Planta (1982)156:374-379
P l a n t a 9 Springer-Verlag 1982
Possible involvement of nodule superoxide dismutase and catalase in leghemoglobin protection A. Puppo, L. Dimitrijevic and J. Rigaud Laboratoire de Biologie V6g6tale, Facult~ des Sciences et des Techniques, Pare Valrose, F-06034 Nice C6dex, France
Abstract. Superoxide anion is able to oxidize oxyleghemoglobin prepared from soybean nodules. Furthermore, ferrileghemoglobin is oxidized to leghemoglobin (IV) by hydrogen peroxide and this irreversible reaction leads to a complete inactivation of the hemoprotein. In scavenging O f and H202, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) are able to limit these oxidations. The occurrence of these enzymes within soybean nodules and their main characteristics are reported here. A general scheme taking into account their roles in leghemoglobin protection in vivo is proposed. Key words: Catalase - Glycine- Leghemoglobin - Root nodule - Superoxide dismutase.
Introduction
Leghemoglobin (Lb), a myoglobin-like hemoprotein, is found exclusively in the active nitrogenfixing root nodules of legumes (Appleby 1974). Its main function in vivo appears to be ensuring an adequate flux of oxygen to the bacteroids (Bergersen 1980). As other oxygen-carrying hemoproteins, Lb is sensitive to autoxidation: we have shown (Puppo et al. 1981) that this reaction results in the formation of both ferriLb and superoxide anion (see Fig. 5). The ferriLb, unable to bind oxygen, is inactive in the nitrogen fixation process (Wittenberg et al. 1974) but can be reduced by a reductase present in the cytosol of nodules (Puppo et al. 1980) or during the simultaneous oxidation of indole-3-acetic acid (Puppo and Rigaud 1979). The superoxide anion and its dismutation product, hydrogen peroxide, are well known to react with heAbbreviations." Lb=leghemoglobin; SOD=superoxide dismutase
moproteins. The aim of this paper is to investigate their respective actions on Lb and to investigate the possibility that superoxide dismutase (SOD) and catalase play a major role in their scavenging. The occurrence and isolation of these enzymes from soybean nodules will also be reported and their role in the protection of Lb, under physiological conditions, will be discussed.
Material and methods Leghemoglobin. Soybeans (Glyc&e max Merr. cv. Amsoy) were grown in a glasshouse and supplied with a nitrogen-free mineral solution, as previously described (Rigaud and Puppo 1975). The nodules (80 g fresh weight) were harvested 30 d after their appearance on the roots. Leghemoglobin (Lb) was purified from the red supernatant derived from nodule homogenates as described previously (Puppo and Rigaud 1975). A further chromatography on a 80.2.5 cm column of Sephadex G-100 was routinely performed in order to avoid the contamination of Lb by endogenous SOD and catalase. FerriLb was obtained from the reaction of K-ferricyanide to a Lb solution followed by chromatography on Sephadex G-15 (Wittenberg et al. 1974).
Superoxide anion generation. The formation of superoxide anion was achieved by spontaneous reoxidation of photoreduced riboflavin (Lynch etal. 1976). Solutions containing 50 gM riboflavin were illuminated with a 20-W fluorescent lamp.
Superoxide dismutase preparation. The red supernatant, obtained after crushing recently harvested nodules (210 g fresh weight) and sedimenting the bacteroids, was submitted to the Tsuchihashi chloroform-ethanol treatment as described by McCord and Fridovich (1969). The precipitate was removed by centrifugation and the resulting supernatant was applied to a DE-52 column (5"25 cm), equilibrated with 50 mM TrisHC1 buffer, pH 7.8. After washing the column, SOD was eluted with the same buffer containing 0.5 M NaC1. The active fractions were pooled, dialyzed overnight, and put onto a column of DEAE-Sephade~ A-50 (2.5.40 cm), equilibrated with 50 mM Tris-HC~ buffer, p ~ 7.8. Elution was accomplished with a linear gradient ~0~4M NaC1 in 50 mM Tris-HC1 buffer, pH 7.8. The enzyme extract was further purified by passage over a column (2.5.80 cm) of Sephadex G-75 in 50 mM TrisHC1 buffer, pH 3.8,
0032-0935/82/0156/0374/$01.20
A. Puppo et al. : Leghemoglobin protection Catalase preparation. (NH4)2SO 4 fractionation was applied to the red supernatant derived from nodule (90 g fresh weight) homogenates. The active fraction (20-40% saturation) was dialyzed and submitted to a chromatography on a DE-52 column (2.5.40 cm), equilibrated with 25 m M Tris-HC1 buffer, pH 8. Catalase was eluted with a linear gradient 0-0.3 M NaC1 in the same buffer. This enzyme was further purified by two successive passages over a column (2.5-80 cm) of Sephadex G-150 in 25 mM Tris-HCl buffer, pH 8. Enzymatic assays. All extracts were tested for SOD activity using the riboflavin/methionine system (Beauchamp and Fridovich 1971). This photochemical procedure is less influenced by the presence of other enzymes than are enzymatic systems (Giannopolitis and Ries 1977a). The mixtures were illuminated in a box wrapped with aluminium foil and fitted with a 20-W fluorescent lamp. The reduction of nitro blue tetrazolium was followed at 560 nm. The results are expressed in units of activity: One unit of SOD is defined as the amount that inhibits the nitro blue tetrazolium photoreduction by 50%, under the assay conditions (Beauchamp and Fridovich 1971). The catalase activity was routinely detected during the purification steps according to Lacoppe and Hofinger (1968). For the purified fractions, it was measured by the method of Beers and Sizer (1952); one unit of catalase is defined as one ]amol HzO 2 degraded per min and mg. Peroxidase activity was appreciated by guaiacol oxidation in the presence of HzO 2 (Gaspar et al. 1969).
Analytical methods. Hydrogen peroxide concentration was determined just before experiments using the iodide assay (Cotton and Dunford 1973). Protein concentrations were measured by the method of Lowry et al. (1951). Spectrophotometric measurements were performed with a Beckman 3600 spectrophotometer. Electrophoresis and molecular weight determination. Polyacrylamide gel electrophoresis was performed according to Davis (1964). A current of 3 mA per gel was applied during the migration and bromophenol blue was used as a marker. SOD was localized by the photochemical assay of Beauchamp and Fridovich (1971). The method of Weber and Osborn (1969) was used for sodium dodeeyI sulfate (SDS) polyacrytamide gel electrophoresis. The proteins were denatured by heating at 60 ~ C, in the presence of SDS 1% and 2-mercaptoethanol 1% ; protein bands were stained by Coomassie blue. The molecular weight of SOD was determined by molecular sieving on a Sephadex G-75 column (2.5.80 cm); the standard proteins were cytochrome e, chymotrypsinogen, bovine SOD, ovalbumin, and nodule peroxidase. Molecular-weight determination of nodule SOD was also performed using sucrosegradient centrifugation (Martin and Ames 1961). A linear sucrose gradient was prepared in 50 mM Tris-HC1 buffer, pH 7.8. The enzyme preparation was centrifuged at 50,000 rpm for 16 h in a Lz-65 B Beckman ultracentrifuge fitted with a SW 65 Ti rotor. Lb, bovine SOD and ovalbmnin were used as markers. Catalase molecular weight was estimated by molecular sieving on a Sephadex G-200 column (2.5.80 cm), using bovine serum albumin, aldolase, bovine liver catalase, and ferritin as standard proteins.
Results
Effect of superoxide anion on oxyleghemoglobin and role of Superoxide dismutase. A solution of oxyLb
375
E
c e,, Lo
O
~
0-------0"
9
--0
|
t..Q gl .1:1
P l a n t a 9 Springer-Verlag 1982
Possible involvement of nodule superoxide dismutase and catalase in leghemoglobin protection A. Puppo, L. Dimitrijevic and J. Rigaud Laboratoire de Biologie V6g6tale, Facult~ des Sciences et des Techniques, Pare Valrose, F-06034 Nice C6dex, France
Abstract. Superoxide anion is able to oxidize oxyleghemoglobin prepared from soybean nodules. Furthermore, ferrileghemoglobin is oxidized to leghemoglobin (IV) by hydrogen peroxide and this irreversible reaction leads to a complete inactivation of the hemoprotein. In scavenging O f and H202, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) are able to limit these oxidations. The occurrence of these enzymes within soybean nodules and their main characteristics are reported here. A general scheme taking into account their roles in leghemoglobin protection in vivo is proposed. Key words: Catalase - Glycine- Leghemoglobin - Root nodule - Superoxide dismutase.
Introduction
Leghemoglobin (Lb), a myoglobin-like hemoprotein, is found exclusively in the active nitrogenfixing root nodules of legumes (Appleby 1974). Its main function in vivo appears to be ensuring an adequate flux of oxygen to the bacteroids (Bergersen 1980). As other oxygen-carrying hemoproteins, Lb is sensitive to autoxidation: we have shown (Puppo et al. 1981) that this reaction results in the formation of both ferriLb and superoxide anion (see Fig. 5). The ferriLb, unable to bind oxygen, is inactive in the nitrogen fixation process (Wittenberg et al. 1974) but can be reduced by a reductase present in the cytosol of nodules (Puppo et al. 1980) or during the simultaneous oxidation of indole-3-acetic acid (Puppo and Rigaud 1979). The superoxide anion and its dismutation product, hydrogen peroxide, are well known to react with heAbbreviations." Lb=leghemoglobin; SOD=superoxide dismutase
moproteins. The aim of this paper is to investigate their respective actions on Lb and to investigate the possibility that superoxide dismutase (SOD) and catalase play a major role in their scavenging. The occurrence and isolation of these enzymes from soybean nodules will also be reported and their role in the protection of Lb, under physiological conditions, will be discussed.
Material and methods Leghemoglobin. Soybeans (Glyc&e max Merr. cv. Amsoy) were grown in a glasshouse and supplied with a nitrogen-free mineral solution, as previously described (Rigaud and Puppo 1975). The nodules (80 g fresh weight) were harvested 30 d after their appearance on the roots. Leghemoglobin (Lb) was purified from the red supernatant derived from nodule homogenates as described previously (Puppo and Rigaud 1975). A further chromatography on a 80.2.5 cm column of Sephadex G-100 was routinely performed in order to avoid the contamination of Lb by endogenous SOD and catalase. FerriLb was obtained from the reaction of K-ferricyanide to a Lb solution followed by chromatography on Sephadex G-15 (Wittenberg et al. 1974).
Superoxide anion generation. The formation of superoxide anion was achieved by spontaneous reoxidation of photoreduced riboflavin (Lynch etal. 1976). Solutions containing 50 gM riboflavin were illuminated with a 20-W fluorescent lamp.
Superoxide dismutase preparation. The red supernatant, obtained after crushing recently harvested nodules (210 g fresh weight) and sedimenting the bacteroids, was submitted to the Tsuchihashi chloroform-ethanol treatment as described by McCord and Fridovich (1969). The precipitate was removed by centrifugation and the resulting supernatant was applied to a DE-52 column (5"25 cm), equilibrated with 50 mM TrisHC1 buffer, pH 7.8. After washing the column, SOD was eluted with the same buffer containing 0.5 M NaC1. The active fractions were pooled, dialyzed overnight, and put onto a column of DEAE-Sephade~ A-50 (2.5.40 cm), equilibrated with 50 mM Tris-HC~ buffer, p ~ 7.8. Elution was accomplished with a linear gradient ~0~4M NaC1 in 50 mM Tris-HC1 buffer, pH 7.8. The enzyme extract was further purified by passage over a column (2.5.80 cm) of Sephadex G-75 in 50 mM TrisHC1 buffer, pH 3.8,
0032-0935/82/0156/0374/$01.20
A. Puppo et al. : Leghemoglobin protection Catalase preparation. (NH4)2SO 4 fractionation was applied to the red supernatant derived from nodule (90 g fresh weight) homogenates. The active fraction (20-40% saturation) was dialyzed and submitted to a chromatography on a DE-52 column (2.5.40 cm), equilibrated with 25 m M Tris-HC1 buffer, pH 8. Catalase was eluted with a linear gradient 0-0.3 M NaC1 in the same buffer. This enzyme was further purified by two successive passages over a column (2.5-80 cm) of Sephadex G-150 in 25 mM Tris-HCl buffer, pH 8. Enzymatic assays. All extracts were tested for SOD activity using the riboflavin/methionine system (Beauchamp and Fridovich 1971). This photochemical procedure is less influenced by the presence of other enzymes than are enzymatic systems (Giannopolitis and Ries 1977a). The mixtures were illuminated in a box wrapped with aluminium foil and fitted with a 20-W fluorescent lamp. The reduction of nitro blue tetrazolium was followed at 560 nm. The results are expressed in units of activity: One unit of SOD is defined as the amount that inhibits the nitro blue tetrazolium photoreduction by 50%, under the assay conditions (Beauchamp and Fridovich 1971). The catalase activity was routinely detected during the purification steps according to Lacoppe and Hofinger (1968). For the purified fractions, it was measured by the method of Beers and Sizer (1952); one unit of catalase is defined as one ]amol HzO 2 degraded per min and mg. Peroxidase activity was appreciated by guaiacol oxidation in the presence of HzO 2 (Gaspar et al. 1969).
Analytical methods. Hydrogen peroxide concentration was determined just before experiments using the iodide assay (Cotton and Dunford 1973). Protein concentrations were measured by the method of Lowry et al. (1951). Spectrophotometric measurements were performed with a Beckman 3600 spectrophotometer. Electrophoresis and molecular weight determination. Polyacrylamide gel electrophoresis was performed according to Davis (1964). A current of 3 mA per gel was applied during the migration and bromophenol blue was used as a marker. SOD was localized by the photochemical assay of Beauchamp and Fridovich (1971). The method of Weber and Osborn (1969) was used for sodium dodeeyI sulfate (SDS) polyacrytamide gel electrophoresis. The proteins were denatured by heating at 60 ~ C, in the presence of SDS 1% and 2-mercaptoethanol 1% ; protein bands were stained by Coomassie blue. The molecular weight of SOD was determined by molecular sieving on a Sephadex G-75 column (2.5.80 cm); the standard proteins were cytochrome e, chymotrypsinogen, bovine SOD, ovalbumin, and nodule peroxidase. Molecular-weight determination of nodule SOD was also performed using sucrosegradient centrifugation (Martin and Ames 1961). A linear sucrose gradient was prepared in 50 mM Tris-HC1 buffer, pH 7.8. The enzyme preparation was centrifuged at 50,000 rpm for 16 h in a Lz-65 B Beckman ultracentrifuge fitted with a SW 65 Ti rotor. Lb, bovine SOD and ovalbmnin were used as markers. Catalase molecular weight was estimated by molecular sieving on a Sephadex G-200 column (2.5.80 cm), using bovine serum albumin, aldolase, bovine liver catalase, and ferritin as standard proteins.
Results
Effect of superoxide anion on oxyleghemoglobin and role of Superoxide dismutase. A solution of oxyLb
375
E
c e,, Lo
O
~
0-------0"
9
--0
|
t..Q gl .1:1
