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Characterization of Phenoloxidase from the Bay Scallop Argopecten irradiansAuthor(s): Jingwei Jiang, Jing Xing, Xiuzhen Sheng and Wenbin ZhanSource: Journal of Shellfish Research, 30(2):273-277. 2011.Published By: National Shellfisheries AssociationDOI: http://dx.doi.org/10.2983/035.030.0212URL: http://www.bioone.org/doi/full/10.2983/035.030.0212

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CHARACTERIZATION OF PHENOLOXIDASE FROM THE BAY SCALLOP

ARGOPECTEN IRRADIANS

JINGWEI JIANG, JING XING,* XIUZHEN SHENG AND WENBIN ZHAN

Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, Qingdao266003, PR China

ABSTRACT Phenoloxidase (PO) is one of the most important factors in the bivalve immune defense system. In the current

study, the PO of the scallopArgopecten irradianswas purified from hemocytes using linear-gradient native PAGE combined with

catechol and Coomassie brilliant blue staining. The results show that purified PO had a molecular mass of 555 kDa in native

PAGEand 55 kDa in denatured PAGE. The analysis of the kinetics indicated that theKm values of PO for L-DOPA, catechol, and

hydroquinone were 0.51 mM/L, 0.23 mM/L, and 0.87 mM/L, respectively, which suggests that the PO was a type of

p-diphenoloxidase. The POhad optimal activities at 40�C and a pHof 8.0, and its activity was greatly enhanced byCa2+ andMg2+,

and inhibited by Fe2+ and Mn2+. In addition, the PO activity was inhibited by sodium sulfite, ascorbic acid, sodium

diethyldithiocarbamate (DETC), cysteine, citric acid, and ethylenediamine tetraacetic acid disodium (EDTA). However, no

inhibition was found when thiourea and sodium azide were used. Based on the inhibition that was induced by EDTA and DETC,

we conclude that the PO of A. irradians was a copper-containing metalloenzyme.

KEY WORDS: phenoloxidase, bay scallop, Argopecten irradians, linear-gradient native PAGE, hemocyte, metalloenzyme

INTRODUCTION

Phenoloxidase (PO), which is generated from the limited

proteolysis of prophenoloxidase (proPO) by a proPO-activat-ing enzyme (Aspan et al. 1995, Soderhall & Cerenius 1998), isresponsible for initiating the conversion of phenols to unstable

quinones, which are nonenzymatically transformed into mela-nin (Asokan et al. 1997, Jiravanichpaisal et al. 2006, Hellio et al.2007). Melanin and a number of intermediate metabolites are

involved in melanization, encapsulation, wound healing, phago-cytosis, and hemocyte locomotion (Vass et al. 1993, Parrinelloet al. 2003, Aladaileh et al. 2007a).

The PO activity in hemolymph (PAH) of bivalve species hasclose relationships with temperature, pH, metal ions, and path-ogenic infections. A gradual increase in the temperature of up to37�C causes a remarkable increase in PAH in the oyster

Saccostrea glomerata (Aladaileh et al. 2007b). In addition, therange in pH from 5.0–6.5 induces a significant and continuousgrowth of PAH in the scallopChlamys farreri (Sun&Li 1999). The

exposure of the oyster Pinctada fucata to copper results ina significantly higher PAH compared with controls, and thisalteration appeared earlier than in other enzymes (Jing et al.

2006). The acute viral necrobiotic virus (AVNV) infection inC. farreri induces a dramatic increase in PAH at 6 h postinfection,andPO is the earliest responding endoenzyme toAVNVamong the7 determined enzymes (Xing et al. 2008). All these results indicate

that PO is one of the most sensitive factors in bivalve immune andphysiological responses. Some researchers have reported thepurification and characterization of PO in the clam Ruditapes

philippinarum (Cong et al. 2005), and themusselsModiolus demissus(Waite &Wilbur 1976) andMytilus edulis (Renwrantz et al. 1996).The current study demonstrated the purification of PO from the

scallop Argopecten irradians, which is one of the most importantcommercial species in North China; the determination of POmolecular mass; the substrate specificity; and the PO activities

related to temperature, pH, divalent metal ions, and inhibitors.

In addition, we provided primary data suggesting the potentialindex of PO in the physiological and immune systems of thescallop.

MATERIALS AND METHODS

Experimental Animals

The adult scallop A. irradians (5.5 ± 0.52 cm in shell length)waspurchased fromQingdao inChina andkept in a recirculatingseawater system in the laboratory until use.

Hemocyte Lysate Supernatant Preparation

Hemolymph was withdrawn from the adductor muscle sinus

using a sterilized syringe and centrifuged at 7003g for 10 min at4�C. The pellet was collected and suspended in phosphate-buffered saline (2.7 mMKCl, 0.137 M NaCl, 1.47 mMKH2PO4,

8.09 mM Na2HPO4�12H2O, PBS, pH 7.6). The samples weresonicated and centrifuged at 15,0003 g for 30 min at 4�C. Theresulting supernatant, whichwas referred to as the hemocyte lysate

supernatant (HLS), was collected and stored at –80�C until use.

PO Purification

The HLS was subjected to electrophoresis with a 5% stacking

gel and a 6–20% linear-gradient separating gel in Tris-Glycinebuffer (0.025 M Tris, 0.2 M glycine, pH 8.0) for 12 h at 3 W usinghigh-molecular weight markers ranging from 67–669 kDa (GE

Healthcare,UK).One lane of the gel was cut and stained using 1%catechol to label the PO-containing bands. Based on the catechol(Songon, China) staining, the unstained lanes were excised for PO-

containing bands, which were sonicated in PBS and centrifuged at17,0003 g for 30 min. The resulting supernatant was condensedusing centrifugal concentrators (Millipore, USA) and successivelysubjected to linear-gradient native PAGE (6–20%). After electro-

phoresis, two lanes of the gel were cut and stained with catecholand Coomassie brilliant blue R-250 (CBB, Biorad, USA), re-spectively, to identify PObands. ThePObands in the unstained gel

were excised, sonicated, and centrifuged as described earlier. The*Corresponding author. E-mail: xingjing@ouc.edu.cn

DOI: 10.2983/035.030.0212

Journal of Shellfish Research, Vol. 30, No. 2, 273–277, 2011.

273

resulting supernatant was condensed and desalted using centrifu-gal concentrators. The purified PO solution was used to evaluate

the PO activity and perform the biochemical analysis. All theprocedures except stainingwere performed at 4�C, and none of thebuffers that were used in the PO purification contained sodiumdodecyl sulfate (SDS) or reducing agents.

PO Activity Assay and Protein Assay

PO activity was determined by L-3,4-dihydroxyphenylala-

nine (L-DOPA) transformation to dopachromes (Soderhall1981) with some modifications. Briefly, 100 mL of the samplewas added to 2.0 mL of 15-mM L-DOPA (Sigma, USA) that

was dissolved in 100 mM Tris-HCl buffer (pH 7.0). Thedopachrome formed was measured spectrophotometrically at490 nm every 3 min for 30 min. The PO activity was estimatedbased on the increment in the rate of absorbance. An increase of

0.001/min was considered 1 U (A490 10–3/min).

The protein concentrations were determined according tothe Bradford method (Bradford 1976), using bovine serum

albumin (BSA; Sigma, USA) as the protein standard.

Molecular Mass of Purified PO

The purified PO was applied to native PAGE (describedlater) and SDS-PAGE. SDS-PAGE was performed using a 5%

stacking gel and a 12% separating gel according to the methodof Laemmli (Laemmli 1970), using standard proteins rangingfrom 14.4–116 kDa as molecular weight markers (Fermentas,

USA). After electrophoresis, the gels were stained with CBB.

Optimal Temperature and pH for Purified PO

To determine the optimal temperature of the purified PO, 100

mLof the purified PO solutionwas incubatedwith 2.0mL15mML-DOPA for 20 min at 10�C, 20�C, 30�C, 40�C, 50�C, and 60�C.PO activities were determined spectrophotometrically at 490 nm.

To determine the optimal pH of purified PO, 100 mL purifiedPO solution was added to 2.0 mL of 15 mM L-DOPA that wasdissolved in 100 mM acetate buffer (pH 5.0, 6.0), Tris-HCl

buffer (pH 7.0, 8.0, 9.0), and carbonate–bicarbonate buffer (pH10.0). PO activities were measured at 490 nm.

Kinetic Parameters

Kinetic parameters were determined using the Lineweaver-

Burk plot method. A total of 100 mL of the purified PO solutionwere added to 2.0 mL of different concentrations of L-DOPA,catechol, hydroquinone (Songon, China), and tyrosine (Son-gon, China) that were dissolved in 100mMTris-HCl buffer (pH

7.0). PO activities were determined spectrophotometrically at490 nm.

Effects of Divalent Metal Ions on the Activity of Purified PO

Mg2+, Zn2+, Mn2+, Fe2+, Cu2+, and Ca2+ from MgSO4,ZnSO4, MnCl2, FeCl2, CuSO4, and CaCl2 (Songon, China)

were used as divalent metal ions. A total of 100 mL purified POsolution was incubated with 100 mL of divalent metal ions atdifferent concentrations dissolved in 100 mM Tris-HCl buffer

(pH 7.0) for 20min at 4�C, followed by the addition of 1.9 mL of15 mM L-DOPA. PO activities were measured spectrophoto-metrically at 490 nm. The control sample was performed by

incubating 100 mL of 100 mM Tris-HCl buffer (pH 7.0) withoutdivalent metal ions.

Effects of Inhibitors on the Activity of Purified PO

Cysteine, ascorbic acid, sodium sulfite, citric acid, thiourea,sodium azide, ethylenediamine tetraacetic acid disodium

(EDTA) and sodium diethyldithiocarbamate (DETC) wereused as PO inhibitors. A total of 100 mL of the purified POsolution was incubated with 100 mL of PO inhibitors dissolved

in 100 mM Tris-HCl buffer (pH 7.0) at different concentra-tions for 20 min at 4�C, followed by the addition of 1.9 mL of15 mM L-DOPA dissolved in 100 mM Tris-HCl buffer (pH

7.0). PO activities were determined spectrophotometrically at490 nm. The control sample was performed by incubating 100mL of 100 mM Tris-HCl buffer (pH 7.0) without the POinhibitor.

Statistical Analysis

All of these experiments were performed in triplicate, and the

data from sectionsOptimal Temperature and pH for Purified POand Effects of Divalent Metal Ions on the Activity of Purified POwere presented as the mean ± SD. Statistical analysis was

performed using SPSS 11.5 (SPSS, USA).

RESULTS

Purification of PO

Argopecten irradians PO purification yielded 0.06 mg POfrom HLS containing approximately 64.72 mg of total protein

with a 40.68% total recovery of activity and a 438.76-foldpurification (Table 1). After linear-gradient native PAGE, HLSexhibited many consecutive protein bands, among which only 1

band reacted to catechol, which was revealed by a mahoganycolor. The purified PO showed a clear band with a molecularmass of 555 kDa in native PAGE, and a single protein band with

the molecular mass of 55 kDa in SDS-PAGE (Fig. 1).

Optimal Temperature and pH for Purified PO

Using L-DOPA as a substrate, the optimal temperature ofpurified POwas 40�Cwith the determined temperature range of10–60�C (Fig. 2A), and the optimal pH was 8.0 with thedetermined pH range of 5.0–10.0 (Fig. 2B).

TABLE 1.

Purification of PO from hemocytes of A. irradians.

Purification

Step

Total

Protein

(mg)

Total

Activity*

(U)

Specific

Activity*

(U/mg)

Recovery

(%)

Purification

Fold

Hemocyte lysate

supernatant

64.72 354.02 5.47 100 1.00

Two-step native

PAGE

0.06 144.00 2,400 40.68 438.76

* PO activity was assayed using L-DOPA (15 mM) as a specific

substrate.

JIANG ET AL.274

Kinetic Parameters

Using the Lineweaver-Burk model, Km values of the PO forL-DOPA, catechol, and hydroquinone were 0.51 mM, 0.23mM, and 0.87 mM, respectively (Fig. 3). However, no reaction

with tyrosine was detected.

Effects of Metal Ions on the Activity of Purified PO

As shown in Figure 4,Mg2+ and Ca2+ stimulated PO activityat all the determined concentrations. Zn2+ stimulated POactivity at concentrations of 5, 10, and 30 mM, but inhibited POactivity at 50 mM. Cu2+ had an obvious stimulatory effect at

5mM, and inhibitory effects at the concentrations of 10, 30, and50 mM. Fe2+ and Mn2+ inhibited PO activity at all the de-termined concentrations.

Effects of Inhibitors on the Activity of Purified PO

The effects of various inhibitors on the activity of purified

PO using L-DOPA as a substrate are shown in Table 2. One

hundred percent inhibition was achieved after PO was incu-bated with 0.23 mM sodium sulfite, 0.45 mM ascorbic acid, 0.45

mMDETC, 1.36 mM cysteine, and 2.27 mM citric acid. A 65%inhibition was determined with 2.27 mM EDTA. However, noinhibition occurred using thiourea and sodium azide.

DISCUSSION

PAH variations associated with the temperature, pH, and

stimulation of microbial polysaccharides have been reported inA. irradians (Jordan et al. 1997, Yang et al. 2006). Here, wepurified PO from hemocytes of the scallop and characterized

purified PO based on the optimal temperature and pH, thekinetic parameters, and the effects of divalent metal ions andinhibitors on enzymatic activity.

In the current study, a 40.68% total recovery of enzymaticactivity and a 438.76-fold purification were obtained, whichsuggests a high efficiency in PO purification using the two-step

native PAGE method despite a low content of PO in A.irradians. Similar to oyster Crassostrea virginica and flukeFasciola gigantica, PO was found in hemocytes using SDS-PAGE and catechol as a substrate (Nellaiappan et al. 1989,

Jordan & Deaton 2005). These results suggest that POs usecatechol as a common substrate in some invertebrates. In termsof molecular mass, A. irradians PO (555 kDa in native PAGE

and 55 kDa in SDS-PAGE) differed from S. glomerata PO (219kDa in native PAGE and 90.6 kDa in SDS-PAGE) (Aladailehet al. 2007b) andM. edulis PO (381 kDa in native PAGE and 39

kDa in SDS-PAGE) (Renwrantz et al. 1996). However, theseresults reflect the POs as aggregates, which are commonlyreported in invertebrates (Aspan & Soderhall 1991, Perdomo-Morales et al. 2007). In addition, the molecular mass of A.

irradians PO in SDS-PAGEwas smaller than that ofC. virginicaPO (133 kDa) and R. philippinarum PO (76.9 kDa) (Cong et al.2005, Jordan & Deaton 2005). Among the reported marine

bivalve POs, A. irradians PO is similar to M. edulis PO, whichhas a high molecular weight under native conditions butcomprises a small subunit.

The optimal temperature of A. irradians PO was similar tothat of S. glomerata PO (37�C) andR. philippinarum PO (40�C).The optimal pH of purified A. irradians PO was 8.0, which

Figure 2. (A) Effects of different temperatures on the activity of purified A. irradians PO. (B) Effects of different pH on the activity of purified A.

irradians PO. The PO activity was assayed using L-DOPA (15 mM) as a specific substrate.

Figure 1. (A) Linear-gradient native PAGE of different samples from A.

irradians. Proteins that were separated using linear-gradient native PAGE

from lane 1 to lane 3 were stained with CBB, whereas lane 4 was stained

with 1% catechol. Lane 1, marker of protein; lane 2, A. irradians HLS;

lane 3, purified A. irradians PO; lane 4, A. irradians HLS stained with

catechol. (B) SDS-PAGE of different samples from A. irradians was

stained with CBB. Lane 1, low-molecular weight protein standards; lane 2,

A. irradians HLS; lane 3, purified A. irradians PO.

PHENOLOXIDASE IN HEMOCYTES OF BAY SCALLOP 275

coincided with that of S. glomerata and M. demissus POs(Aladaileh et al. 2007b, Cong et al. 2005, Waite & Wilbur

1976), and was similar to C. virginica PO (6.0–7.5) (Jordan &Deaton 2005). These results imply that there is similarity inmarine bivalve PO in terms of depending on pHand temperature.

There are three types of PO in invertebrates (Barret 1987):

p-diphenoloxidase (E.C.1.10.3.2; p-diphenol:O2 oxidoreductase),o-diphenoloxidase (E.C.1.10.3.1 diphenol: O2 oxidoreductase),and monophenoloxidase (E.C.1.14.18.1 monophenol, L-DOPA:

O2 oxidoreductase). The PO from A. irradians effectivelyoxidized L-DOPA, catechol, and hydroquinone, but failed tooxidize tyrosine. These results imply that this enzyme is a

p-diphenoloxidase that is similar to the PO inC. virginica and thesoluble-form PO in F. gigantica, which shows more affinity fordiphenols and paraphenols than monophenols (Nellaiappan

et al. 1989, Jordan & Deaton 2005). In addition, Km valuesindicated that A. irradians PO had the highest affinity forcatechol and a higher affinity for L-DOPA than hydroquinone.

The activity of A. irradians PO was strongly increased in thepresence of Ca2+ andMg2+. The higher concentration of Ca2+ or

Mg2+ resulted in a stronger enhancement of PO activity thatwas dose dependent, which suggests that Ca2+ and Mg2+ playpositive roles during PO catalysis. Ca2+-mediated enhancementof PO activity has been reported in the silkworm Bombyx mori

(Ashida et al. 1983), the cockroach Blaberus craniifer (Leonardet al. 1985), and the moth Galleria mellonella (Dunphy 1991).Mg2+-mediated enhancement of PO activity has been demon-

strated in the pyralid Ostrinia furnacalis (Feng et al. 2008). Inaddition, Zn2+ and Cu2+ had opposite effects at a high concen-tration (50 mM) and a low concentration (5 mM), and showed

no regular trends during the experiments, which indicates thattheir effects were dose independent. However, Mn2+ and Fe2+

presented consistent inhibition at all concentrations throughout

the experiments. The possible explanations for these resultsincluded metal ions that may activate electrophile and nucleo-phile binding, and release electrons to modulate PO activity

TABLE 2.

Effects of inhibitors on the activity of purifiedA. irradiansPO.

Inhibitor Concentration (mM)

Inhibition rate

(%)

Cysteine 1.36 100

0.45 78

0.23 17

Ascorbic acid 0.45 100

0.23 28

sodium sulfite 0.23 100

citric acid 2.27 100

1.36 94

0.45 44

0.23 28

Thiourea 2.27 0

Sodium azide 2.27 0

Ethylenediamine tetraacetic

acid disodium

2.27 65

1.36 35

0.45 18

0.23 12

Sodium diethyldithiocarbamate 0.45 100

0.23 82

Figure 4. Effects of 6 metal ions on the activity of purified PO from A.

irradians. The PO activity was assayed using L-DOPA (15 mM) as

a specific substrate.

Figure 3. Kinetic analysis of the purified PO from A. irradians. The Km values were determined using L-DOPA, catechol and hydroquinone as

substrates, and were calculated according to the Lineweaver-Burk model.

JIANG ET AL.276

(Stryer 1995), or divalent cations that may change the second-ary structure of certain peptides of PO to influence PO activity

(Feng et al. 2008). However, the detailed mechanisms requirefurther investigation.

In addition to the common inhibitors for oxidase, such asascorbic acid, sodium sulfite, citric acid, and cysteine, EDTA

and DETC—a divalent cation and specific copper chelator,respectively—may inhibit A. irradians PO activity. These resultsimply that this PO is a copper-containing metalloenzyme similar

to the four marine bivalvesC. virginica (Jordan &Deaton 2005),R. philippinarum (Cong et al. 2005),S. glomerata (Aladaileh et al.

2007b), andM. demissus (Waite &Wilbur 1976), and the flukeF.gigantica (Nellaiappan et al. 1989).

In the current study, we purified PO from the bay scallop,further study on gene cloning andmonoclonal antibody produc-tion using this PO will be helpful to investigate PO as a potentialindex in physiological and immune system of this scallop.

ACKNOWLEDGMENTS

Project 30901112 was supported by National Natural ScienceFoundation of China.

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