Short communication
Purification and partial characterization of Cu,Zn superoxide
dismuta from haemolymph of Oriental river
prawn Macrobrachium nipponen
Cui-Luan Yao a ,An-Li Wang b,⁎,Zhi-Yong Wang a,⁎,Wei-Na Wang b ,Ru-Yong Sun b
a
Key Laboratory of Science and Technology for Aquaculture and Food Safety,Fujian Province University,
Fisheries College,Jimei University,Xiamen 361021,China
b
College of Life Science,South China Normal University,Guangzhou 510631,China
Received 28January 2007;received in revid form 20April 2007;accepted 20April 2007
Abstract
The copper plus zinc superoxide dismuta (Cu,Zn-SOD)was purified from haemolymph of the Oriental river prawn,Macrobrachium nipponen and partially characterized.Partial protein precipitation in crude extract was affected by using heat treatment and (NH 4)2SO 4fractionated precipitation methods.Fractionation of superoxide dismuta was performed by DEAE-cellulo 32ion-exchange chromatography and followed by CM-cellulo cation-exchange chromatography.The molecular weight of it was about 66.1kDa,as judged by SDS polyacrylamide gel electrophoresis.The enzyme was nsitive to cyanide and H 2O 2,and contained 1.08±0.14atom of copper and 0.98±0.11zinc per subunit shown in atomic absorption spectroscopy,which revealed that purified SOD was Cu,Zn superoxide dismuta.The purified enzyme had an absorption peak of 269nm in ultraviolet region and the enzyme remained stable at 25–45°C within 60min.But it was rapidly inactivated at higher temperature (50°C).The activity of purified shrimp Cu,Zn-SOD was remained stable over the range pH 5.8–8.3.Treated with 10mM mercaptoethanol,the enzyme activity significantly incread.However,the enzyme activity was obviously inhibited by 10mM CaCl 2,ZnCl 2,SDS,EDTA –Na 2and 1mM and 10mM K 2Cr 2O 7.The results showed that it might be a kind of EC-SOD.And it was the first report of some characterizations of this EC-SOD in M.nipponen .©2007Published by Elvier B.V .
Keywords:Superoxide dismuta;Macrobrachium nipponen ;Haemolymph;Purification;Characterization
1.Introduction
Reactive oxygen species (ROS),which include hydroxyl radical,superoxide anion,hydrogen peroxide,and singlet oxygen,are physiologically generated in a ries of biochemical reactions within cellular compart-ments and increa in physiological conditions that result in oxidative stress,dia and immune defen reactions (Dirks et al.,1982).The incread levels of ROS may lead to irreversible cell damage and eventually to
cell
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⁎Corresponding authors.A.-L.Wang is to be contacted at College of Life Science,South China Normal University,Guangzhou 510631,China.Tel./fax:+862085216862.Z.-Y .Wang,Key Laboratory of Science and Technology for Aquaculture and Food Safety of Fujian Province University,Fisheries College,Jimei University,Xiamen 361021,China.Tel./fax:+865926183816.
E-mail address:wanganl@scnu.edu (A.-L.Wang),zywang@jmu.edu (Z.-Y .Wang).
0044-8486/$-e front matter ©2007Published by Elvier B.V .doi:10.1016/j.aquaculture.2007.04.068
death.Superoxide dismutas(SODs,EC1.15.1.1)play a crucial role in the defen against oxidative cell damage,through catalyzing the breakdown of superoxide anion to oxygen and hydrogen peroxide(McCord and Fridovich,1998).
小型宠物狗Superoxide dismuta is a metalloprotein with a redox metal at its active site.The catalytic mechanism involves quential oxidation and reduction of the metal ion,which provides electrostatic channeling of super-oxide radicals to the active site.Copper plus zinc or mangane or iron is the most familiar type as the prosthetic groups are found in SODs.The Cu and Zn enzyme,the most studied form Cu,Zn-SOD,is characteristic of the cytosols of eukaryotes,fungi,and mammalian cells and is very nsitive to cyanide and H2O2(Weisiger and Fridovich,1973;Kwiatowshi and Kaniuga,1986;Hatzinikolaou et al.,1997;Palma et al., 1997).Mn-SODs were mainly found in prokaryotes and in mitochondria of eukaryotes,and is innsitive to cyanide and H2O2(Kawaguchi et al.,1989).Fe-SOD is usually found in prokaryotes and in chloroplasts of some plants,and is not nsitive to cyanide but is inhibited by H2O2(Asada et al.,1980).Bad on the different nsitivity to cyanide and H2O2,the metal prosthetic groups in SOD could be identified.
SOD activity has been reported to be prent in many shrimp species and play an important role in immunity (Neves et al.,2000).In shrimp immune respon, phagocytosis is associated with the produ
ction of ROS to kill the foreign microorganism(Bachèrèet al.,1995; Muñoz et al.,2000).The antioxidant SOD converts this microbiocidal metabolite superoxide anion into oxygen and hydrogen peroxide that pass freely through membranes.SOD activity correlated cloly with immune stimulation,dia,and healthy status of the prawn (Mohankumar and Ramasamy,2006;Wang and Chen, 2006;Cheng et al.,2007).Several prawn Mn-SODs were cloned,which include Fenneropenaeus chinensis(Zhang et al.,2007),Macrobrachium ronbergii(Cheng et al., 2006a,b)and Litopenaeus vannamei(Gómez-Anduro et al.,2006);the calculated molecular weight is about 24kDa,showing a similarity with a Mn-SOD that was purified from prawn M.nipponen(Yao et al.,2004). Johansson et al.(1999)demonstrated that a cell-surface Cu,Zn-SOD was a binding protein for peroxinectin in crayfish and might mediate or regulate the cell adhesion and phagocytosis.An extracellular Cu,Zn-SOD(EC-Cu, Zn-SOD)has been cloned in crayfish Pacifastacus leniuscufus(Johansson et al.,1999).Brouwer et al. (2003)demonstrated that a EC-Cu,Zn-SOD in haemo-cyte of blue crab Callinectes sapidus played an important role in oxygen transport(Brouwer et al.,2003).
M.nippone is a freshwater or brackish prawn that is often a commercially important species in China,Japan and Vietnam(Uno,1971;Wang et al.,2002).A Mn-SOD was purified from it and partial ch
aracterization was studied(Yao et al.,2004).To gain new insights into the characterization of SODs and its role in immuno-modulation in prawn and to better understand its physiological function in immune respon,a Cu,Zn-SOD from M.nipponen haemolymph was purified and biochemically characterized in this study.
2.Materials and methods
2.1.Protein purification
Whole prawns,M.nippone,5–8g in body weight, were collected from Lake Bai Yangdian,Hebei Province, China.Only intermolt prawns were lected in this experiment.Two hundred microlitres of haemolymph was collected from the ventral sinus of each prawn into a1ml syringe(26gauge)containing400μl anticoagulant (sodium chloride0.45M,gluco0.1M,sodium citrate 30mM,citric acid26mM,EDTA20mM,pH4.5, Söderhäll and Smith,1983).About600ml of fresh prawn haemolymph diluted with anticoagulant was homoge-nized immediately.Unless otherwi stated,all purifica-tion procedures were carried out at0–4°C.The homogenate was centrifuged at20,000g for20min (Beckman,J2-21,USA).The supernatant was subjected to heat treatment at60°C for1min then to ice treatment for1min.The same procedure was repeated five times and then followed by cen清平调其二
trifugation at20,000g for 20min.The supernatant was brought to65%saturation of ammonium sulfate by gradually adding solid(NH4)2SO4 and was stirred for2h.After recovery at20,000g of centrifugation,the supernatant was subjected to90% ammonium sulfate,and the pellet that was collected at 20,000g of centrifugation was resuspended in and dialyzed into10mM sodium phosphate buffer,pH7.8. After dialysis,the sample was applied to a column (2×10cm)of DEAE-cellulo that had been previously equilibrated with10mM of the same buffer,then eluted with a linear gradient of10–200mM pH7.8sodium phosphate buffer at a flow rate of0.3ml/min.The eluted fractions containing high SOD activity were collected, pooled and dialyzed against10mM phosphate buffer,pH 5.8.After dialysis the pooled fractions were applied to a CM-cellulo column and elution with the same buffer to purify SOD further.After loading the sample on the column,the column was eluted by10–200mM sodium phosphate buffer(pH7.8)at a flow rate of0.3ml/min. Tho fractions with SOD activity were pooled.Fractions
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containing SOD activity were analyzed with SDS polyacrylamide gel electrophoresis(SDS-PAGE),and fractions with the least amount of contaminants were pooled.
2.2.Protein assay
Total protein concentration was measured using the Bradford(1976)method with bovine rum albumin as the standard.
2.3.Enzyme assays
SOD activity in solution was determined using the method of Marklund and Marklund(1974)bad on the autoxidation of pyrogallol and modified from Jing and Zhao(1995).SDS-PAGE was performed with6%stacking gels and10%parating gel described by Laemmli(1970) using the middle-range molecular weight standards: phosphoryla B(97.4kDa),BSA(66.2kDa),ovalbumin (45.0kDa),carbonic anhydra(31.0kDa),and lysozyme (14.4kDa).Cu,Zn-SOD bands were located by staining the gels with Coomassie blue R-250and silver.
2.4.Determination of metal content
Metal content of purified M.nipponen SOD was determined by a microcuvette atomic absorption method after the enzyme was dialyzed extensively against 10mM phosphate buffer,pH7.4,containing1mM ethylenediaminetetraacetic acid and followed by buffer lacking ethylenediaminetetraacetic acid.问客
2.5.Absorption spectra in ultraviolet region
Ultraviolet absorption measurement was carried out with a UV-1200model ultraviolet and visible region spectrophotometer.
2.6.Heat stability assay
Heat stability experiments had been carried out at25, 35,45,55,65,75and85°C in aled glass vials containing 20μl of enzyme in50mM sodium phosphate buffer,pH 7.8for60min respectively,immediately cooled on ice and assayed for residual activity at25°C using the same method as above.
王可一2.7.pH stability assay
Stability and activity of the purified enzyme were determined by add10μl of enzyme in20μl50mM pH 4.3–5.3acetate(sodium acetate,acetate),pH5.8–7.8 PBS,pH8.3–8.8Tris–HCl and pH9.3–10.3carbonate buffer(sodium carbonate,sodium bicarbonate)respec-tively,at25°C,to study the effect of pH value of the reaction mixture.
2.8.Effect of chemicals on SOD activities
Chemical inactivation experiments were carried out in10and1mM CuSO4,CaCl2,ZnCl2,K2Cr2O7,ED
TA, SDS and mercaptoethanol,respectively,in aled glass vials containing20μl of enzyme in50mM sodium phosphate buffer,pH7.8.Each was with three duplicates and the mixtures were withdrawn at appropriate times and immediately assayed for enzyme activity at25°C. The data was analyzed using Origin7.0software.
3.Results
The purified enzyme was obtained through the following steps:heat treatment,(NH4)2SO4
precipitation, Fig.1.Chromatography elution profile.A.DEAE-cellulo chroma-tography elution profile.The column was eluted with a linear gradient (10–200mM)sodium phosphate buffer,pH7.8.SOD-containing fractions eluted at a sodium phosphate buffer concentration of30mM.
B.The CM-cellulo elution profile.The column was eluted with a linear gradient(10–200mM)phosphate buffer solution,pH5.8.SOD-containing fractions eluted at a phosphate buffer solution concentration of35mM.
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DEAE-cellulo anion-exchange chromatography and CM-cellulo cation-exchange chromatography.Heat treatment and (NH 4)2SO 4salting out were effective methods to remove most of the protein.SOD was eluted from the DEAE-cellulo column at a concentration of 30mM and 50mM pH 7.8sodium phosphate buffers respectively.Then the first fraction was applied to the CM-cellulo column and SOD was eluted at a concentration of 35mM pH 5.8phosphate buffer solution.The elution profile of the DEAE-cellulo and CM-cellulo purified SOD is shown in Fig.1A and B.The results of the purification of Cu,Zn-SOD from prawn M.nipponen haemolymph are sum
marized in Table 1.The final enzyme was purified 17.87fold from the initial homogenate,and its specific activity was 140.44U/mg.
Gel electrophoresis of the CM cellular elute revealed a single band of protein at 66.1kDa (Fig.2A,B).
No activity was determined for M.nipponen Cu,Zn-SOD in the prence of 2mM KCN or H 2O 2in the assay medium,which is consistent with the contention that Cu,Zn-SOD is nsitive to the inhibitors.
Atomic absorption spectroscopy revealed that puri-fied SOD is compod of one subunit having 1.07±0.13and 0.95±0.12atoms of Cu and Zn elements.
The purified enzyme had an absorption peak with a maximum at 269nm in the ultraviolet region.The spectra are reminiscent of the comparable spectra for other Cu,Zn-SODs.
The thermal stability variations of Cu,Zn-SOD were investigated by incubating the appropriate enzyme at 25,35,45,55,65,75and 85°C for 60min.The enzyme was stable to incubation between 25and 45°C within 60min,while above this temperature,it was inactivated moderately.The pH dependence of the enzyme stability was measured from the residual activity at standard activity assa
y conditions after 1.5h of preincubation at 25°C,at range of pH 4.3–10.3buffer systems.The purified enzyme remained comparatively stable in the region of pH 5.8–8.3,but was inactivated slowly out of this range.Treatment of the enzyme with 10mM mercaptoetha-nol significantly incread the enzyme activity (P b 0.05).However,the enzyme activity was obviously inhibited by 10mM CaCl 2,ZnCl 2,SDS,EDTA –Na 2and 1mM and 10mM K 2Cr 2O 7(P b 0.05).SOD activity did not show significant variation after incubated with 1mM CaCl 2,ZnCl 2,SDS,CuSO 4,EDTA –Na 2,mercaptoethanol and 1mM and 10mM CuSO 4(P N 0.05).The results are shown in Table 2.4.Discussion
In general,there are two types of SOD included in Mn-SOD and Cu,Zn-SOD in the prawn.Mn-SOD was
Table 1
Purification of Cu,Zn-SOD from shrimp M.nipponen haemolymph Purification step
Total protein (mg)Total activity (U)
Specific activity (U/mg protein)Purification (fold)Crude extract 8126380
7.861Thermal treatment 132580443.96 5.5990%(NH 4)2SO 445.8257856.297.16DEAE-3213.5013
2097.7812.44CM-cellulo 4.5
632
140.44
17.87
Fig.2.Purification of SOD as determined with SDS-PAGE.(A)Electrophoresis was carried out using 10%polyacrylamide gel:line 1,crude extracts;line 2,heat treatment;line 3,ammonium sulfate precipitation;line 4,enzyme solution after DEAE-cellulo column;line 5,enzyme solution after CM-cellulo column.(B)Line 1,molecular mass makers (in kilodaltons);molecular standard;line 2,SDS-PAGE of CM-cellulo elution column of SOD activity.The protein samples were stained with silver.
Table 2
Effect of different inorganic and organic chemicals on the enzyme stability,each test with three duplicated and the data was analyzed by using Origin7.0software Medium
Concentration 1mM
风吹过的夏天10mM CaCl 281%±3.8%78%±5.3%CuSO 495%±5.1%93%±4.6%ZnCl 2103.1%±7.8%72%±6.2%K 2Cr 2O 750%±4.2%10%±3.3%SDS
政府五大职能85%±5.3%65%±4.9%EDTANa 285%±4.9%70%±4.2%Et-SH
95%±3.2%
151%±9.9%
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known as cytosolic MnSOD(cytMnSOD)and mito-chondrial MnSOD(mtMnSOD).The former lacks a mitochondrial transit peptide and is retained in cytosol. The latter is transported to the mitochondria with the help of mitochondrial transit peptide after translation (Brouwer et al.,1997,2003).CytCu,Zn-SOD and extracellular SOD(EC-SOD)are the two common forms of Cu,Zn-SOD(Marklund,1982).CytCu,Zn-SOD is a hou-keeping enzyme that is esntial for intracellular antioxidant defence and occurs in all eukaryotes;however,EC-SOD is far less conrved than cytCu,Zn-SOD(Fattman et al.,2000).
Johansson et al.(1999)demonstrated that an extracellular Cu,Zn-SOD played as a binding protein for a cell-adhesive factor,peroxinectin,in crayfish.The binding of this Cu,Zn-SOD to peroxinectin might mediate or regulate cell adhesion and phagocytosis in shrimp immunity.We have purified and partially characterized a Mn-SOD from M.nipponen(Yao et al.,2004),which showed some similar characteriza-tions with other Mn-SOD.In this study,a Cu,Zn-SOD was purified from haemolymph of Oriental river prawn M.nipponen.Compared with other Cu,Zn-SODs,it showed some different char
acterizations.The specific activity of the purified enzyme was found to be140.44IU/ mg,corresponding to a17.87-fold purification.The isolated SOD showed a single band and had a molecular weight of about66.1kDa,which is different from the cytCu,Zn-SOD that consists of the same two subunits, each with a molecular weight of16kDa.However,an EC-SOD of crayfish was parated with a high molecular weight of about25kDa(Johansson et al.,1999).The 135kDa EC-SOD and270kDa EC-SOD were obtained from human plasma,and a240–260kDa EC-SOD was obtained from hamster(Sies and Stahl,1995;Petern et al.,2004;Due et al.,2006).However,few of the EC-SOD from different species showed similar molecular weights (Johansson et al.,1999;Akita et al.,2007).And we speculate that this Cu,Zn-SOD was obtained from M. nipponen might belong to EC-SODs.
The M.nipponen Cu,Zn-SOD showed a lower specific activity than the enzyme level reported for most other species,which may indicate that some enzymes lost their activity during the purification process(Fitch and Ayala,1994),or that their half-lives are shorter;as a result,the activity decread remarkably in vitro.More so,probably,it is an adaptation of the aquatic species to water where oxygen is less than the species onland (Heremems,1982).The purified SOD was thermostable between25°C and45°C within60min.Above45°C, the thermostability of the enzyme was much lower.The purified en
zyme remained comparatively stable in the region of pH5.8–8.3,but was inactivated slowly out of this range.Most Cu,Zn-SODs have a broad optimal temperature and pH range.Compared with other SODs, the enzyme is relatively stable.However,the stability of most EC-SODs is unknown.
The interaction of common positive charges and the hydrophobic central on Cu,Zn-SOD with the negative head groups leads to strongly steric strains and stability loss.Almost half of the enzyme activity was lost after treatment by K2Cr2O7;it was probably due to K2Cr2O7as a strong oxidant(E0=1.33V standard electrode voltage). The prence of10mM EDTA,Ca2+,Zn2+and SDS decread the enzyme activity and might be due to the fact that they can combine with the activity central of the enzyme to decrea the activity of the enzyme.The enzyme activity was incread significantly by10mMβ-mercaptoethanol indicating that–SH groups in the Cu, Zn-SOD structure play an important role for activity and suggested that it can protect the enzyme to some extent. The purified Cu,Zn-SOD was stable in a medium containing Cu2+,and1mM SDS,EDTA and Zn2+,which had no obvious effect on Cu,Zn-SOD activity,suggesting that this Cu,Zn-SOD is stable due to the metal prosthetic group to some extent(Donnelly et al.,1989).
The Cu,Zn-SOD,purified from haemolymph of M. nipponen,with a higher molecular weight than that of other cytCu,Zn-SODs and minor similar properties, might belong to a class of EC-SOD.EC-S
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OD was shown to be the predominant SOD in extracellular fluids in former studies(Marklund et al.,1982),however,most studies focud on cytCu,Zn-SOD and cytMnSOD or mtMnSOD,and less is known about the biochemical properties of EC-SOD.So,more of their characteriza-tions and functions need to be further studied. Acknowledgements
This rearch was supported in part by the National Natural Science Foundation of China(30671605and 30671628),Major State Basic Rearch Development Program of China(973program,2006CB101804), GDSF(5005909and021098)and Project supported by the Foundation for Innovative Rearch Team of Jimei University,China(2006A001).
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