Archives of Biochemistry and BiophysicsVol. 378, No. 1, June 1, pp. 107–115, 2000doi:10.1006/abbi.2000.1821, available online at http://www.idealibrary.com on

Purification and Characterization of Chorion Peroxidasefrom Aedes aegypti Eggs

Qian Han, Guoyu Li, and Jianyong Li1

Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Champaign–Urbana, 2001 SouthLincoln Avenue, Urbana, Illinois 61802

Received July 7, 1999, and in revised form March 21, 2000

Previous study has shown that a peroxidase is presentin the mature eggs of Aedes aegypti mosquitoes, and theenzyme is involved in the formation of a rigid and insol-uble chorion by catalyzing chorion protein crosslinkingthrough dityrosine formation. In this study, chorion per-oxidase was solubilized from egg chorion by 1% SDS and2 M urea and purified by various chromatographic tech-niques. The enzyme has a relative molecular mass of63,000 as estimated by SDS–PAGE. Spectral analysis ofthe enzyme revealed the presence of the Soret band witha lmax at 415 nm, indicating that chorion peroxidase is ahemoprotein. Treatment of the native enzyme with H2O2

in excess in the absence of reducing agents shifted theSoret band from 415 to 422 nm, and reduction of thenative enzyme with sodium hydrosulfite under anaero-bic conditions changed the Soret band from 415 to 446nm. These results show that the chorion peroxidase be-haves similarly to other peroxidases under oxidativeand reductive conditions, respectively. Compared toother peroxidases, the chorion peroxidase, however, isextremely resistant to denaturing agents, such as SDSand organic solvents. For example, chorion peroxidaseremained active for several weeks in 1% SDS, whilehorseradish peroxidase irreversibly lost all its activityin 2 h under the same conditions. Comparative analysisbetween mosquito chorion peroxidase and horseradishperoxidase showed that the specific activity of chorionperoxidase to tyrosine was at least 100 times greaterthan that of horseradish peroxidase to tyrosine. Chorionperoxidase is also capable of catalyzing polypeptide andchorion protein crosslinking through dityrosine forma-tion during in vitro assays. Our data suggest that thecharacteristics of the chorion peroxidase in mosquitoesclosely reflect its functions in chorion formation andhardening. © 2000 Academic Press

1

To whom correspondence should be addressed. Fax: (217) 244-7421. E-mail: jli2@uiuc.edu.

0003-9861/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

Key Words: Aedes aegypti; chorion peroxidase; dity-rosine; protein crosslinking.

The chorion of newly oviposited mosquito eggs iswhite and soft, but turns black and becomes rigid andinsoluble 2–3 h after oviposition in a moist environ-ment. These changes, termed chorion hardening in ourprevious reports, increase tremendously the resistanceof the eggs to desiccation and other environmentaladversities. Floodwater mosquitoes, such as Aedes ae-gypti, lay their eggs on substrates at the edge of water,and the eggs hatch only after being flooded into waterfollowing adequate rainfall. Therefore, the time forthese eggs to survive in the environment before hatch-ing varies enormously depending on weather condi-tions. Consequently, the ability to resist desiccation iscritical for the survival of these eggs before beingflooded for hatching.

Two interrelated biochemical events, phenol oxidase/dopa decarboxylase catalyzed chorion melanizationand peroxidase-mediated chorion protein crosslinkingreaction, contribute to the formation of a rigid andprotective chorion in A. aegypti (1, 2). In peroxidase-catalyzed protein crosslinking, the enzyme catalyzesoxidation of tyrosine residues on proteins to tyrosineradicals which interact to form dityrosine, therebyleading to chorion protein crosslinking (3). The involve-ment of peroxidase in chorion protein crosslinking in A.aegypti is based on the progressive increase of peroxi-dase activity in the developing eggs, the localization ofthe enzyme in the chorion layer of the mature eggs, andthe detection of dityrosine in the hydrolysate of thehardened egg chorion (2).

Although the presence of a specific chorion peroxi-dase and its involvement in chorion protein crosslink-

ing have been established, the biochemical character-

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istics of the enzyme remain poorly understood. Perox-idase activity has been detected in Drosophila eggs4–7). However, no other information about the Dro-ophila egg peroxidase is available. To understand thehorion peroxidase, we initiated the purification andharacterization of the enzyme from A. aegypti eggs. Inhis report, we describe the techniques used for thesolation of chorion peroxidase and characteristics ofhe enzyme in relation to its biological functions inhorion formation and hardening in A. aegypti mosqui-oes.

MATERIALS AND METHODS

Chemicals. Diaminobenzidine, EDTA, guaiacol, H2O2, horserad-sh peroxidase (HRP,2 EC 1.11.1.7), b-ME, PMSF, sodium hydrosul-te, tropolone, L-tyrosine, angiotensin II (Asp-Arg-Val-Tyr-Ile-His-

Pro- Phe), BSA, and quaternary ammonium cellulose (Q-cellulose)were from Sigma (St. Louis, MO). Hydroxyapatite, polyvinylidenedifluoride (PVDF sheet, 0.2 mm), 10% acrylamide ready gel, andCoomassie blue R-250 were purchased from Bio-Rad (Hercules, CA).Dityrosine was synthesized by a described method (3).

Mosquito rearing, blood feeding, and ovary collection. Aedes ae-gypti black-eyed Liverpool strain mosquitoes were reared as de-scribed previously (1). Blood feeding of female mosquitoes and ovarydissection were the same as described in our previous report (2).

Purification of Chorion PeroxidaseChorion isolation and solubilization. About 25,000 ovary pairs

(dissected from female mosquitoes 3 days after a blood meal) werehomogenized using a Polytron homogenizer (England) in Buffer A(10 mM phosphate, 1 mM PMSF, and 5 mM EDTA, pH 6.5). EDTA atthe applied concentration in Buffer A had no negative effect onchorion peroxidase activity. The homogenate was centrifuged at1500g for 10 min and supernatant, containing released proteins,ipids, and other components from oocytic materials, was separated.he chorion sediments were washed three times with Buffer A andreated with 1% SDS and 2 M urea prepared in Buffer A. Theixture was sonicated for 5 min and centrifuged at 120,000g for 20in at 2°C to obtain supernatant. The chorion sediments were

reated another three times by the same procedure, and superna-ants, containing released chorion peroxidase and other chorion pro-eins, were pooled, dialyzed against Buffer A, and then used as thenitial material for chorion peroxidase purification.

Peroxidase activity assay. Chorion peroxidase activity was as-ayed primarily with guaiacol and H2O2 as substrates (8). Typically,ml of a reaction mixture containing 6 mM guaiacol, 0.8 mM H2O2,

nd varying amounts of solubilized chorion proteins or pooled activeeroxidase fractions was incubated at 30°C. Absorbance increase at35 nm due to guaiacol oxidation was continuously monitored for 3in using a Hitachi Model U-2001 double-beam spectrophotometer

quipped with a water-jacketed six-cell positioner (San Jose, CA).eroxidase activity was defined arbitrarily as units of absorbance

ncrease at 435 nm in 1 ml of reaction mixture per minute measuredn a cuvette with d 5 1 cm. Protein content was determined usinghe colorimetric method of Lowry (9).

Q-cellulose column chromatography. A Q-cellulose column (2.5 30 cm) was equilibrated with 10 mM phosphate buffer (pH 6.5) at

2 Abbreviations used: AA, amino acid; b-ME, mercaptoethanol;BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic

acid; HRP, horseradish peroxidase; PMSF, phenylmethylsulfonylfluoride.

°C, and the dialyzed chorion protein sample (approximately 150 ml)as applied to the column. Proteins were eluted with 600 ml of a

inear NaCl gradient (0–500 mM) prepared in Buffer A. Active frac-ions were pooled and concentrated using a stirred cell and a YM 30embrane with molecular weight cutoff at 30,000 (Millipore).Hydroxyapatite chromatography. The concentrated peroxidase

ractions were chromatographed on a hydroxyapatite column (1.5 30 cm) that was equilibrated with Buffer A, and proteins were elutedith 300 ml of a linear NaCl gradient (0–500 mM). The peroxidase

ractions were pooled and concentrated using a 15-ml concentratorith a 30,000 molecular weight cutoff membrane (Millipore).Mono-Q chromatography. A Mono-Q anion exchange column (7 3

5 mm, Pharmacia) was equilibrated with 10 mM phosphate bufferpH 6.5) using a Hitachi gradient HPLC system. Concentrated per-xidase fractions (0.4 ml) were injected into the column. Proteinsere eluted with a linear NaCl gradient (0–500 mM) during a0-min period at a flow rate of 1 ml min21. The fractions correspond-

ing to individual protein peaks (monitored at 280 nm) were collectedmanually, and attention was paid to avoid overlap among the differ-ent protein peaks. The fraction with peroxidase activity was concen-trated and rechromatographed using the same Mono-Q column.

Electrophoresis. Purified chorion peroxidase was mixed withtreatment buffer (1:1) with or without 2% b-ME for 15 min and thenanalyzed using SDS–PAGE with 10% acrylamide gel. Some enzymewas also heated in boiling water for 5 min in the presence or absenceof b-ME prior to electrophoresis to determine the effect of heatreatment on its migration in polyacrylamide gel. After electrophore-is, the gel was cut vertically into sections that were stained eitherith Coomassie blue or with 5 mM tropolone or 5 mM diaminoben-

idine in the presence of 1 mM H2O2.The effects of heat and b-MEtreatment on the mobility of the chorion peroxidase in the gels wereevaluated by the migration distances of the treated enzyme in com-parison with those of untreated enzymes after electrophoresis.

Characterization of Chorion PeroxidaseN-Terminal AA sequence and AA composition. The partial N-

terminal AA sequence of chorion peroxidase was determined usingan Applied Biosystems Procise (Department of Biochemistry, Mich-igan State University). The AA composition was determined at theBiotechnology Center of the University of Illinois (Champaign, IL).

Spectral analysis. The spectrum of the native enzyme in both theUV and visible regions was measured using a Hitachi Model U-2001double-beam spectrophotometer. Changes of redox states in chorionperoxidase during its oxidation and reduction by H2O2 and sodium

ydrosulfite, respectively, were based on spectral shifts of the Soretand and other minor peaks in the visible region as described forRP (10, 11).Optimum reaction conditions. The optimum pH and H2O2 con-

centration for chorion peroxidase were determined using guaiacol asa substrate. Guaiacol, H2O2, and chorion peroxidase were preparedin different buffers, including 100 mM phosphate buffer (pH 6–7.5),Tris buffer (pH 8–8.5), and 2-amino-2-methanol/1-propanol buffer(pH 9–10). Guaiacol and peroxidase were mixed prior to the additionof H2O2, and the reaction was initiated by adding 0.1 ml of H2O2 (8

M) into 0.9 ml of guaiacol/peroxidase mixture. The optimum con-entration for H2O2 was assayed by mixing 0.1 ml of H2O2 solution

(1–40 mM) into 0.9 ml of guaiacol/peroxidase mixture prepared inTris buffer (pH 8). For both assays, peroxidase activity was based onabsorbance increase at 435 nm during 3-min incubation at 30°C. Theamount of chorion peroxidase used in the reaction mixture was 0.2mg, and the final concentration of guaiacol in the reaction mixturewas 8 mM.

Resistance to SDS. Purified chorion peroxidase was treated with

1.5% SDS in 100 mM phosphate buffer (pH 8). At 1, 5, 10, 20, 30, and120 min after SDS treatment, 0.2 ml of the treated enzyme was

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109FUNCTION OF CHORION PEROXIDASE FROM Aedes aegypti EGGS

mixed with 0.8 ml of substrate preparation containing 9.25 mMguaiacol and 1 mM H2O2. The enzyme activity was monitored spec-trophotometrically at 435 nm. The effect of SDS on chorion peroxi-dase activity was based on absorbance increase at 435 nm during3-min incubation in the reaction mixtures in comparison with thatrecorded for the untreated enzyme. SDS was not removed from thetreated enzyme. Therefore, the final reaction mixture contained 0.3%of SDS. The effect of SDS on HRP was determined in the samemanner, and the difference in resistance to SDS between the chorionperoxidase and HRP was compared.

Substrate specificities. The commonly used peroxidase sub-trates, including guaiacol, diaminobenzidine, and tyrosine, alsoere the substrates of chorion peroxidase. Among these substrates,uaiacol and tyrosine were used as representatives, and the specificctivities of the chorion peroxidase to these two compounds wereetermined and compared to those of HRP to guaiacol and tyrosinender identical assay conditions. Production of dityrosine duringhorion peroxidase and HRP catalyzed tyrosine oxidation was basedn the increase in absorbance at 315 nm, where the dityrosineroduct had an absorption coefficient of 6.3 3 1023 M21 cm21 (12).

Peroxidase-mediated protein crosslinking through dityrosine for-mation. The ability of chorion peroxidase to catalyze proteincrosslinking through dityrosine formation was determined usingangiotensin II (a tyrosine-containing polypeptide), BSA, and someisolated chorion protein that was devoid of peroxidase activity. A1-ml reaction mixture consisting of 1 or 2 mg of chorion peroxidase,.8 mM H2O2, and 1.0 mg of angiotensin II or 5 mg of chorion protein

or BSA was incubated at 30°C. Peptide or protein crosslinkingthrough dityrosine formation was based on the detection of dity-rosine in the hydrolysates using HPLC-ED after the above reactionmixtures were hydrolyzed in 6 M HCl at 110°C for 20 h. Identifica-tion of dityrosine in the hydrolysate was based on both its retentiontime and oxidation potentials during HPLC-ED analysis in compar-ison with those generated using dityrosine standard under identicalconditions (2).

RESULTS

Purification of chorion peroxidase

Chorion isolation and chorion peroxidase solubiliza-tion. When dissected mature eggs were homogenizedbriefly in Buffer A containing 5 mM EDTA, the oocyticmaterials within the chorion, including proteins, lip-ids, and other components for embryo development,were released into the supernatant. Homogenizationbroke the intact chorion into pieces; however, this pro-cess did not result in a substantial solubilization ofchorion peroxidase, because the majority of the enzymeactivity (90%) remained associated with the chorionpieces.

Treatment of chorion sediments with 1% SDS and 2M urea in Buffer A plus sonication was effective tosolubilize the enzyme. The majority of the enzyme ac-tivity (.90%) was released after the chorion sediments

ere treated for four times using 1% SDS and 2 M ureawith sonication.

Purification of chorion peroxidase. Purification ofchorion peroxidase from the solubilized chorion pro-teins was achieved by Q-cellulose, hydroxyapatite, andMono-Q column chromatographies. Chorion peroxidase

activity was eluted within broad gradient ranges from

165 to 430 mM and from 100 to 185 mM NaCl duringconventional Q-cellulose and hydroxyapatite chro-matographies, respectively. After hydroxyapatite chro-matography, the concentrated peroxidase fractionswere separated by Mono-Q anion exchange HPLC. Twopeaks, one major with a retention time of 14.8 min andone minor with a retention time of 10.3 min, showedperoxidase activity (Fig. 1A). Due to the limitedamount of sample available, no further effort was madeto purify the minor peroxidase peak. After the secondstep of Mono-Q column chromatography, peroxidasebecame the only detected protein (Fig. 1B). The chorionperoxidase was purified 128-fold, with a yield of 35.4%(Table I).

SDS–PAGE analysis of the purified chorion peroxi-dase, treated in boiling water in the presence of b-MEprior to electrophoresis, showed the enzyme with an M r

of 63,000 (Fig. 2, lane A2). Heat treatment of the en-zyme under reducing conditions also completely abol-

FIG. 1. Mono-Q anion exchange chromatography. Concentratedperoxidase fractions, obtained after conventional Q-cellulose andhydroxyapatite chromatographies, were separated by anion ex-change HPLC with a Mono-Q column (7 3 65 mm) using a linearNaCl gradient. The peroxidase peak was collected, concentrated, andrechromatographed on the same Mono-Q column under identicalconditions. Chromatogram A shows the protein elution profile of thesample during the first Mono-Q column chromatography. Chromato-gram B illustrates the protein profile of the concentrated peroxidaseduring the second Mono-Q column chromatography. Both the 14.8-min major peak and the 10.3-min minor peak in A show peroxidaseactivity (see Results for details), but only the major peroxidase peak(14.8 min) was isolated and used for subsequent enzyme character-ization.

ished its activity because the peroxidase band was not

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110 HAN, LI, AND LI

stained positive for peroxidase activity (not shown). Incontrast, the enzyme was stained easily with tropo-lone, diaminobenzidine, guaiacol, and dopa in the pres-ence of H2O2 (Fig. 2, lane B1) when it was not reducedand heated prior to electrophoresis. In addition, theuntreated enzyme migrated faster on the gel than theheated and reduced enzyme (see Fig. 2, lanes A2 andB1). Treatment of b-ME alone (i.e., without heating)did not completely abolish its activity because the per-oxidase band on the polyacrylamide gel remained de-tectable by substrate staining, but it did not reach thesame staining intensity (Fig. 2, lane B2) as that ofunreduced enzyme (Fig. 2, lane B1). b-ME treatmentalso reduced the mobility of the enzyme in the gelduring electrophoresis.

Characterization of Chorion Peroxidase

Spectral characteristics. Spectral analysis of cho-rion peroxidase showed two major absorption peakswith lmax at 280 and 415 nm (Fig. 3A), respectively, and

TA

Purification of Chorion Per

Fraction Volume (ml) Protein(s)

olubilized chorion proteins 150.0 2.A-cellulose fraction 30.0 0.ydroxyapatite fraction 20.0 0.irst Mono-Q fraction 2.8 1.econd Mono-Q fraction 1.4 0.

a Measurement of protein(s) in each fraction was performed by thb The specific activity of the chorion peroxidase is expressed as un

FIG. 2. SDS–PAGE analysis. Purified chorion peroxidase was an-alyzed using SDS–PAGE with either Coomassie blue or substratestaining as described under Materials and Methods. Lanes A1 andA2 are protein molecular mass standards and purified chorion per-oxidase, respectively, stained with Coomassie blue. Lanes B1 and B2are purified chorion peroxidase stained with dopa in the presence of

H2O2. Lane A2, with heated and reduced enzyme; lane B1, withuntreated enzyme; lane B2, with b-ME reduced enzyme.

four minor peaks at 495, 545, 582, and 648 nm (Fig.3B), respectively. The detection of the 415-nm peak,commonly termed the Soret band, suggests that cho-rion peroxidase is a heme-containing protein. Additionof H2O2 caused a shift of the Soret band from 415 to 422nm (Fig. 4A) and the disappearance of the 495- and648-nm peaks with concomitant formation of two newminor peaks at 546 and 586 nm. The spectral shift ofthe Soret band in chorion peroxidase was similar tothat observed for HRP under identical assay condi-tions, in which the Soret band of HRP was shifted from403 to 417 nm (not shown). Reduction of the nativechorion peroxidase by sodium hydrosulfite shifted the

I

idase from Mosquito Eggs

g/mg) Specific activityb Fold Recovery (%)

5 1 100.0115 23 78.4245 49 67.8340 68 47.1640 128 35.4

ethod of Lowry (9), using bovine serum albumin as a standard.of absorbance of tetraguaiacol min21 mg21 of protein.

FIG. 3. Spectrum of the native chorion peroxidase. (A) Spectrum ofthe native chorion peroxidase in both the UV and visible regions; (B)

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minor peaks at 495, 545, 582, and 648 nm in the visible region athigh sensitivity.

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111FUNCTION OF CHORION PEROXIDASE FROM Aedes aegypti EGGS

Soret band from 415 to 446 nm under anaerobic con-ditions (Fig. 4B), which was also similar to that ob-served for HRP under the same conditions (not shown).

Partial N-terminal AA sequence and AA composition.The partial N-terminal AA sequence of the chorionperoxidase, obtained by Edman digestion, was -LPN-VPPNNLS-YRTI-DE. This partial sequence showed es-sentially no similarity to N-terminal sequences of HRP(13), sea urchin ovoperoxidase (14), and the derived AAsequence of a putative peroxidase from Drosophila(15). Residues 1, 12, and 17 of the N-terminal sequenceof the chorion peroxidase were not identified. Therewere fewer Ser, Ala, Arg, Val, and Phe residues and

FIG. 4. Spectral changes of chorion peroxidase in relation to itsredox states. (A) Spectral shift of the Soret band from 415 to 422 nm,with the concomitant disappearance of the 495- and 648-nm minorpeaks and formation of two new minor peaks at 546 and 586 nm forchorion peroxidase, after mixing 5.4 mM chorion peroxidase with 6.2mM H2O2 in 0.1 M acetate buffer (pH 5.4). (B) Spectral shift of the

oret band from 415 to 446 nm and disappearance of the minor 495-nd 648-nm peaks and concomitant formation of two new minoreaks at 558 and 596 nm for chorion peroxidase after the enzyme (5.4

mM) was treated with 2 mM sodium hydrosulfite in 0.1 M acetate(pH, 4.0) at anaerobic conditions. The solid line illustrates the spec-trum of the native enzyme and the dashed line shows the spectralchange of the enzyme in the presence of H2O2 or sodium hydrosulfite,respectively. Inserts in A and B show the spectral changes of theminor peaks at higher sensitivity.

more Lys, Tyr, and Gly residues in chorion peroxidase

than in HRP and sea urchin ovoperoxidase. Table IIshows the amino acid composition of chorion peroxi-dase in comparison with those reported for HRP (13)and sea urchin ovoperoxidase (14), respectively.

Optimal H2O2 concentration and pH for chorion per-xidase activity. Chorion peroxidase displayed itsighest activity at pH 8 with guaiacol as a reducinggent (Fig. 5A). Therefore, the subsequent enzyme as-ays were conducted in Tris buffer at pH 8. Oxidationf guaiacol by chorion peroxidase was not observed inhe absence of H2O2, but a high concentration of H2O2

greatly decreased its specific activity (Fig. 5B). Theoptimal concentration of H2O2 was around 0.8 mM forchorion peroxidase (see Fig. 5B).

Resistance to SDS and other denaturing agents.Chorion peroxidase was resistant to SDS. The releasedperoxidase remained active for a long time in 1% SDSsolution. For example, no noticeable decrease in perox-idase activity was observed when solubilized chorionprotein remained in 1% SDS and 2 M urea for monthsat 4°C (not shown). However, when the purified cho-rion peroxidase was treated with 1.5% SDS for 1–120min prior to mixing with guaiacol and H2O2, somedecrease in enzyme activity was observed in the SDS-treated enzyme as compared to the activity level of theuntreated enzyme (Fig. 6A). Interestingly, whentreated briefly (#5 min) with SDS prior to mixing withsubstrate preparation, the enzyme displayed a rela-

TABLE II

Amino Acid Composition of Chorion Peroxidasefrom A. aegypti, Sea Urchin Ovoperoxidase,

and Horseradish Peroxidase

Amino acid CPO OPOa HRPb

Asx 86 129 156Glx 94 95 65Ser 57 76 81His 13 23 10Gly 76 73 55Thr 62 44 81Ala 59 74 75Arg 51 86 68Tyr 27 22 16Val 37 60 55Met 12 18 13Phe 43 68 65Ile 37 45 42Leu 83 82 114Lys 65 21 20Pro 66 63 55Cys NA 22 26Trp NA 25 3

Note. Values are shown in residues/1000 residues. The values ofOPO and HRP were recalculated from the original data.

a

From Nomura and Suzuki (14).b From Welinder (13).

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112 HAN, LI, AND LI

tively large decrease (about 15%) in activity, whileprolonged incubation of the enzyme in SDS prior toactivity assay did not result in further decreases inactivity (see Fig. 6A). The enzyme remained activeafter having been in 1.5% SDS solution for days atroom temperature. In contrast, when HRP was treatedwith 1.5% SDS, rapid inactivation of HRP became ap-parent within minutes and the decrease in activitycorrelated with the time of SDS exposure (Fig. 6B).Dialysis of the SDS-treated HRP did not lead to anyrecovery of its activity (not shown).

Chorion peroxidase was also resistant to methanoland acetic acid. After SDS–PAGE (without boiling andb-ME treatment of the enzyme) and a staining (0.1%Coomassie blue, 40% methanol, and 5% acetic acid inwater) and destaining process (50% methanol and 5%acetic acid in water), the Coomassie blue stained per-oxidase band on the gel was overtaken rapidly by thebrown-colored oxidation products upon mixing withtropolone and H2O2 (not shown).

Substrate specificity. Chorion peroxidase re-sponded actively to commonly used peroxidase sub-strates, such as guaiacol, diaminobenzidine, and ty-

FIG. 5. Effect of pH and H2O2 concentration on chorion peroxidaseactivity. The reaction mixtures containing guaiacol, H2O2, and cho-ion peroxidase were prepared as described under Materials andethods. (A) Peroxidase activity under different pH conditions; (B)

eroxidase activity in the presence of different concentrations of2O2.

rosine. The specific activities of chorion peroxidase e

were determined with guaiacol and tyrosine as reduc-ing agents and compared to those of HRP to these twocompounds under identical assay conditions (Figs. 7Aand 7B). The calculated specific activity of chorion per-oxidase to guaiacol (128 U min21 mg21) was in the sameorder of magnitude as that of HRP (66 U min21 mg21).In contrast, the specific activity of chorion peroxidaseto tyrosine (10.4 mmol min21) was two orders of mag-nitude greater than that of HRP to tyrosine (0.05 mmolmin21 mg21).

Peroxidase-mediated protein crosslinking throughdityrosine formation. Both tyrosine and dityrosinewere electrochemically active, but they were resolvedcompletely under the applied HPLC-ED conditions(Fig. 8A). When angiotensin II was incubated at 30°Cin the presence of chorion peroxidase and H2O2, treatedwith 0.4 M trifluoroacetic acid for 15 min after incuba-tion to stop the reaction, lyophilized under vacuum,hydrolyzed in 6 M HCl at 110°C, and analyzed usingHPLC-ED, dityrosine was detected in the hydrolysateand the amount of dityrosine was approximately pro-portional to the incubation times (Figs. 8B–8D). WhenBSA or isolated chorion protein, after being incubatedat 30°C for 1 h in the presence of chorion peroxidasend H2O2, was analyzed using procedures identical to

those for angiotensin II reaction mixtures, dityrosinewas also detected in the hydrolysates (Figs. 8E and

FIG. 6. Effect of SDS on chorion peroxidase and HRP activities.Details for the treatment of the enzymes with SDS and the subse-quent enzyme activity assays are described under Materials andMethods. A and B illustrate the change of enzyme activity in relationto the time of SDS exposure for chorion peroxidase and HRP, respec-

tively. The x-axis indicates time period of SDS treatment prior tonzyme activity assay.

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113FUNCTION OF CHORION PEROXIDASE FROM Aedes aegypti EGGS

8F). In contrast, essentially no dityrosine was detectedin a chorion protein reaction mixture in the absence ofH2O2 (not shown).

DISCUSSION

Data from our previous study demonstrated thepresence of a peroxidase within the chorion of A. ae-gypti eggs and the involvement of the enzyme in cho-rion protein crosslinking during chorion hardening (2).However, little is known about the biochemical char-acteristics of this enzyme. In this study, mosquito cho-rion peroxidase was successfully purified, and somebasic biochemical and biophysical characteristics of theenzyme, such as its hemoprotein nature, relative mo-lecular mass, optimal reaction conditions, partial N-terminal AA sequence and AA composition, formationof intermediates in the presence of H2O2 and hydrosul-fite, substrate specificity, and resistance to denaturingagents, were established. Peroxidase-catalyzed egg-shell or chorion hardening is likely to be a critical eventin all oviparous insects, yet no peroxidase from theeggs of any insects has been purified and character-ized. Therefore, data generated in this study providethe basis for future research related to mosquito cho-

FIG. 7. Specific activities of chorion peroxidase and HRP toguaiacol and tyrosine. (A) Absorbance increase at 435 nm in 1 ml ofreaction mixture containing 6 mM guaiacol, 0.8 mM H2O2, and 0.2 mgof chorion peroxidase (line 1) or 0.4 mg of HRP (line 2). (B) Absor-bance increase in 1 ml of reaction mixture containing 1.4 mM ty-rosine, 0.8 mM H2O2, and 1 mg of chorion peroxidase (line 1) or 2 mgf HRP (line 2). The amounts of chorion peroxidase and HRP used inand B, respectively, were different, but the ratio of chorion perox-

dase to HRP (1:2) in A was the same as in B.

rion peroxidase or egg peroxidase from other insects.

The detection of the 415-nm Soret band indicated theenzyme was a hemoprotein, and the requirement ofH2O2 as the oxidizing agent for the enzyme establishedthe peroxidatic nature of the reactions it catalyzes.HRP is the most extensively studied enzyme in bio-chemistry. In the catalytic cycle, the resting ferric HRPis oxidized by H2O2 to an active form, designated com-pound I, that is two oxidizing equivalents above itsresting state (16). Two sequential one-electron oxida-tions of reducing agents by the enzyme, involving anintermediate species termed compound II, return theenzyme to its resting form (16–18). Formation of com-pound I and compound II for HRP was mainly based onspectral shift of the Soret band in the presence of H2O2

(16). It was difficult to detect compound I because itwas unstable and decomposed rapidly to compound II(17–18). With H2O2 in excess, compound I and/or IIwas/were further oxidized to compound III (17). Thelmax of the Soret band was similar between compoundII and compound III in HRP except for the difference oftheir extinction coefficients (16). Therefore, the spec-tral shift from 415 to 422 nm for chorion peroxidaseduring H2O2 treatment was likely due to accumulationof both compound II and compound III in the reactionmixture. The similarity in spectral shift between cho-rion peroxidase and HRP during H2O2 oxidation sug-gests that the same catalytic cycle for HRP applies tochorion peroxidase mediated reactions.

An exceptional physical characteristic of the mos-quito chorion peroxidase is its resistance toward dena-turing agents such as SDS and organic solvents. Inter-nal hydrophobic interaction of proteins is one of the keyattractive forces maintaining their three-dimensionalstructure. Detergents and organic solvents interactwith hydrophobic residues, which disrupt the tertiarystructure of the protein molecules, leading to theirdenaturation. Compared to HRP, chorion peroxidase isextremely resistant to detergents and organic solvents.Although some decrease in activity was observed inSDS-treated peroxidase, it seemed apparent that thedecrease in activity was not due to peroxidase inacti-vation because prolonged incubation of the enzyme inSDS did not lead to further decrease in its activity. Ifthe activity decrease was due to inactivation of thechorion peroxidase, a steady decline in activity wouldhave been observed as seen for HRP (Fig. 5B). SDS wasnot removed from the treated enzyme prior to mixingwith substrate preparation during the assays. Thepresence of SDS (0.3% final concentration) likely in-creased greatly the viscosity of the reaction mixture,which might affect the turnover of the enzyme, therebydecreasing its specific activity.

Resistance of chorion peroxidase to denaturing con-ditions was also supported by results from SDS–PAGEof the enzyme. SDS denatures proteins and also binds

strongly to protein, with approximately one SDS mol-

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s atn p

114 HAN, LI, AND LI

ecule per two amino acid residues (or 1.4 g of SDS/g ofprotein). Because the charge density and conformationare essentially constant for all proteins, their separa-tion in SDS–PAGE is due basically to the sieving effectof the gel. Undoubtedly, SDS binds to chorion peroxi-dase, but the enzyme is far from being completelydenatured or linearized by SDS because of its easydetection by substrate staining after SDS–PAGE.However, it seems that boiling of the enzyme in thepresence of both b-ME and SDS leads to a completedenaturation of the chorion peroxidase, which is sup-ported by the disappearance of the enzyme activity andthe detection of a much sharper band of the treatedenzyme. Disulfide bonds are important for maintainingthe three-dimensional structures of peroxidases (19),which may also be true for chorion peroxidase. Thismight explain the effects of b-ME on the partial dena-

FIG. 8. Protein crosslinking through dityrosine formation. Angioteneroxidase and H2O2 and incubated at 30°C. Protein crosslinking th

hydrolysate of the reaction mixtures. Chromatograms illustrate the dof dityrosine in the hydrolysates of angiotensin II reaction mixturedityrosine in the hydrolysate of BSA reaction mixture (E) and chorio

turation and inactivation of chorion peroxidase.

Another distinctive characteristic of the chorion per-oxidase as compared to HRP is its high specific activitytoward tyrosine. For example, there is about a twofolddifference between chorion peroxidase and HRP toguaiacol (a commonly used substrate for peroxidaseassay), but the specific activity of the chorion peroxi-dase to tyrosine is more than 100-fold greater than thatof the same HRP to tyrosine (see Fig. 6). The highactivity of chorion peroxidase to tyrosine, the ability ofthis enzyme to catalyze polypeptide and chorion pro-tein crosslinking through dityrosine formation, and thedetection of dityrosine in the hardened egg chorion (2)provide strong evidence for the active role of the cho-rion peroxidase in chorion hardening through dity-rosine formation in A. aegypti eggs. In mosquitoes,peroxidase-catalyzed chorion protein crosslinking oc-curs in the environment because dityrosine is not de-

II, BSA, or isolated chorion protein was mixed with purified choriongh dityrosine formation was based on detection of dityrosine in thection of tyrosine and dityrosine standards (A), the relative amounts0 (B), 5 (C), and 15 min (D) after incubation, and the amounts ofrotein reaction mixture during 60 min incubation (F), respectively.

sinrouete

tected from the dissected or newly oviposited eggs. In

1

11

11

1

1

1

1

115FUNCTION OF CHORION PEROXIDASE FROM Aedes aegypti EGGS

contrast, protein crosslinking in Drosophila eggs oc-curs prior to oviposition (3–6). Therefore, the environ-ment for mosquito chorion peroxidase to display itsfunction is different from that of other peroxidases,including Drosophila egg peroxidase. Consequently,environmental pressures or selection might explainwhy chorion peroxidase is extremely resistant to harshconditions, such as its strong resistance to denatur-ation by SDS and organic solvents. However, we areunable to provide a clear explanation about the mech-anism for the strong resistance of mosquito chorionperoxidase to denaturing agents at this time due todifficulties in obtaining an adequate amount of enzymefor further structural analysis. We also have no preciseanswer for the extremely high activity of the enzyme totyrosine as compared to other peroxidases. The unam-biguous answers for these unique characters of thechorion peroxidase rely ultimately on a thorough un-derstanding of the detailed structure of the enzyme,including its primary sequence, its posttranslationmodification, and its three-dimensional structure.

ACKNOWLEDGMENT

This work is supported by NIH Grant AI 37789.

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