enzyme activity of 0.9 pmol of L-ascorbic acid oxidizedimg proteinimin. Protein was determined by the method of Lowry et al. (4). E?lzyme assays. L-Ascorbic acid peroxidase was assayed at 32°C in a reaction mixture (2 ml) containing 25 mM Verona1 buffer (pH 6.2), 0.4 mM sodium Lascorbate, 0.11 mM hydrogen peroxide, and the enzyme solution. The peroxidase activity with pyrogallol or guaiacol as electron donor was assayed by the method of Chance and Maehly (5). The peroxidase activity with glutathione (6), cytochrome c (7), NADH (8), NADPH (9), palmitic acid (lo), or iodide (11) was assayed by the respectively reported methods. The peroxidase activity with reductones (triose reductone and reductic acid) was assayed in a reaction mixture (2 ml) containing 25 mM veronal buffer (pH 6.0), a 0.2 mM reductone, 0.15 mM hydrogen peroxide, and the enzyme, by measuring directly the decrease of the absorbances of triose reductone and reductic acid at 310 and 300 nm, respectively. The oxidase activity with triose reductone or reductic acid was assayed in a reaction mixture (2 ml) containing25 mM veronal buffer (pH 6.0), a 0.2 mM reductone, 0.2 mM MnCl,, 0.1 mM 00039861/80/050121-07$02.00/o Copyright 0 1980 by Academic Press, Inc. .411rights of reproduction in any form reserved.

122

SHIGEOKA, NAKANO, AND KITAOKA

)n-cresol, and enzyme, by measuring spectrophotometrically the decrease of the reductone and also by measuring oxygen consumption with an oxygen electrode. The oxidase activity with NADH and NADPH was assayed by the method of Akazawa and Conn (12). Indoleacetic acid oxidase activity was assayed by the method of Shin and Nakamura (13). Molecular weight determination by gel filtration. Molecular weight of the enzyme was determined by the method of Andrews (14) using a column (1.5 x 90 cm) of Sephadex G-150. Chromatography was carried out at 4°C at a flow rate of 3.6 ml/h using 25 mM Verona1 buffer (pH 6.8) containing 30%. sucrose as an eluant. The column was calibrated with glucose oxidase (Aspergill7h.s niger), lactate dehydrogenase (hog muscle), ovalbumin, trypsin inhibitor (soybean), and cytochrome c (horse heart). Polyacrylamide gel electrophoresis. Disc electrophoresis in polyacrylamide gel was performed as described by Davis (15) using ‘7.5% polyacrylamide gel. Electrophoresis was carried out at a constant current (2 mA/gel) with bromophenol blue as a migration marker. Proteins in the gel were stained with Coomassie brilliant blue R-250 and destained in a 5% acetic acid. Absorption spectra. Absorption spectra of L-ascorbic acid peroxidase and its derivatives were obtained at the scanning speed of 30 nm/min with an autorecording spectrophotometer (Hitachi 356) with 0.5.cm light path quartz cells. Chemicals. t-Butyl hydroperoxide was kindly supplied by Dr. N. Oshino, Nihon Schering K. K., Osaka. Triose reductone and reductic acid were gifts of Dr. M. Takagi, University of Osaka Prefecture. Cytochrome c (horse heart) and glutathione reductase (yeast) were purchased from Sigma Chemical Company. ll-‘“ClPalmitic acid was obtained from Commissariat a I’Energie Atomique, France. RESULTS

Puti$cation of L-Ascorbic Peyoxidase

Acid

librated with the same buffer as employed in the CM-cellulose chromatography. After washing with 40 ml of the buffer, the column was eluted with a linear concentration gradient (O-O.5 M) of potassium chloride and active fractions were brought to 60% saturation of ammonium sulfate. The precipitate was dissolved in the above buffer and chromatographed on a Sephadex G-150 column (2.0 x 100 cm). The active fractions were again applied onto a column (2.0 x 15 cm) of DEAE-cellulose and the column eluted with a O-O.4 M gradient of potassium chloride. The elution pattern (Fig. 1) showed only one peak of peroxidase activity when L-ascorbic acid or pyrogallol was used as an electron donor. The active fractions were dialyzed for 3 h against 25 mM Verona1buffer (pH 6.8) containing 30% sucrose. The dialyzed enzyme solution was applied onto a CMcellulose column (2.0 x 15 cm) and elutecl with the above Verona1buffer. The peroxidative activity was found only in the void volume (total 14 ml). The enzyme preparation thus obtained had been purified 282-fold over the crude enzyme, giving finally 8.6% recovery of the peroxidase activity. Polyacrylamide gel electrophoresis of the purified enzyme showed one major and one minor faint protein band. The peroxidase activity resided only in the main band as determined by the activity in l-mm slices of the gel column. The major band contained about 90% of the total protein. Determination of Molecular by Gel Filtration

Weight

Figure 2 shows plots of the elution volume (Ve) from a Sephadex G-150 column against logarithms of the molecular weight of the standard protein and L-ascorbic acid peroxidase. The molecular weight of this enzyme was calculated to be about 76,000.

Typical purification steps of the Euglena L-ascorbic acid peroxidase are summarized in Table I. The crude enzyme was centrifuged at 105,OOOg for 60 min with an MSE Superspeed ‘75 centrifuge, and the supernatant was applied onto a CM’-cellulose column (2.5 x 40 cm) and eluted with 25 mM Verona1 Stability and Optimum pH and Temperature buffer (pH 6.8) containing 30% sucrose. The active fractions (total 108 ml), collected This enzyme was so labile that it was in the void volume, were applied onto a almost inactivated at 40°C for 5 min in DEAE-cellulose column (2.5 x 40 cm) equi- ordinary buffer. However, L-ascorbic acid peroxidase which was preincubated in 30% sucrose and 0.05 mM ferrous sulfate was ’ Abbreviation used: CM, carboxymethyl.

L-ASCORBIC

ACID-SPECIFIC

PEROXIDASE

TABLE PURIFICATION

Step (1) Crude extract (2) Ultracentrifugation (3) DEAF,-cellulose (4) (i-L),SO, (60% saturation) (5) Sephadex G-150 (6) DEAE-cellulose (7) C&cellulose

ACID

PEROXIDASE

Specific activity (wnolimg proteinimin)

Total activity (fimoVmin)

2674 1121 89 42 x.2 3. 1 0.82

stabilized and retained 88.1% activity after the above treatment. The maximum activity of the enzyme was maintained up to 37°C between pH 6.0 and 7.9 and the enzyme lost the activity almost completely at 52°C. The enzyme showed an optimum pH at 6.2 and optimum temperature at 32-34°C. Szdxtrate

I

OF L-ASCORBIC

Total protein (mg)

Specjficity

For purified Euglerza L-ascorbic acid peroxidase L-ascorbic acid was the best electron donor, giving a specific activity of 253.7 pmol of L-ascorbic acid oxidizedlmg protein/min (Table II). Against this value, D-araboascorbic acid, reductic acid, iodide, pyrogallol, and guaiacol showed 56.0, 7.1, 2.3, 73.1, and 8.0% activities as the electron donors, respectively; glutathione showed less than 1.1% activity and cytochrome c, NADH, NADPH, palmitic acid, and triose

123

IN E,rgletra

Yield (S7,)

2407 ‘690 1291

0.9 2.4 14.5

100 112 .53.6

l’% _* 680 474 208

29.9 82.9 153.0 253.7

52.2 28.3 19.7 8.6

reductone did not serve for the enzyme as electron donors at all. The peroxidase reduced t-butyl hydroperoxide and cumene hydroperoxide in the specific activities 172.8 and 132.2 pmol/mg protein/min, respectively, when L-ascorbic acid was employed as the electron donor. These values correspond to 68.1 and 52.1%, respectively, against the value found when hydrogen peroxide was reduced by the enzyme. The peroxidase did not reduce these hydroperoxides when glutathione was used as an electron donor. L-Ascorbic acid peroxidase showed an oxidase activity significantly only with triose reductone in a specific activity of 235.7

I l-2

I

I 1.6

I

, 2.0

I

5

I 24

VelVo

FIG. 1. Elution chromatography.

pattern

in DEAE-cellulose

column

FIG. 2. Molecular weight estimation by gel filtration. A Sephadex G-150 column (1.6 x 90 cm) was calibrated with known protein standards, including glucose oxidase (186,000) (I), lactate dehydrogenase (109,000) (2), ovalbumin (43,500) (3), trypsin inhibitor (21,600) (4), and cytochrome c (12,400) (5). (0) Euglena L-ascorbic acid peroxidase.

124

SHIGEOKA, TABLE

NAKANO.

II

SUBSTRATESPECIFICITY Hydrogen

Electron donor L-Ascorbic acid D-Araboascorbic acid Triose reductone Reductic acid Iodide Glutathione Ferrocytochrome c NADH NADPH Palmitic acid Pyrogallol Guaiacol

Specific activity (I*moVmg proteinimin) 253.7 142.1 0 18.0 5.8 2.8 0 0 0 0 185.5 20.3

peroxide

Relative activity” (%I 100 56.0 0 7.1 2.3 1.1 0 0 0 0 73.1 8.0

0 The peroxidase activity for L-ascorbic acid (electron donor) and hydrogen peroxide (acceptor) was shown as 100% of activity.

AND KITAOKA

slightly but when the enzyme was incubated with EDTA (1 mM) at 37°C for 3 min in the absence of sucrose and ferrous sulfate there was nearly complete inhibition of the enzyme activity, indicating that participation of metal ion is essential for the activity of the peroxidase. The enzyme was inhibited 96.4 and 91.5% by cyanide and azide (both 1 mM), respectively. As shown in Table IV cyanide inhibits the enzyme competitively against hydrogen peroxide while azide uncompetitively. The apparent K, values for cyanide and azide are 1.8 x 1O-6 and 5.9 X lop5 M, respectively. Reaction Mechanism

The procedure employed was essentially the method by Cleland (16). Using double reciprocal plots of L-ascorbic acid concentration versus reaction velocity, the enzyme systems gave parallel lines (Fig. 3) indicating that the reaction proceeds by a ping-pong mechanism. Secondary plots of slopes or intercepts allowed determination of the kinetic constants: the K,,8 for L-ascorbic acid was 0.41 InM and that for hydrogen peroxide 0.056 mM. When pyrogallol was used as the electron donor, the K, values for pyrogallol and hydrogen peroxide were 9.6 and 0.22 mM, respectively.

pmol/mg protein/min, when Mn” and mcresol were present. The oxidase activity was found feebly with NADH, NADPH, and indoleacetic acid also, in specific activities of 0.5, 0.5, and 0.8 pmollmg protein/ min, respectively. With L-ascorbic acid or reductic acid no oxidase activity was found Absorption Spectra even in the presence of Mn*+ and phenolic In these experiments absorption spectra compounds. were obtained only in the Soret region Effects of Metal Ions and Some Compounds

In 1 InM concentration, Hg2+ showed complete while Mn*+ and Br- showed marked inhibition on the L-ascorbic acid peroxidase activity. A13+,Zn’+, Mg2+, Ca’+, Ni2+, Li+, F-, and I- also inhibited the enzyme to some extent, while Co2+, K+, Cl-, and Se0,2- had no significant effects on the enzyme activity. Table III shows effects of some compounds on the peroxidase activity. Euglena peroxidase was hardly inhibited by sulfhydryl inhibitors like p-chloromercuribenzoate and N-ethylmaleimide, indicating that sulfhydryl group is not concerned with the active center of the enzyme protein. The addition of EDTA inhibited the enzyme

TABLE

III

EFFECTS OFVARIOUS COMPOUNDS" Relative Addition None EDTA p-Chloromercuribenzoate N-Ethylmaleimide NaN, KCN

0.5

mM

100 84.1 94.4 95.6 9.2 4.1

activity

(%a)

1.0 mM 100 83.1 92.8 8.5 3.6

” The compounds were added to the final concentrations indicated, except to 0.05 mM with p-ehloromercuribenzoate, in the standard assay system.

L-ASCORBIC

ACID-SPECIFIC

PEROXIDASE

TABLE KINETICS

OF INHIBITION

L-Ascorbic

Inhibitor KCN NaNj,

Ki value 8.3 x 10” M 8.5 x 10m5M

IV BY CYANIDE

AND AZIDE

Hydrogen

acid Type of inhibition

peroxide Type of inhibition

K, value

Uncompetitive Competitive

because the amount of the purified enzyme was much smaller than that required for covering all ordinary regions. L-Ascorbic acid peroxidase gives an absorption peak at 407 nm indicating it is a hemoprotein. When 7.4 nmol of hydrogen peroxide was added to the enzyme (0.21 mg), the spectrum was converted to a spectrum with a peak at 414 nm, which resembles closely those of the peroxidase-hydrogen peroxide complexes of other well-known heme-containing enzymes (7, 11). The addition of an excess (2 pmol) of Lascorbic acid to the peroxidasehydrogen peroxide complex changed the spectrum to the one with a peak at 408 nm similar to the original spectrum of L-ascorbic acid peroxidase. Complexes of L-ascorbic acid peroxidase with cyanide and azide (both 6 pmol) showed the Soret peaks at 421 and 412 nm, respectively, in good agreement with those reported for hemoproteins (7,17).

125

IN Euglena

1.8 X 10” M 5.9 X lo-5 M

Competitive Uncompetitive

acid is used as the electron donor as when pyrogallol is used. The peroxidase activities with L-ascorbic acid and pyrogallol were recovered in the same fractions in the CM-cellulose and DEAE-cellulose column chromatographies during the purification of the enzyme (Fig. l), and they were found as one peak in the same position in polyacrylamide gel electrophoresis (1). The results indicate that a single protein is certainly responsible for the two activities and that only a single peroxidase is contained in E. grucilis cells. L-Ascorbic acid is an electron donor with a relatively weak activity, among many other compounds, for many nonspecific peroxidases, for example horseradish peroxidase (2), but no peroxidase has been known which requires specifically L-ascorbic acid as the physiological electron donor. Groden and Berk (18, 19) have reported the occurrence of an L-ascorbic acid-specific peroxi-

DISCUSSION

A highly purified preparation of a peroxidase from an extract of E. gracilis was obtained by a seven-step procedure. This enzyme required L-ascorbic acid virtually as the sole natural electron donor; it utilized glutathione only to a much lesser extent. NADH, NADPH, cytochrome c, palmitic acid and triose reductone were not electron donors for the peroxidase activity in both crude and purified preparations. Among artificial substances pyrogallol and D-araboascorbic acid had considerable activities as electron donors. The K,,) values show that the peroxidase has 23 times as high an affinity toward L-ascorbic acid as toward pyrogallol, and its affinity toward hydrogen peroxide is 4 times as high when L-ascorbic

I I I-Ascorbic

rc,*

(mK’1

l/H202(mM-‘I

FIG. 3. Double reciprocal plots of L-ascorbic acid peroxidase activity in various concentrations of both L-ascorbic acid and hydrogen peroxide (a) Double reciprocal plots of initial velocity against variable Lascorbic acid concentrations at several fixed hydrogen peroxide concentrations. lH,O,] were 0.0110 (line l), 0.0168 (line 2), 0.0221 (line 3), 0.0294 (line 4), 0.0442 (line 5), and 0.0735 (line 6) mM. (b) Replots ofintercepts against reciprocal [H,O,].

126

SHIGEOKA,

NAKANO,

dase in spinach chloroplasts. The Euglena peroxidase occurs in the cytosol (1). Besides being distinct from previously known peroxidases with regard to the specificity of electron donor requirement, the Euglena L-ascorbic acid peroxidase has revealed unique properties. The molecular weight of the enzyme was estimated to be 76,000 by gel filtration. The value is similar to the one of rat liver glutathione peroxidase (ZO),and different from those of other specific peroxidases and nonspecific peroxidases. Another characteristic property ofEuglena peroxidase is its instability in ordinary buffer. The presence of 30% sucrose and 0.05 lllM ferrous sulfate stabilizes the enzyme; stannous chloride has also such a stabilizing action to a lesser extent. Such stability characteristics have not been reported on any previously known peroxidases (2). Plant peroxidases exert an oxidase activity also with such compounds as reduced pyridine nucleotides, while the Euglena peroxidase showed a significant oxidase activity only with triose reductone. The oxidase activities of the Euglena enzyme with NADH, NADPH, and indoleacetic acid were very low unlike other peroxidases such as horseradish peroxidase (12). Double reciprocal plots were linear and constituted a family of parallel lines indicating the enzyme proceeds by a ping-pong mechanism. The same mechanism has been reported on some peroxidases such as turnip peroxidase (21, 22), rat lung glutathione peroxidase (23), and cytochrome c peroxidase (24). The L-ascorbic acid peroxidase in E. gracilis is a hemoprotein like many other peroxidases (2, 7), since it is inhibited by cyanide and azide, the inhibitors specific for heme-containing enzymes. The absorption spectra of the Euglena L-ascorbic acid peroxidase and its complex with hydrogen peroxide resemble closely the corresponding spectra of other hemoprotein-peroxidases (7, ll), supporting the assumption that the Euglena peroxidase is a hemoprotein. The spectral maxima of the complex of the Euglena peroxidase with cyanide and azide are similar to those of cytochrome c peroxidase (7) and horseradish peroxidase (17).

AND KITAOKA

Glutathione peroxidase, with glutathione as the specific electron donor, reduces a variety of organic hydroperoxides as well as hydrogen peroxide (25). The similar reactivity has been found with the Euglena L-ascorbic acid peroxidase with L-ascorbic acid, but not glutathione, as the specific electron donor. It is well known that peroxidation of lipids in cell membrane causes loss of integrity of the membrane and inactivation of the membrane-bound enzymes (26). L-Ascorbic acid peroxidase in E. gracilis accordingly works for protection of cell membrane, like glutathione peroxidase in rat tissues (27,28), by reducing the peroxide compounds generated endogenously from unsaturated fatty acids. E. gracilis contains abundant polyunsaturated fatty acids (29). This action should be the second most important function of this enzyme, next to its action of destroying hydrogen peroxide in E. gracilis which lacks catalase (1). The experimental results herein reported have clearly shown that the peroxidase occurring in E. gracilis is a hemoprotein and has properties some of which are common to other peroxidases but it is distinct from any previously known specific and nonspecific peroxidases in its specific requirement of L-ascorbic acid as electron donor, stability characteristics, and other properties. It may be warranted that the Euglena peroxidase is classified as a new type of peroxidase. ACKNOWLEDGMENTS We wish to thank Dr. U. Morita, for his useful discussion.

Kyoto University,

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