/ . Biochem. 83, 849-857 (1978)
A Study'orj the Reconstitution of Iron-Superoxide Dismutase from Pseudomonds ovalis1 .
Fumiyuki YAMAKURA Department of Chemistry, School of Medicine, Juntendo University, Narashino, Chiba 275 Received for publication, September 24, 1977
Iron-free aposuperoxide dismutase was prepared by two methods from a native enzyme from Pseudomonas ovalis. The first method was the incubation of the native enzyme with sodium carbonate buffer (pH 11.0) containing dithiothreitol and EDTA under anaerobic conditions with subsequent dialysis to return the pH to 7.8. The second method was the incubation of the native enzyme in 3-(N-morpholino)-propanesulfonic acid-NaOH buffer (pH 7.0) containing urea, dithiothreitol, and EDTA under anaerobic conditions with subsequent dialysis to remove the urea. The reconstituted enzyme was obtained by incubation of the apoenzyme with the corresponding buffer described above, with addition of ferrous ammonium sulfate under anaerobic conditions. The apoenzyme had neither any significant iron content nor enzymatic activity. The reconstituted enzyme had the same amount of iron and the same enzymatic activity as the native one. The apoenzyme had no visible absorption, but the reconstituted enzyme had the same visible absorption as the native enzyme. The four sulfhydryl groups of the apoenzyme showed high reactivity with 5,5'-dithiobis-(2-nitrobenzoic acid), while those of the native and reconstituted enzymes had little reactivity. The fluorescence intensity of the apoenzyme was about three times that of the native and reconstituted enzymes. Circular dichroism spectra of the apo- and reconstituted enzymes in the 200-250 nm region were similar in shape to that of the native enzyme. In the 250-340 nm region, the circular dichroism spectrum of the apoenzyme was different from that of the native enzyme; this difference was greatly reduced in§ the reconstituted enzyme. The electron paramagnetic resonance spectrum of the reconstituted enzyme showed the same signal prameters as the native enzyme. Thermal inactivation kinetics of the reconstituted enzyme were compared with those of the native enzyme; the reconstituted enzyme was slightly less stable than the native enzyme.
Superoxide dismutase [EC 1.15.1.1], which catalyzes the dismutation of superoxide free radicals, O t ~+ O t "+2 H+—•H,Ot+O,, was first discovered by 1
McCord and Fridovich in 1969 (7, 2). This enzyme has been proposed to be an important- component of the defences which have evolved to deal
This work was presented in a thesis in partial fulfillment of the requirements of Rikkyo (St. Paul's) University for the doctoral degree. Vol. 83, No. 3, 1978
849
850
with the damaging actions of the superoxide radical in organisms (7). Superoxide dismutase is widely distributed among oxygen-metabolizing organisms and some obligate anaerobes (3-5). Two evolutionally distinct classes of superoxide dismutase, namely, Mnor Fe-containing and Cu,Zn-containing enzymes, have been observed in many organisms. With a few exceptions (6-10), superoxide dismutases isolated from prokaryotes and from mitochondria contain manganase or iron (J, 11-23), whereas Cu.Zn-enzymes have been found only in eulcaryotes (1, 24). Since McCord and Fridovich (2) reported the preparation of apo- and reconstituted Cu.Znsuperoxide dismutases, many studies have been carried out on the properties of apo- and reconstituted Cu.Zn-superoxide dismutases (1, 25, 26). It has been shown that copper is essential for enzymatic activity, and that copper and zinc are located in very close proximity to each other. In the case of Mn- or Fe-superoxide dismutase, in contrast, there have been few attempts to prepare apo- and reconstituted enzymes. Ose and Fridovich (27) reported the reversible removal of Mn from Mn-superoxide dismutase of Escherichia coli and the replacement of Mn with Co, Zn, or Ni. Brock et al. (28) and Sato et al. (14) also reported the preparation of stable apoenzyme and reconstitution of Mn-superoxide dismutase from Bacillus stearothermophilus and Thermus aquaticus, respectively. Very recently, Anastasi et al. (22) reported a purification of Fe-superoxide dismutase from Bacillus megaterium and some spectroscopic studies on the apoenzyme, which had been prepared by dissolution of acid-denaturated enzyme in buffer at neutral pH. In previous studies, we isolated and characterized Fe-superoxide dismutase from Pseudomonas ovalis (20, 29, 30). The Fe-enzyme has a molecular weight of 40,000 and is composed of two subunits of equal size (20). The iron content of the purified enzyme varied from 1.1 to 1.5 g atoms per mol of enzyme. The present report describes the first study on the preparation of Fe-free aposuperoxide dismutase, the reconstitution of the Fe-enzyme, and characterization of apo- and reconstituted Fe-superoxide dismutase. Preliminary accounts of a portion of this work have already appeared (30).
F. YAMAKURA
MATERIALS AND METHODS Materials—Xanthine oxidase [EC 1.2.3.1] and cytochrome c were products of Boehringer Mannheim. Xanthine, ovalbumin, bovine serum albumin, chymotrypsinogen, and myoglobin were obtained from Sigma. All other materials were of the highest grade commercially available. Preparation of Fe-Superoxide Dismutase—Fesuperoxide dismutase was isolated from Pseudomonas ovalis according to the procedure reported previously (20). Enzyme Assay—Superoxide dismutase activity was measured by the method of McCord and Fridovich (2), with a slight modification (20). For the studies on the thermal stability, the enzymatic activity was determined by the method of Nishikimi et al. (31). Determination of Protein Concentration—Protein concentration was determined by the microbiuret method of Itzhaki and Gill (52), using bovine serum albumin as a standard. The concentration of the native enzyme was determined from the absorbance at 280 nm using an extinction coefficient of 80,600 M"1 -cm"1
(20).
Metal Analysis—Iron was determined by the method of Massey (33), except that balho-phenanthroline was used as a colorimetric reagent. Estimation of Molecular Weight—The molecular weights of the apo- and reconstituted enzymes were estimated by gel filtration according to Andrews (34). A column of Sephadex G-100 (1.5x87 cm) was eluted with 50 mM potassium phosphate buffer (pH 7.8) containing 0.1M KC1. The column was calibrated with the following standards: bovine serum albumin, ovalbumin, chymotrypsinogen, and myoglobin. Subunit molecular weights were estimated by the method of Weber and Osborn (35), using bovine serum albumin, ovalbumin, chymotrypsinogen, myoglobin, and cytochrome c as standard proteins. Disc Gel Electrophoresis—Polyacrylamide gel electrophoresis was performed with 6% gel (pH 8.9) at 5°C according to Davis (56). Superoxide dismutase activity on the gel was detected by the photochemical method of Beauchamp and-Fridovich (37). In order to analyze the relationship between major and minor protein bands, mobilities of the apo- and reconstituted superoxide dismutase /. Biochem.
RECONSTITUTION OF Fe-SUPEROXIDE DISMUTASE on polyacrylamidc gels (6, 7.5, 9, and 10.5%) were examined by the method of Hedrick and Smith (38). Reactivity of Sulfhydryl Groups—Reactivity of sulfhydryl groups was examined spectrophotometrically using 5,5'-dithiobis-(2-nitrobenzoic acid) as described by Ellman (59). Spectral Measurements—Fluorescence emission spectra were measured with a Hitachi 204 spectrophotofluorometer, but were not corrected for the wavelength dependence of the instrument's sensitivity. Absorption spectra were obtained using a Hitachi 124 spectrophotometer. CD spectra were measured with a JASCO J-20 automatic recording spectropolarimeter. CD measurements were carried out with a 0.5 cm light-path cell and a protein concentration of 30-35 ^M from 250 to 340 nm, and with a 0.063 cm light-path cell and a protein concentration of 11-12 ^M from 200 to 250 nm. In the calculation of the mean residue ellipticity, [6], mean residue weight was taken as 110.6, a value which was calculated from the amino acid composition described in the previous report (20). EPR spectra were measured at 77°K with a Nihondenshi EPR spectrometer, model JES-ME3X, equipped with a 100 kHz field modulator.
851
gas using a Thunberg tube. This solution was dialyzed against 0.2 M sodium carbonate buffer (pH 9.2) containing 1 mM EDTA and 1 mM dithiothreitol for 5-12 h at 4CC, and subsequently against 50 mM potassium phosphate buffer (pH 7.8) containing 0.5 mM dithiothreitol for 18 h at 4°C under anaerobic conditions, bubbling nitrogen gas through the dialysis buffers. The enzyme was diluted ten-fold with distilled water and applied to a DEAE-cellulose column (0.9x4 cm) previously equilibrated with 2.5 mM potassium phosphate buffer (pH 7.8). The apoenzyme was eluted by 50 mM potassium phosphate buffer (pH 7.8), and used for the reconstitution study. The apoenzyme (14 mg) was incubated for 25 min at 30°C with 8 ml of 0.2 M sodium carbonate buffer (pH 11.0) containing 12 mM ferrous ammonium sulfate and 10 mM dithiothreitol under anaerobic conditions. Then the solution was dialyzed against 0.2 M sodium carbonate buffer (pH 9.2) containing 1 mM dithiothreitol and 1.2mM ferrous ammonium sulfate for 5-12 h at 4°C under anaerobic conditions. After further dialysis against two changes of 50 mM potassium phosphate buffer (pH 7.8), reconstituted enzyme was recovered using a DEAE-cellulose column as described above. Determination of Thermal Inactivation Charac-The yields of the apo- and reconstituted enzymes teristics—Test tubes containing about 0.3 I*M en- were about 75% and 70%, respectively. Reconzyme were sealed with cellophane and immersed in stitution of the Fe-enzyme did not occur on ada water bath at various temperatures. The re- dition of ferrous ammonium sulfate to the aposidual activity were measured at various times by enzyme in buffer at pH 7.8. the assay method of Nishikimi el at. (31). (B) Urea-denaturation method: The purified Fe-superoxide dismutase (30 mg) was incubated for 2 h at 45°C in 8 ml of 0.12 M 3-(N-morphoIino)RESULTS propanesulfonic acid-NaOH buffer (pH 7.0) conPreparation of Aposuperoxide Dismutase and taining 7.8 M urea, 10 mM dithiothreitol, and 2 mM Reconstitution of the Holoenzyme—The methods EDTA. This solution was dialyzed against the developed for the preparation of apo- and recon- same buffer at 30 mM containing 1 mM dithiothreitol stituted enzymes are described here in detail. Two and 1 mM EDTA for 5-6 h at 20°C, and subsequently against 50 mM potassium phosphate buffer methods were developed, as follows: {A) Alkaline treatment method: The purified (pH 7.8) containing 0.5 mM dithiothreitol for 18 h Fe-superoxide dismutase (30 mg) was dialyzed at 4°C. All procedures before this last step were overnight against 700 volumes of distilled water. carried out under anaerobic conditions in the same The dialyzed enzyme was incubated for 25 min at way as the alkaline treatment method. The apo30°C in 5 ml of 0.2 M sodium carbonate buffer enzyme was recovered using a DEAE-cellulose (pHll.O) containing 2 mM EDTA and 10 mM column as described above. dithiothreitol after the pH of the mixture had been The apoenzyme (10 mg) was incubated for adjusted to 11 by addition of 1 N NaOH. The 90 min at 45°C in 16 ml of 0.12 M 3-(N-morphoincubation was carried out under anaerobic con- lino)-propanesulfonic acid-NaOH buffer (pH 7.0) ditions by evacuation and flushing with nitrogen containing 12 mM ferrous ammonium sulfate, 7.8 M Vol. 83, No. 3, 1978
852
ID;
Fig. 1. Polyacrylamide gel electrophoresis of native, apo-, and reconstituted enzymes, (a) Native enzyme, 44 eg; (b) apoenzyme, 40 ^g; (c) reconstituted enzyme, 39 fig; and (d) reconstituted enzyme, 4 fig were each subjected to polyacrylamide gel electrophoresis at pH 8.9. The gels (a, b, c) were stained for protein with Amido Black 10B; the achromatic zones indicate enzymatic activity (d). 1—
1
1 -
r
_i
^ = 5 ^ ^
on ° 0.5 LATIVE 1
urea, and 10 mM dithiothreitol under anaerobic conditions. Then the solution was dialyzed against 30 mM 3-(N-morpholino)-propanesu]fonic acidNaOH buffer (pH 7.0) containing 7.8 M urea, 12 mM ferrous ammonium sulfate, and 10 mM dithiothreitol under anaerobic conditions. Next, the solution was dialyzed overnight against 30 mM 3 - (N - morphol ino) - propanesulfonic acid-NaOH buffer (pH 7.0) containing 1.2 mM ferrous ammonium sulfate and 1 mM dithiothreitol at 4°C under anaerobic conditions. After dialysis against two changes of 50 mM potassium phosphate buffer (pH 7.8), the reconstituted enzyme was recovered on a DEAE-cellulose column. The yields of the apoand reconstituted enzymes by this method were about 70% and 55%, respectively. Unless otherwise stated, the enzyme preparations used in the present work were prepared by the alkaline treatment method. Characterization of Apo- and Reconstituted Enzymes—Electrophoreses: Figure 1 shows the electrophoretic patterns of apo- and reconstituted superoxide dismutase on polyacrylamide gels. One major and one minor protein bands were observed in both the apo- and reconstituted enzymes. Both protein bands of the reconstituted enzyme coincided with the zones of enzymatic activity, which were located by the photochemical method. The major protein bands of the apo- and reconstituted enzymes showed the same mobility as the native enzyme (Fig. 1). It appeared that the major and minor protein bands of the apo- and reconstituted enzymes were "charge" isomers, according to the analytical method of Hedrick and Smith (38) (Fig. 2). Molecular weight: The apo- and reconstituted enzymes were found to have a molecular weight of 40,000 ±1,500 by the gel nitration technique. The apo- and reconstituted enzymes migrated as a single band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis and subunit molecular weights were estimated to be in the range of 19,5OO±5OO. These results indicate that both the apo- and reconstituted enzymes are composed of two subunits of equal size, as in the case of the native enzyme (20). Iron contents and activities: Iron contents and enzymatic activities of the apo- and reconstituted enzymes prepared by either the alkaline treatment or urea-denaturation method are shown
F. YAMAKURA
MINOR PROTEIN BAND!
-
MAJOR PROTEIN BAND
LU
cr
i
3 GEL
l
10 CONCENTRATION
t
15
Fig. 2. The effects of different gel concentrations on the mobility of apo- and reconstituted enzymes. O, Major protein band; • , minor protein band. in Table I. The apoenzymes had neither any significant Fe content nor enzymatic activity. The reconstituted enzymes had about 1.1 to 1.3 g atoms of Fe per mol of enzyme. Enzymatic activities of the reconstituted enzymes were almost the same as that of the native enzyme. These results show that the iron is essential for Fe-superoxide dismutase activity. Absorption spectra: Figure 3 shows the optical absorption spectra of the apo- and reconstituted enzymes. In the visible range from 320 to 600 run, the broad absorption observed in the native enzyme / . Biochem.
RECONSTITUTION OF Fe-SUPEROXIDE DISMUTASE
853
TABLE I. Properties of apo- and reconstituted enzymes. Fe» (g atoms/mol)
Specific activity
1. Native enzyme (Alkaline method) 2. Apoenzyme 3. Reconstituted enzyme 4. Reconstituted enzyme
1.12±0.06
3,200±151
0.035
0.085 ±0.022 1.13±0.O4 1.27 ±0.03
100±23 3,400+380 3,300±280
0.0025 0.035 0.036
(Urea method) 5. Apoenzyme 6. Reconstituted enzyme
0.113±0.01 1.06±0.1
A Study'orj the Reconstitution of Iron-Superoxide Dismutase from Pseudomonds ovalis1 .
Fumiyuki YAMAKURA Department of Chemistry, School of Medicine, Juntendo University, Narashino, Chiba 275 Received for publication, September 24, 1977
Iron-free aposuperoxide dismutase was prepared by two methods from a native enzyme from Pseudomonas ovalis. The first method was the incubation of the native enzyme with sodium carbonate buffer (pH 11.0) containing dithiothreitol and EDTA under anaerobic conditions with subsequent dialysis to return the pH to 7.8. The second method was the incubation of the native enzyme in 3-(N-morpholino)-propanesulfonic acid-NaOH buffer (pH 7.0) containing urea, dithiothreitol, and EDTA under anaerobic conditions with subsequent dialysis to remove the urea. The reconstituted enzyme was obtained by incubation of the apoenzyme with the corresponding buffer described above, with addition of ferrous ammonium sulfate under anaerobic conditions. The apoenzyme had neither any significant iron content nor enzymatic activity. The reconstituted enzyme had the same amount of iron and the same enzymatic activity as the native one. The apoenzyme had no visible absorption, but the reconstituted enzyme had the same visible absorption as the native enzyme. The four sulfhydryl groups of the apoenzyme showed high reactivity with 5,5'-dithiobis-(2-nitrobenzoic acid), while those of the native and reconstituted enzymes had little reactivity. The fluorescence intensity of the apoenzyme was about three times that of the native and reconstituted enzymes. Circular dichroism spectra of the apo- and reconstituted enzymes in the 200-250 nm region were similar in shape to that of the native enzyme. In the 250-340 nm region, the circular dichroism spectrum of the apoenzyme was different from that of the native enzyme; this difference was greatly reduced in§ the reconstituted enzyme. The electron paramagnetic resonance spectrum of the reconstituted enzyme showed the same signal prameters as the native enzyme. Thermal inactivation kinetics of the reconstituted enzyme were compared with those of the native enzyme; the reconstituted enzyme was slightly less stable than the native enzyme.
Superoxide dismutase [EC 1.15.1.1], which catalyzes the dismutation of superoxide free radicals, O t ~+ O t "+2 H+—•H,Ot+O,, was first discovered by 1
McCord and Fridovich in 1969 (7, 2). This enzyme has been proposed to be an important- component of the defences which have evolved to deal
This work was presented in a thesis in partial fulfillment of the requirements of Rikkyo (St. Paul's) University for the doctoral degree. Vol. 83, No. 3, 1978
849
850
with the damaging actions of the superoxide radical in organisms (7). Superoxide dismutase is widely distributed among oxygen-metabolizing organisms and some obligate anaerobes (3-5). Two evolutionally distinct classes of superoxide dismutase, namely, Mnor Fe-containing and Cu,Zn-containing enzymes, have been observed in many organisms. With a few exceptions (6-10), superoxide dismutases isolated from prokaryotes and from mitochondria contain manganase or iron (J, 11-23), whereas Cu.Zn-enzymes have been found only in eulcaryotes (1, 24). Since McCord and Fridovich (2) reported the preparation of apo- and reconstituted Cu.Znsuperoxide dismutases, many studies have been carried out on the properties of apo- and reconstituted Cu.Zn-superoxide dismutases (1, 25, 26). It has been shown that copper is essential for enzymatic activity, and that copper and zinc are located in very close proximity to each other. In the case of Mn- or Fe-superoxide dismutase, in contrast, there have been few attempts to prepare apo- and reconstituted enzymes. Ose and Fridovich (27) reported the reversible removal of Mn from Mn-superoxide dismutase of Escherichia coli and the replacement of Mn with Co, Zn, or Ni. Brock et al. (28) and Sato et al. (14) also reported the preparation of stable apoenzyme and reconstitution of Mn-superoxide dismutase from Bacillus stearothermophilus and Thermus aquaticus, respectively. Very recently, Anastasi et al. (22) reported a purification of Fe-superoxide dismutase from Bacillus megaterium and some spectroscopic studies on the apoenzyme, which had been prepared by dissolution of acid-denaturated enzyme in buffer at neutral pH. In previous studies, we isolated and characterized Fe-superoxide dismutase from Pseudomonas ovalis (20, 29, 30). The Fe-enzyme has a molecular weight of 40,000 and is composed of two subunits of equal size (20). The iron content of the purified enzyme varied from 1.1 to 1.5 g atoms per mol of enzyme. The present report describes the first study on the preparation of Fe-free aposuperoxide dismutase, the reconstitution of the Fe-enzyme, and characterization of apo- and reconstituted Fe-superoxide dismutase. Preliminary accounts of a portion of this work have already appeared (30).
F. YAMAKURA
MATERIALS AND METHODS Materials—Xanthine oxidase [EC 1.2.3.1] and cytochrome c were products of Boehringer Mannheim. Xanthine, ovalbumin, bovine serum albumin, chymotrypsinogen, and myoglobin were obtained from Sigma. All other materials were of the highest grade commercially available. Preparation of Fe-Superoxide Dismutase—Fesuperoxide dismutase was isolated from Pseudomonas ovalis according to the procedure reported previously (20). Enzyme Assay—Superoxide dismutase activity was measured by the method of McCord and Fridovich (2), with a slight modification (20). For the studies on the thermal stability, the enzymatic activity was determined by the method of Nishikimi et al. (31). Determination of Protein Concentration—Protein concentration was determined by the microbiuret method of Itzhaki and Gill (52), using bovine serum albumin as a standard. The concentration of the native enzyme was determined from the absorbance at 280 nm using an extinction coefficient of 80,600 M"1 -cm"1
(20).
Metal Analysis—Iron was determined by the method of Massey (33), except that balho-phenanthroline was used as a colorimetric reagent. Estimation of Molecular Weight—The molecular weights of the apo- and reconstituted enzymes were estimated by gel filtration according to Andrews (34). A column of Sephadex G-100 (1.5x87 cm) was eluted with 50 mM potassium phosphate buffer (pH 7.8) containing 0.1M KC1. The column was calibrated with the following standards: bovine serum albumin, ovalbumin, chymotrypsinogen, and myoglobin. Subunit molecular weights were estimated by the method of Weber and Osborn (35), using bovine serum albumin, ovalbumin, chymotrypsinogen, myoglobin, and cytochrome c as standard proteins. Disc Gel Electrophoresis—Polyacrylamide gel electrophoresis was performed with 6% gel (pH 8.9) at 5°C according to Davis (56). Superoxide dismutase activity on the gel was detected by the photochemical method of Beauchamp and-Fridovich (37). In order to analyze the relationship between major and minor protein bands, mobilities of the apo- and reconstituted superoxide dismutase /. Biochem.
RECONSTITUTION OF Fe-SUPEROXIDE DISMUTASE on polyacrylamidc gels (6, 7.5, 9, and 10.5%) were examined by the method of Hedrick and Smith (38). Reactivity of Sulfhydryl Groups—Reactivity of sulfhydryl groups was examined spectrophotometrically using 5,5'-dithiobis-(2-nitrobenzoic acid) as described by Ellman (59). Spectral Measurements—Fluorescence emission spectra were measured with a Hitachi 204 spectrophotofluorometer, but were not corrected for the wavelength dependence of the instrument's sensitivity. Absorption spectra were obtained using a Hitachi 124 spectrophotometer. CD spectra were measured with a JASCO J-20 automatic recording spectropolarimeter. CD measurements were carried out with a 0.5 cm light-path cell and a protein concentration of 30-35 ^M from 250 to 340 nm, and with a 0.063 cm light-path cell and a protein concentration of 11-12 ^M from 200 to 250 nm. In the calculation of the mean residue ellipticity, [6], mean residue weight was taken as 110.6, a value which was calculated from the amino acid composition described in the previous report (20). EPR spectra were measured at 77°K with a Nihondenshi EPR spectrometer, model JES-ME3X, equipped with a 100 kHz field modulator.
851
gas using a Thunberg tube. This solution was dialyzed against 0.2 M sodium carbonate buffer (pH 9.2) containing 1 mM EDTA and 1 mM dithiothreitol for 5-12 h at 4CC, and subsequently against 50 mM potassium phosphate buffer (pH 7.8) containing 0.5 mM dithiothreitol for 18 h at 4°C under anaerobic conditions, bubbling nitrogen gas through the dialysis buffers. The enzyme was diluted ten-fold with distilled water and applied to a DEAE-cellulose column (0.9x4 cm) previously equilibrated with 2.5 mM potassium phosphate buffer (pH 7.8). The apoenzyme was eluted by 50 mM potassium phosphate buffer (pH 7.8), and used for the reconstitution study. The apoenzyme (14 mg) was incubated for 25 min at 30°C with 8 ml of 0.2 M sodium carbonate buffer (pH 11.0) containing 12 mM ferrous ammonium sulfate and 10 mM dithiothreitol under anaerobic conditions. Then the solution was dialyzed against 0.2 M sodium carbonate buffer (pH 9.2) containing 1 mM dithiothreitol and 1.2mM ferrous ammonium sulfate for 5-12 h at 4°C under anaerobic conditions. After further dialysis against two changes of 50 mM potassium phosphate buffer (pH 7.8), reconstituted enzyme was recovered using a DEAE-cellulose column as described above. Determination of Thermal Inactivation Charac-The yields of the apo- and reconstituted enzymes teristics—Test tubes containing about 0.3 I*M en- were about 75% and 70%, respectively. Reconzyme were sealed with cellophane and immersed in stitution of the Fe-enzyme did not occur on ada water bath at various temperatures. The re- dition of ferrous ammonium sulfate to the aposidual activity were measured at various times by enzyme in buffer at pH 7.8. the assay method of Nishikimi el at. (31). (B) Urea-denaturation method: The purified Fe-superoxide dismutase (30 mg) was incubated for 2 h at 45°C in 8 ml of 0.12 M 3-(N-morphoIino)RESULTS propanesulfonic acid-NaOH buffer (pH 7.0) conPreparation of Aposuperoxide Dismutase and taining 7.8 M urea, 10 mM dithiothreitol, and 2 mM Reconstitution of the Holoenzyme—The methods EDTA. This solution was dialyzed against the developed for the preparation of apo- and recon- same buffer at 30 mM containing 1 mM dithiothreitol stituted enzymes are described here in detail. Two and 1 mM EDTA for 5-6 h at 20°C, and subsequently against 50 mM potassium phosphate buffer methods were developed, as follows: {A) Alkaline treatment method: The purified (pH 7.8) containing 0.5 mM dithiothreitol for 18 h Fe-superoxide dismutase (30 mg) was dialyzed at 4°C. All procedures before this last step were overnight against 700 volumes of distilled water. carried out under anaerobic conditions in the same The dialyzed enzyme was incubated for 25 min at way as the alkaline treatment method. The apo30°C in 5 ml of 0.2 M sodium carbonate buffer enzyme was recovered using a DEAE-cellulose (pHll.O) containing 2 mM EDTA and 10 mM column as described above. dithiothreitol after the pH of the mixture had been The apoenzyme (10 mg) was incubated for adjusted to 11 by addition of 1 N NaOH. The 90 min at 45°C in 16 ml of 0.12 M 3-(N-morphoincubation was carried out under anaerobic con- lino)-propanesulfonic acid-NaOH buffer (pH 7.0) ditions by evacuation and flushing with nitrogen containing 12 mM ferrous ammonium sulfate, 7.8 M Vol. 83, No. 3, 1978
852
ID;
Fig. 1. Polyacrylamide gel electrophoresis of native, apo-, and reconstituted enzymes, (a) Native enzyme, 44 eg; (b) apoenzyme, 40 ^g; (c) reconstituted enzyme, 39 fig; and (d) reconstituted enzyme, 4 fig were each subjected to polyacrylamide gel electrophoresis at pH 8.9. The gels (a, b, c) were stained for protein with Amido Black 10B; the achromatic zones indicate enzymatic activity (d). 1—
1
1 -
r
_i
^ = 5 ^ ^
on ° 0.5 LATIVE 1
urea, and 10 mM dithiothreitol under anaerobic conditions. Then the solution was dialyzed against 30 mM 3-(N-morpholino)-propanesu]fonic acidNaOH buffer (pH 7.0) containing 7.8 M urea, 12 mM ferrous ammonium sulfate, and 10 mM dithiothreitol under anaerobic conditions. Next, the solution was dialyzed overnight against 30 mM 3 - (N - morphol ino) - propanesulfonic acid-NaOH buffer (pH 7.0) containing 1.2 mM ferrous ammonium sulfate and 1 mM dithiothreitol at 4°C under anaerobic conditions. After dialysis against two changes of 50 mM potassium phosphate buffer (pH 7.8), the reconstituted enzyme was recovered on a DEAE-cellulose column. The yields of the apoand reconstituted enzymes by this method were about 70% and 55%, respectively. Unless otherwise stated, the enzyme preparations used in the present work were prepared by the alkaline treatment method. Characterization of Apo- and Reconstituted Enzymes—Electrophoreses: Figure 1 shows the electrophoretic patterns of apo- and reconstituted superoxide dismutase on polyacrylamide gels. One major and one minor protein bands were observed in both the apo- and reconstituted enzymes. Both protein bands of the reconstituted enzyme coincided with the zones of enzymatic activity, which were located by the photochemical method. The major protein bands of the apo- and reconstituted enzymes showed the same mobility as the native enzyme (Fig. 1). It appeared that the major and minor protein bands of the apo- and reconstituted enzymes were "charge" isomers, according to the analytical method of Hedrick and Smith (38) (Fig. 2). Molecular weight: The apo- and reconstituted enzymes were found to have a molecular weight of 40,000 ±1,500 by the gel nitration technique. The apo- and reconstituted enzymes migrated as a single band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis and subunit molecular weights were estimated to be in the range of 19,5OO±5OO. These results indicate that both the apo- and reconstituted enzymes are composed of two subunits of equal size, as in the case of the native enzyme (20). Iron contents and activities: Iron contents and enzymatic activities of the apo- and reconstituted enzymes prepared by either the alkaline treatment or urea-denaturation method are shown
F. YAMAKURA
MINOR PROTEIN BAND!
-
MAJOR PROTEIN BAND
LU
cr
i
3 GEL
l
10 CONCENTRATION
t
15
Fig. 2. The effects of different gel concentrations on the mobility of apo- and reconstituted enzymes. O, Major protein band; • , minor protein band. in Table I. The apoenzymes had neither any significant Fe content nor enzymatic activity. The reconstituted enzymes had about 1.1 to 1.3 g atoms of Fe per mol of enzyme. Enzymatic activities of the reconstituted enzymes were almost the same as that of the native enzyme. These results show that the iron is essential for Fe-superoxide dismutase activity. Absorption spectra: Figure 3 shows the optical absorption spectra of the apo- and reconstituted enzymes. In the visible range from 320 to 600 run, the broad absorption observed in the native enzyme / . Biochem.
RECONSTITUTION OF Fe-SUPEROXIDE DISMUTASE
853
TABLE I. Properties of apo- and reconstituted enzymes. Fe» (g atoms/mol)
Specific activity
1. Native enzyme (Alkaline method) 2. Apoenzyme 3. Reconstituted enzyme 4. Reconstituted enzyme
1.12±0.06
3,200±151
0.035
0.085 ±0.022 1.13±0.O4 1.27 ±0.03
100±23 3,400+380 3,300±280
0.0025 0.035 0.036
(Urea method) 5. Apoenzyme 6. Reconstituted enzyme
0.113±0.01 1.06±0.1
