Dismutation Properties of Purified and GDA Modified CuZnSOD from Chicken Heart

Abstract

Superoxide dismutase (SOD, 1.15.1.1) from chicken heart has been purified 139-fold with specific activity of 2130 IU/mg. Purified SOD has a molecular weight 31.0 ± 1.0 kDa and is composed of two equally sized subunits each having 1.1 ± 0.03 and 0.97 ± 0.02 atoms of Cu and Zn elements, respectively. Purified CuZnSOD modified by covalent attachment of the glutaraldehyde (GDA) in presence and absence of bovine serum albumin (BSA). The optimum conditions were obtained with a series of modification reactions as 0.25 mg/mL CuZnSOD in 50 mM phosphate buffer, pH 7.5 containing 3% GDA in presence and absence of 0.25 mg/mL BSA. The highest recovery activity of modified SODs was determined as 23.4 and 18.5% for the designated SOD-I and SOD-II derivatives, respectively. The recovery activity of SOD-I reached 28.6% while SOD-II didn’t change significantly and determined as 19% after the reaction with 1% ethylendiamine. The activity variations of native and modified CuZnSODs were investigated depending on the pH and temperature. Optimum pH values for native and modified SOD-I, -II were determined as 8.8, 8.3, and 8.2, respectively. The native and modified SODs have the same optimum temperatures approximately as 35°C. The pH- and thermal-stability properties of modified SODs were found to be better than native SOD, in the pH range of 6.5–8.5 at 25°C after 6 h, and up to 40°C at pH 7.4 after 3 h incubation period. Inhibitory effects of ditiothreitol (DTT), β-mercaptoethanol, and iodoacetamide were not observed on the native and modified SODs activities after 5 h incubation period. Phenylmethylsulfonylfloride (PMSF), H₂O₂, and EDTA were caused by slight inhibition on the enzyme activities.

• Superoxide dismutase
• Chicken heart
• Purification
• GDA modification

Introduction

In all aerobic organisms, many metabolic pathways have different reactive oxygen species, such as superoxide, hydroxyl, and peroxyl radicals (Fridovich, [1986a], [1995]; McCord, [1993]). The intracellulaar antioxidant enzyme systems such as superoxide dismutase, catalase, and glutathione peroxidase and the other antioxidants play an important role in preventing various pathological diseases such as chronic inflammatory autoimmune diseases, ischemia/reperfusion injury, cancer, arteriosclerosis and aging (Ahmad, [1995]).

Superoxide dismutase as an antioxidant enzyme serves as the first line of enzymatic defense with dismutation of superoxide anion radical , which is formed during the univalent reduction of oxygen to hydrogen peroxidase. Superoxide dismutases that are metalloproteins contain iron or manganese or copper plus zinc as the prosthetic groups (Fridovich, [1986b]). CuZnSODs were isolated from various eukaryotic sources and surprisingly in an increasing although limited number of bacteria. MnSODs were found in prokaryotes and in mitochondria of eukaryotes (Öztürk et al., [1999]). FeSOD has been found so far only in prokaryotic organisms such as Escherichia coli and algae and recently in the eukaryote Euglena gracilis (Benov and Fridovich, [1995]; Kardinahl et al., [1996]; Misra and Fridovich, [1977]).

SODs play an essential role in allowing organisms to survive in the presence of oxygen. Purification as well as modification in soluble form of antioxidant enzymes has become an approach applicable to the solution of the various problems in biological science and therapy (Bannister et al., [1987]; Caliceti et al., [1996]; Veronese et al., [2002]). The selection of modification strategy is important to reach the best overall recovery catalytic activity, effectiveness of catalyst utilization, stability and regeneration capacity, and cost. In recent years, more research has been performed especially on the modification of antioxidant enzymes with polyethylene glycol and their derivatives in soluble form in order to investigate kinetic parameters used, activity assay conditions, and therapeutic applicability (Bullock et al., [1997]; He et al., [1993]; Tang et al., [1993]).

The purpose of this study is to characterize dismutation capacity variations and stability properties of native and GDA modified CuZnSOD in relation to pH, temperature, and type of some substances after purification from chicken heart.

Materials and Methods

Purification of Superoxide Dismutase from Chicken Heart

Fresh chicken heart (Ross PM3), obtained from a local abattoir, was washed, trimmed of fat and connective tissue, removed of blood immediately, and stored at −20°C until needed. After thawing overnight at 4°C, 5.5 g of heart muscle was diced very thinly and homogenized at 2°C in 10 mL precooled 20 mM potassium phosphate buffer, pH 6.0. The homogenization procedure was performed at 8000 rpm for 1.5 min, at 9500 rpm for 1.5 min, and at 13,500 rpm for 1 min respectively. Homogenate was clarified by centrifugation at 18,000 rpm at 2°C for 15 min. Unless otherwise specified, all subsequent steps were performed at 4°C. The proteins in supernatant with 15.36/Ulmg SOD activity were precipitated fractionally with 80% (NH₄)₂SO₄ (w/w) for 1 h and then centrifuged at 18,000 rpm for 15 min. After (NH₄)₂SO₄ precipitation, the precipitate is resuspended into the initial volume by 0.02 M potassium phosphate buffer, pH 6.0 and then haemoglobin was removed from the extract by adding 0.4 mL ethanol, and 0.05 mL chloroform, precooled in a freezer, for each mL of supernatant. After 15 to 30 min, the mixture was centrifuged at 18,000 rpm for 15 min. And then, the supernatant was applied to a CM-cellulose column (1.6 × 30 cm), which had been equilibrated with 0.02 M potassium phosphate buffer pH 6.0. A linear gradient of 0.02–0.1 M, potassium phosphate buffer, pH 6.0, and 0.10–1.0 M NaCl solution, in a total volume of 200 mL was applied at a flow rate of 1.2 mL/min. Fractions having SOD activity were pooled and concentrated using 10,000 NMWC ultra filter and were then subjected to a gel filtration chromatography in 0.02 M potassium phosphate buffer, pH 6.0 on a column of Sephadex G-75 (2.5 × 90 cm). Purified SOD was dialyzed against 0.02 M potassium phosphate buffer, pH 7.4 then concentrated with 10,000 NMWC ultrafilter. The final specific activity of the enzyme was 2130 IU/mg.

Modification of Chicken Heart Superoxide Dismutase

Modification of purified SOD with GDA were carried out with 0.25 mg/mL SOD in the presence and absence of 0.25 mg/mL BSA in 0.05 M phosphate buffer pH 7.4 containing 3% GDA in a total volume 2 mL by shaking slowly at 4°C for 5 h. Modified SOD + BSA and SOD derivatives with red-brown color in soluble form were removed by ultrafiltration with 100,000 NMWC membrane by washing with 0.05 M phosphate buffer pH 7.4 until no determined protein and SOD activity in the effluent. After washing, modified SODs in upper phase membrane were cream-colored. Elsewhere in this article the modified SOD + BSA and SOD have been referred to as preparations SOD-I and SOD-II, respectively.

The recovery reactive groups of GDA on the SOD-I and SOD-II were blocked with 1% ethylendiamine (w/v) in the modification condition for 1 h. After ultrafiltration, modified SODs were stored in 0.05 M phosphate buffer pH 7.4 at 4°C. The recovery activities of SOD-I and SOD-II were determined as 28.6 and 19.0%, respectively.

Activity Measurements for Native and Immobilized Superoxide Dismutase

The SOD activity assay system was based on the inhibitory effect of SOD on the spontaneous autoxidation of 6-hydroxydopamine (Crosti et al., [1987]). One unit of the native and immobilized SOD is defined as that amount which requires 50% inhibition of the initial rate of 6-OHDA autoxidation. Specific activity is defined as the units of activity/mg of native or modified protein.

Protein Determination

Protein determination was estimated by the method of Lowry using a crystalline/bovine serum albumin as the standard (Lowry et al., [1951]).

Molecular Weight Determination of Purified Superoxide Dismutase

The molecular weight of the purified enzyme was determined using gel filtration on a Sephadex G-75 column (2.5 × 90 cm) equilibrated with 0.02 M potassium phosphate buffer, pH 6.0. BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa), and aprotinin (6.5 kDa) were used as molecular weight standards. The subunit molecular weight was estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of β-mercaptoethanol. SDS-PAGE was performed according to the method of Laemmli using a vertical slab gel apparatus. BSA (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-P-dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), α-lactalbumin (14.2 kDa) were used as SDS electrophoresis molecular-weight standards (Laemmli, [1970]).

Determination of Metal Content

The protein was dialyzed extensively against 10 mM phosphate buffer, pH 7.4, containing 1mM EDTA, followed by buffer lacking EDTA, and then subjected to micro-cuvette atomic absorption analysis.

Data in the manuscript is the average of five measurements and the standard deviation is less than 5%.

Results

The Properties of Purified Chicken Heart SOD

At the end of the purification procedure, the chicken SOD was purified 138.6-fold with a specific activity of enzyme of 2130 IU/mg.

The molecular weight of the purified enzyme was found as 31.0 ± 1.0 kDa by Sephadex G-75 gel filtration column (Fig. 1). The molecular weight of the subunits of chicken heart superoxide dismutase was estimated as 15.5 ± 0.5 kDa by SDS-PAGE electrophoresis (Fig. 2).

Figure 1. Gel filtration of chicken heart CuZnSOD fractions on Sephadex G-75 at pH 6.0: (a) Protein absorption (-○-) and CuZnSOD activity (-•-) variations; (b) Calibration curve of Sephadex G-75 column with using molecular weight markers.

Figure 2. Separation differences in SDS-PAGE. (a) Molecular weight markers. (b) purified chicken heart CuZnSOD.

According to atomic absorption spectroscopy, purified chicken heart SOD contained 1.1 ± 0.03 atoms of Cu and 0.97 ± 0.02 atoms of Zn per subunit. The result shows that the enzyme has one Cu and one Zn atom per subunit.

The Modification of Purified SOD

Modification reactions were achieved in two steps at 4°C for 5 h with a total volume of 2 mL by using different amounts of SOD and GDA in 0.05 M phosphate buffer pH 7.4 at the constant amounts of 3% GDA and 0.25 mg/mL SOD respectively. A series of modifications were also repeated in the presence of 0.25 mg/mL BSA in both conditions. Activity variations of the modified SODs in relation to the amount of native SOD and GDA were shown in Fig. 3.

Figure 3. Activity variations depend on the SOD concentration in 3% GDA (––) and GDA concentration in 0.25 mg/mL SOD (- - - -) containing modification medium at 25°C: (○, SOD + 0.25 mg/mL BSA); (•, SOD).

As can be seen from Fig. 3, retained activity values of SOD-I and SOD-II derivatives were reached to highest values with 0.25 mg/mL SOD containing of 3% GDA in the presence and absence of 0.25 mg/mL as 27.5% and 19% respectively.

The effect of pH on the retained SOD activity was investigated in the pH range 6.0–8.0 of phosphate buffer at the optimized SOD and GDA values.

Figure 4 shows that the pH was affected dramatically on the activity of modified SODs. The optimum immobilization pH of SOD-I and -II was observed as 7.5 and 7.3, respectively. All modification reactions were performed in average value of pH 7.4.

Figure 4. Activity variations depend on the pH in 0.25 mg/mL SOD and 3% GDA containing modification medium at 25°C: (○, SOD + 0.25 mg/mL BSA); (•, SOD).

Characterization of Immobilized SODs

pH-Dependent Activity Variations

pH-dependent activity variations of native and immobilized SODs were investigated at 25°C, using the following pH buffers, 0.1 M phosphate buffer in pH 7.0–8.0 and 0.1 M borate buffer in pH 8.1–9.7 range (Fig. 5).

Figure 5. pH dependent activity variations of native-and modified SODs at 25°C: (○, native SOD); (•, SOD-I and ▴, SOD-II).

As can be seen from Fig. 5, pH activity curve of native SOD was increased up to pH 8.8 and then decreased sharply after 9.0. The optimum pH values for modified SOD-I and -II were shifted to 8.3 and 8.2, respectively.

pH Stability Properties

Native and modified SOD-I and SOD-II enzymes were incubated in 0.1 M potassium phosphate (pH 6.5–8.0) and borate (pH 8.1–9.5) buffers for 6 h at 25°C. The retained activity values were determined after incubation under the standard activity assay condition.

As can be seen in Fig. 6, modified SOD derivatives were more stable than native SOD in the wide pH 6.5–8.5 range.

Figure 6. pH stability variations of native and modified SODs at 25°C for 6 h: (○, native SOD); (•, SOD-I and ▴, SOD-II).

Temperature Dependent Activity Variations

Activity variations in relation to the temperature were investigated in the range of 20–45°C in 0.02 M phosphate buffer, pH 7.4 and constant O₂ concentration (8.2 mg/L). The solubility of oxygen at the different temperature was controlled by O₂-meter by passing pure O₂ and N₂ through buffer solution before adding 6-OHDA. Optimum temperature of native and modified SOD-I and -II was found to be similar values which were approximately 35°C (Fig. 7).

Figure 7. Temperature dependent activity variations of native and modified SODs at pH 7.4 (○, native SOD); (•, SOD-I and ▴, SOD-II).

Temperature Stability Properties

Native and modified SODs were incubated in 0.02 M phosphate buffer, pH 7.4 at 45, 55, 65, and 75°C for 3 h. The retaining activity values obtained after incubation for a period of 3 h showed that native and modified SODs did not lose their activities up to 45°C. As can be seen from Fig. 8, the retained activity values for native and modified SOD-I and SOD-II were 32, 80, and 70%, respectively.

Figure 8. Temperature stability variations of native and modified SODs at pH 7.4 for 3 h: (○, native SOD);(•, SOD-I and ▴, SOD-II).

Effect of Some Substances on the Dismutation Capacity of Native and Modified CuZnSODs

Native and modified CuZnSODs were incubated in 0.02 M potassium phosphate buffer, pH 7.4 containing substances such as dithiothreitol (DTT), β-mercaptoethanol, iodoacetamide, phenylmethylsulfonylfloride (PMSF), EDTA at 2 mM and H₂O₂ at 1 mM during the period of 5 h at 25°C. According to the retained activity values, DTT, PMSF, β-mercaptoethanol and iodoacetamide didn’t show inhibition effect on the native and modified CuZnSODs. On the other hand, these CuZnSODs were inhibited in the similar values at 2 mM EDTA and 1 mM H₂O₂ as 25 and 50%, respectively.

Discussion

SOD is one of the essential enzymes for aerobic cells because of detoxification capacity with dismutation of superoxide anion radical formed oxygen. Therefore, isolation, purification, and immobilization SODs have been studied by many researchers (Öztürk Ürek and Tarhan, [2001]; Öztürk et al., [1999]; Vig et al., [1989]). At the end of the achieved purification procedure, the chicken heart SOD was purified 138.7-fold with a specific activity of 2130 IU/mg. According to the results, purified chicken heart SOD is dimeric enzyme, made of approximately 15.5 ± 0.5 kDa identical subunit, each containing Cu (II) and Zn (II) ion like as bovine and chicken liver CuZnSOD (Öztürk Ürek and Tarhan, [2001]; Vig et al., [1989]). Cu (II) is important in the catalytic cycle, while Zn (II) stabilizes the protein structure. The molecular weight of chicken heart CuZnSOD was found to be similar to the enzyme from chicken liver, erythrocyte, bovine heart, erythrocyte and carp liver (Kumugai et al., [1994]; Michalski and Prowse, [1991]; Öztürk Ürek and Tarhan, [2001]; Vig et al., [1989]; Weisiger and Fridovich, [1973]).

Chemical modifications of purified proteins or enzymes with synthetic or natural macromolecules have found wide applications for biomedical and biotechnological purposes to catalyze specific reactions under very mild conditions (Caliceti et al., [1996]; Muizelaar et al., [1993]; Nakaoka et al., [1997]). Modifications of purified chicken heart SOD by covalent attachment of GDA chains produce a complex product which displays heterogeneity in both the size and charge of the resulting species. Chemical modifications of purified CuZnSOD to reach the maximum retained activity with minimum steric effect and diffusion factor were occurred in optimized SOD/GDA ratio and pH in the presence and absence of BSA.

The activity variations of purified SOD depend on pH having a maximum in alkaline region at 8.8. The optimum pH values of purified SODs from different sources were 8.8 and 8.9 (Öztürk Ürek and Tarhan, [2001]; Öztürk et al., [1999]). The rim of the active-center of SOD enzyme contains four hydrophilic amino acid residues including two glutamate residues and a lysine residue; guide to the positive side chain of an arginine residue in the substrate-binding pocket (Getzoff et al., [1992]). The copper atom in the active-center lies at the bottom of a deep channel in the SOD enzyme. An electric field around the superoxide dismutase active-center enhances the rate of formation of the ES complex about 30-fold. Electrostatic effects allow superoxide dismutase, and perhaps other enzymes, to catalyze reactions much faster than a random collision would. The activity variations of CuZnSODs in the range of alkaline pH reflect the functional role of charged amino acid residues, in particular lysine and cysteine (Getzoff et al., [1983]). According to the results, the optimum pH values of modified SODs were shifted to the acidic region when compared to native CuZnSOD. In the first step of the achieved GDA modification caused an increase in total negative charge on SOD-I and -II when compared to native enzyme. In the second step, SOD-I and -II have supported with positive charge after the block reactions between etylendiamine and retained reactive carbonyl groups of GDA. This blocking reaction on the enzyme surface especially SOD-I caused to increasing of affinity toward superoxide anion substrate. These polycationic characters of SOD-I and -II also caused an accumulation of more hydroxyl ions in the micro-environment will cause the dismutation of to take place at a lower pH and the optimum pH to shift to the acidic region. This effect was observed more strongly in SOD-1, including BSA.

pH as well as temperature-related activity variations of modified SOD-I, which also include supporting protein BSA, provided to increase hydrophilic character, have showed a wider range compared to native and modified SOD-II. The results show that GDA–modified CuZnSODs were more stable in the wider range of pH and up to 55°C when compared to native enzyme. According to the reports, CuZnSODs are generally stable at neutral pH and up to 40°C (Kumugai et al., [1994]; Michalski and Prowse, [1991]; Öztürk Ürek and Tarhan, [2001]; Vig et al., [1989]). However, the pH dependence of the FeSOD stability of Aerobacter aerogenes remained comparatively stable at alkaline pH in the range of 7.0–11.0 but was rapidly inactivated below pH 7.0 (Kim et al., [1991]). Also, SOD modified by PEG-phenylchloroformates derivative was stable at neutral pH (Veronese et al., [1985]).

The observation of any effect of DTT, PMSF, β-mercaptoethanol, and iodoacetamide on the enzyme activity shows that cysteine, methionine, serine, threonine, and histidine amino acid residues have any important role on the purified CuZnSOD activity. The retained CuZnSOD activity was determined as 75% after the incubation in 2 mM EDTA. Some of the research indicates that MnSOD shows that metal ions present in the enzyme structure are more resistant for the occurring of the complex with EDTA (Ramanaiah and Venkaiah, [1992]). The activity of half-life of purified SOD was determined for 1 mM H₂O₂. The inactivation of the CuZnSOD has been attributed to the reduction of the enzyme-bound Cu²⁺ to Cu¹⁺ by H₂O₂, followed by a Fenton-type reaction of Cu^l+ with additional H₂O₂ to form Cu²⁺-OH. This could oxidatively attack an adjacent histidine amino acid residue of the enzyme (Mavelli et al., [1983]).

Purified CuZnSOD from chicken heart after the GDA modification gained more stability properties. Providing more stability properties of SOD with modification in soluble form is of great importance in view of widespread investigation towards clinical applications. The pathophysiological and immunodiagnosis potential of this modified SOD will be investigated in further studies.