Purification and partial characterization of Mn

superoxide dismutase from muscle tissue of

the shrimp Macrobrachium nipponense

Cui-Luan Yao a,b ,c ,An-Li Wang d ,*,Wei-Na Wang d ,Ru-Yong Sun d a Key laboratory of Experimental Marine Biology,Institute of Oceanology,Chinese Academy of Sciences,

7Nanhai Road,Qingdao 266071,PR China

b College of Life Science,Hebei University,Baoding 071002,PR China

c Graduate School,Chinese Academy of Sciences,Beijing 100089,PR China

d Colleg

e o

f Life Science,South China Normal University,Guangzhou 510631,PR China

Received 1May 2004;received in revised form 18August 2004;accepted 18August 2004

Abstract

Superoxide dismutase (SOD;EC 1.15.1.1)is an enzyme that protects against oxidative stress from superoxide radicals in living cells.This enzyme had been isolated,purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense .The purification was achieved by heat treatment,ammonium sulfate fractionated precipitation and column chromato-graph on DEAE-cellulose 32.Some physiological and biochemical characterization of it was tested.The molecular weight of it was about 21.7kDa,as judged by SDS–polyacrylamide gel electrophoresis.The purified enzyme had an absorption peak of 278nm in ultraviolet region,and the enzyme remained stable at 25–458C within 90min.However,it was rapidly inactivated at higher temperature.Treatment of the enzyme with 1mM ZnCl 2,SDS and 1mM or 10mM mercaptoethanol showed some increasing activity.However,the enzyme activity was obviously inhibited by 10mM CaCl 2,CuSO 4,ZnCl 2and 1mM CaCl 2and 10mM K 2Cr 2O 7.SOD activity did not show significantly variation after incubated with 1mM CaCl 2,EDTA and 10mM SDS.The enzyme was insensitive to cyanide and contained 1.03F 0.14atoms of manganese per subunit

*Corresponding author.Tel.:+862085216862;fax:+862085216862.

E-mail address:wanganl@https://www.360docs.net/doc/4b6612099.html, (A.-L.Wang).

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shown in atomic absorption spectroscopy,which revealed that purified SOD was Mn superoxide dismutase.

Keywords:Superoxide dismutase;Purification;Characterization;Macrobrachium nipponense ;Muscle

1.Introduction

Shrimp production is a worldwide economic activity especially in developing countries.The intensification of farming over the last few decades has been accompanied by the development of infectious disease from viral,bacterial,and fungal origin (Roch,1999).Shrimp disease has become an important limiting factor in the shrimp farming industry.However,shrimps have been shown to possess a primitive immune system that relies mainly on phagocytosis,encapsulation and agglutination (Smith and So ¨derha ¨ll,1986).But the knowledge of shrimp pathogenesis,especially the disease that is related with the virus,was not well developed.Vertebrates,including humans,have developed an immune defense that includes an adaptive memory with immunoglobulin antibodies that protect them against pathogenic microorganisms.Invertebrates do not have antibodies,but they possess some immune proteins belonging to the immunoglobulin superfamily (Lanz Mendoza and Faye,1996)which protect them against pathogens (Holmblad and So ¨derha ¨ll,1999).

Superoxide dismutase (SOD;EC 1.15.1.1)is a kind of metal enzyme,which occurs in virtually all O 2respiring organisms.SOD plays an important role in defending against superoxide anion toxicity and radioactive risk and the prevention of aging as it can eliminate oxygen free radicals in the body.In these years,it is found that SOD activity is related with some disease,such as tumor and inflammation (Kroll et al.,1995).It had been proposed to play important roles in various biological events such as neuronal survival (Nakano et al.,1995)and signal transduction (Tu et al.,1997).Johansson et al.(1999)reported an extracellular CuZn-superoxide dismutase in crayfish that could bind to peroxinectin.While an organism is subjected to stresses such as chemical,physical and biological (i.e.pathogen infection)upon sudden a shortage of oxygen,abnormal oxidative reactions in the aerobic pathway result in the formation of excess amounts of singlet oxygen (Tanielian and Chaineaux,1978)and the subsequently generated free radicals.These radicals can impair proteins,carbohydrates,lipids and nucleotides (Yu,1994),which are important parts of cellular constituents,including enzymes,membranes and DNA.Radical damage can be important because it can proceed as a chain reaction.Consequently,mortality can occur due to severe destruction by massive radicals generated from acute stresses or long-term chronic stresses.The more superoxide radicals need to be reacted,the higher the SOD value is.SOD has been widely used in finfish related to nutrient requirements,health enhancement,pollution stress monitoring,pesticide effects,disease indication and thermal or osmotic stress in shrimp (Compa-Cordova et al.,2002;Downs et al.,2001;Wang et al.,2004b ).

SOD is classified into three distinct groups according to the metal cofactor:Fe-SOD is found in prokaryotes and in plants,Mn-SOD in prokaryotes and in the mitochondria of

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C.-L.Yao et al./Aquaculture241(2004)621–631623 eukaryotes,and CuZn-SOD in the cytosol and extracellular compartments of eukaryotes and also in the periplasm of Gram negative bacteria(McCord and Fridovich,1969). However,a novel nickel SOD(Ni-SOD)is found in Streptomyces species recently(Youn et al.,1996).The SOD type can be identified by its susceptibility to inhibition by potassium cyanide(Steinman,1982).

Macrobrachium nipponense is a commercially important species found in brackish and freshwaters throughout China(from North China to Taiwan),Japan and Vietnam(Uno, 1971;Wang et al.,2002).Some physiological and biochemical parameters variation and immune response of it had been studied(Wang and Li,1993;Wang et al.,2002,2003a,b, 2004a,b).The present study was aimed at isolation and biochemical characterization of M. nipponense SOD,in order to better understand its physiological function in immune response.

2.Materials and methods

2.1.Protein purification

Whole shrimp,M.nipponense,were collected from Lake Bai Yangdian,Hebei Province,China.About200g of fresh muscle tissue was homogenized immediately in500 ml of50mM precooled sodium phosphate buffer,pH6.5,containing10mM EDTA. Unless otherwise stated,all purification procedures were carried out at48C.The homogenate was centrifuged at20,000?g for30min.The supernatant was subjected to heat treatment at658C for2min then to ice treatment for2min.The same procedure was repeated five times and then followed by centrifugation at20,000?g for30min.The supernatant was added with ammonium sulphate to70%saturation.After recovery at 20,000?g of centrifugation,the supernatant was subjected to90%ammonium sulphate, and the pellet that was collected at20,000?g of centrifugation was resuspended in and dialysed into10mM sodium phosphate buffer,pH7.8.

After dialysis,the sample was applied to a column(1?24cm)of DEAE-cellulose that had been previously equilibrated with10mM of the same buffer,elution with a10–200 mM sodium phosphate buffer pH7.8;linear gradient was carried out at a flow rate of0.3 ml/min,and collection of the SOD activity fraction was made.Fractions containing SOD activity were analyzed with nondenaturing polyacrylamide gel electrophoresis(PAGE)and 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 serum albumin as the standard.

2.3.Enzyme assays

SOD activity in solution was determined using the method of Marklund and Marklund (1974)based on the autoxidation of pyrogallol and modified by Jing and Zhao(1995).

SDS–polyarylamide gel eletrophoresis (SDS-PAGE)was performed with 6%stacking gels and 12%separating gel described by Laemmli (1970)using the middle-range molecular mass standards.Mn-SOD bands were located by staining the gels with Coomassie blue R-250and silver.Nondenaturing PAGE was performed with 8%acrylamide gels in the same system except SDS and reducing agent was omitted from all buffers,and the samples were not boiled prior to electrophoresis.

2.4.SOD activity stain

Following nondenaturing PAGE,the gel was socked in 100ml of a solution containing 21mg nitro-blue tetrazoliun,1.2mg riboflavin and 220A l TEMED in water for 30min in the dark.After incubation,the gel was transferred to water and developed over sunlight.The activity of Mn-SOD was measured in the detection system with 2mM KCN.

2.5.Determination of metal content

Metal content of purified M.nipponense SOD was determined by a microcuvette atomic absorption method after the enzyme was dialyzed extensively against 10mM phosphate buffer,pH 7.4,containing 1mM ethylenediaminetetraacetic acid and followed by buffer lacking ethylenediaminetetraacetic acid.

2.6.Absorption spectra in ultraviolet region

Ultraviolet absorption measurement was carried out with a UV-1200model ultraviolet and visible region spectrophotometer.

2.7.Amino acid analysis

Amino Acid composition was measured by acid hydrolysis (6N HC1for 24h at 1108C)and was carried out with Hitachi-135-50model amino acid autoanalysis instrument.Tryptophan was not determined.

2.8.Heat stability assay

Heat stability experiments had been carried out at 25,35,45,55,65,75and 858C in sealed glass vials containing 20A l of enzyme in 50mM sodium phosphate buffer,pH 7.8.The sample was collected at 30,60,90and 120min,respectively,immediately cooled on ice and assayed for residual activity at 258C using the same method as above.

2.9.Effect of chemicals on SOD activities

Chemical inactivation experiments had been carried out in 10and 1mM CuSO 4,CaCl 2,ZnCl 2,K 2Cr 2O 7,EDTA,SDS and mercaptoethanol,respectively,in sealed glass vials containing 20A l of enzyme in 50mM sodium phosphate buffer,pH 7.8.The mixtures

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were withdrawn at appropriate times and immediately assayed for enzyme activity at 258C.

3.Results

Heat treatment and (NH 4)2SO 4salting out were effective methods to remove most of the protein.SOD was eluted from the DEAE-cellulose column at a concentration of 75mM sodium phosphate buffer.The elution profile of the DEAE-purified SOD is shown in Fig.1.The results of the purification of Mn-SOD from shrimp M.nipponense muscle tissue are summarized in Table 1.The final enzyme was purified 218.84-fold from the initial homogenate,and its specific activity was 96.29U/mg.

No activity loss was determined for M.nipponense Mn-SOD in the presence of 2mM KCN in the assay medium,which is consistent with the general reports.Gel electrophoresis of the SOD activity elutes showed single band,and it showed

SOD Fig.1.DEAE-cellulose chromatography elution profile.The column was eluted with a linear gradient (10–200mM)sodium phosphate buffer,pH 7.8.SOD-containing fractions eluted at a sodium phosphate buffer concentration of 75mM.

Table 1

Purification of Mn-SOD from shrimp M.nipponense muscle tissue

Purification step

Total protein (mg)Total activity (U)Specific activity (U/mg protein)Purification (fold)Crude extract

17,10073500.441Thermal treatment

31956804 2.13 4.890%(NH 4)2SO 4

55.8257846.20105DEAE-32 6.7565096.29218.84

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Fig.2.Nondenaturing PAGE,activity staining of nondenaturing gels and SDS-PAGE of purified Mn-SOD.(a) Line1,90%ammonium sulphate salting out treatment,line2,DEAE-cellulose elution of SOD activity.The protein samples were stained with Coomassie blue R-250.(b)Line1,gel stained for crude extract SOD without KCN;line2,KCN was included in the SOD stain solution.(c)Line1,SDS-PAGE of DEAE-cellulose elution of SOD activity;line2,molecular standard.The protein samples were stained with

silver.

Fig.3.Ultraviolet absorption spectra.

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activity in activity staining (Fig.2a,b).It showed one band with molecular weight about 21.7kDa tested by SDS-PAGE (Fig.2c).

Atomic absorption spectroscopy revealed that purified SOD contained 1.03F 0.14atoms of manganese per subunit.The purified enzyme had an absorption peak with a maximum at 278nm in the ultraviolet region.The spectra are reminiscent of the comparable spectra for other Mn-SODs (Fig.3).

Amino acid analysis data taken represented triplicate analysis and were not corrected for labile amino acids or peptide bonds resistant to hydrolysis.Tryptophan was not determined.Given the subunit molecular mass of 21,700and assuming a quantitative recovery of amino acids from the hydrolysis procedure,we calculated the residues per subunit by dividing the nanomoles of each amino acid by the total nanomoles of protein (Table 2).

Thermal stability of Mn-SOD was studied by incubating the appropriate enzyme at 25,35,45,55,65,75and 858C at different intervals.The enzyme was stable to incubation between 25and 458C within 30min,while above this temperature,it was inactivated moderately.The residual enzyme activity is shown in Fig.4.

Table 2

Amino acid composition of Mn-SOD from M.nipponense

Amino acid

Amino acid mol%per subunit Mn-SOD Amino acid Amino acid mol%per subunit Mn-SOD Asp

27Met 1Thr

1Ile 8Ser

6Leu 9Glu

16Tyr 5Gly

9Phe 8Ala

13Lys 13Val 13Arg

Fig.4.Thermal stability of M.nipponense Mn-SOD.(a)Effect of different temperature on (30min)enzyme stability.(b)Effect of some temperature (in different time)on enzyme stability.

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Treatment of the enzyme with 1mM ZnCl 2,SDS and 1or 10mM mercaptoethanol increased the enzyme activity.However,the enzyme activity was obviously inhibited by 10mM CaCl 2,CuSO 4,ZnCl 2and 1mM CaCl 2and 10mM K 2Cr 2O 7.SOD activity did not show significant variation after incubated with 1mM CaCl 2,EDTA and 10mM SDS.The results are shown in Table 3.

4.Discussion

SOD is responsible for the elimination of the superoxide anion and is an important defense enzyme against the damaging effects of oxidant stress in an organism.It constitutes the first line of defense against oxidative damage by catalyzing the dismutation of the superoxide radical to molecular oxygen and hydrogen peroxide.In addition,hydrogen peroxide is degraded by catalases and peroxidase (Holmblad and So ¨derha ¨ll,1999).SOD activity has been reported to be present in many shrimp species and play an important role in immunity (Neves et al.,2000).An extracellular CuZn was found as a binding protein for a cell-adhesive factor,peroxinectin,in crayfish.The binding of this CuZn-SOD to peroxinectin may mediate or regulate cell adhesion and phagocytosis in shrimp immunity (Johansson et al.,1999).However,there are few data about Mn-SOD’s characterization.Our study was aimed at the purification and partial characterization of SOD in the muscle extracts of M.nipponense .The isolation and purification of Mn-SOD from shrimp muscle was performed from homogenate by heat treatment and fractionated precipitation with 70%and 90%(NH 4)2SO 4,then DEAE-cellulose ion exchange chromatography.According to this procedure,the specific activity was found to be 96.3IU/mg,corresponding to a 219-fold purification.The isolated SOD showed single band and had a molecular weight of 21.7kDa on SDS-PAGE,which is different from the EC-SOD of crayfish (Johansson et al.,1999)but in good agreement with values given for Mn-SODs from other species (Fridovich,1995).The specific activity of the purified enzyme from M.nipponense was lower than the enzyme level reported for most other species,which may indicate that some enzyme lost its activity during the heat treatment process (Wispe et al.,1989;Neupert,1997;White et al.,1993;Fitch and Ayala,1994),or suggesting that their half-life is shorter;as a result,the activity decrease remarkably in vitro.More so,probably,it is an

Table3

Effect of different chemicals on SOD relative activity (%)

Medium

Concentration 1mM 10mM CaCl 2

91.2545.63CuSO 4

77.5763.87ZnCl 2

109.5127.4K 2Cr 2O 7

27.38 5.6SDS

125.592.3EDTA-Na 2

9588.5Mercaptoethanol 123.2118.63

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C.-L.Yao et al./Aquaculture241(2004)621–631629 adaptation of the aquatic species to water where oxygen is less than onland (Heremems,1982).

In general,Mn-SODs are characterized from different sources that contain two or four subunits(Escuyer et al.,1996;Ravindranath and Fridovich,1975).The cyanide-insensitive M.nipponense SOD may be a homodimer with an identical molecular weight of21.7kDa and contained one atom of Mn per subunit.It is remarkably similar to comparable enzymes isolated from other species.

The purified SOD showed optimum temperature of358C and was thermostable up to 458C within30min.The effect of temperature on some Mn-SODs activity showed an increase in enzyme activity with increase in temperature up to378C(Wilde and Yu,1998). Above458C,the thermostability of the enzyme was much lower.

The interaction of common positive charges and the hydrophobic zones on Mn-SOD with the negative head groups leads to strongly steric strains and stability loss.Almost all of the enzyme activity was lost after treatment by high concentration of K2Cr2O7;it was probably due to K2Cr2O7as a strong oxidant(E0=1.33V standard electrode voltage).h-Mercaptoethanol had no obvious effects on the stability of the enzyme,indicating that–SH groups in the Mn-SOD structure do not play an important role for activity and suggested that it can protect the enzyme to some extent.The purified Mn-SOD was stable in a medium containing SDS and EDTA,which had no effect on CuZn-SOD(Donnelly et al., 1989),suggesting that Mn-SOD is stable due to the metal prosthetic group.

The manganese-containing SOD,purified from M.nipponense,belongs to a class of SOD enzymes having remarkably similar properties and is distinguishable by only minor differences,which indicate little evolutionary divergence of Mn-SOD from M.nipponense and other eukaryotic Mn-SOD enzymes.

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