Peroxidase as the Major Protein Constituent in Areca Nut and Identification of Its Natural Substrates

12 Pages·2013·2.88 MB·English

HindawiPublishingCorporation
Evidence-BasedComplementaryandAlternativeMedicine
Volume2013,ArticleID412851,12pages
http://dx.doi.org/10.1155/2013/412851
Research Article
Peroxidase as the Major Protein Constituent in Areca Nut and
Identification of Its Natural Substrates
Yu-ChingLiu,1,2Chao-JungChen,3,4Miau-RongLee,5MiLi,6Wen-TsongHsieh,7
Jing-GungChung,8,9andHeng-ChienHo5
1DepartmentofMedicalResearch,ChinaMedicalUniversityHospital,Taichung40402,Taiwan
2DepartmentofVeterinaryMedicine,NationalChungHsingUniversity,Taichung40227,Taiwan
3GraduateInstituteofIntegratedMedicine,ChinaMedicalUniversity,Taichung40402,Taiwan
4ProteomicsResearchCoreLab,ChinaMedicalUniversity,Taichung40402,Taiwan
5DepartmentofBiochemistry,ChinaMedicalUniversity,Taichung40402,Taiwan
6WholesalerEnterpriseCompany,Taipei23941,Taiwan
7DepartmentofPharmacology,ChinaMedicalUniversity,Taichung40402,Taiwan
8DepartmentofBiologicalScienceandTechnology,ChinaMedicalUniversity,Taichung40402,Taiwan
9DepartmentofBiotechnology,AsiaUniversity,Taichung41354,Taiwan
CorrespondenceshouldbeaddressedtoJing-GungChung;[email protected]
andHeng-ChienHo;[email protected]
Received6June2013;Accepted4September2013
AcademicEditor:Jen-HweyChiu
Copyright©2013Yu-ChingLiuetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Numerous reports illustrate the diverse effects of chewing the areca nut, most of which are harmful and have been shown to
beassociatedwithoralcancer.Nearlyallofthestudiesarefocusedontheextractand/orlowmolecularweightingredientsin
thearecanut.Thepurposeofthisreportistoidentifythemajorproteincomponentinthearecanut.Afterammoniumsulfate
fractionation,theconcentratedarecanutextractissubjectedtoDEAE-cellulosechromatography.Acoloredproteiniselutedat
lowNaClconcentrationandtheapparentlyhomogeneouseluentrepresentsthemajorproteincomponentcomparedtotheareca
nutextract.Thecoloredproteinsharespartialsequenceidentitywiththeroyalpalmtreeperoxidaseanditsperoxidaseactivity
is confirmed using an established assay. In the study, the natural substrates of areca nut peroxidase are identified as catechin,
epicatechin,andprocyanidinB1.Thetwoformersubstratesaresimilarlyoxidizedtoforma576Daproductwithconcomitant
removaloffourhydrogenatoms.Interestingly,oxidationofprocyanidinB1occursonlyinthepresenceofcatechinorepicatechin
andanadditionalproductwithan864Damolecularmass.Inaddition,procyanidinB1isidentifiedasaperoxidasesubstratefor
thefirsttime.
1.Introduction primarycauseofdeathandoccurredatanaverageageof55
years.Epidemiologicalstudiesandstatisticalanalysesclearly
According to the Taiwan Cancer Registry annual report demonstratedthatthedevelopmentoforalcancerinTaiwan
(DepartmentofHealth,ExecutiveYuan,Taiwan)in2009,the washighlyassociatedwithchewingthearecanut,smoking,
annualincidenceoforalmalignancywas6,480caseswitha andwineconsumption[1].Chewingthearecanutwrapped
medianageof53years,anincreaseof699casesfrom2008. in the Piper betle leaf, which is coated with slaked lime, is
Oralcancerhadbecomethefifthleadingcauseoftumorsin popular in Taiwan, especially in the southeastern region. It
Taiwan, preceded by malignancies of the colon, liver, lung, hasbeenestimatedthattherewereatleasttwomillionoutof
andbreast.Morethan90%ofthenewincidences(5927cases) 23millionresidentsinTaiwanwhowerearecanutchewers.
in2009wereinmales,whoengageinchewingthearecanut. Approximately 90% of oral cancer patients in Taiwan have
FormalesinTaiwan,thismalignancyrepresentedthefourth participatedinthispractice.
2 Evidence-BasedComplementaryandAlternativeMedicine
ThearecanutistheseedofthepalmplantArecacatechuL. MALDI-TOF-TOFMS(UltraflexIIITOF/TOF,BrukerDal-
and is rich in alkaloids, including arecoline, arecaidine, tonics, Inc.) equipped with a smartbeam laser. For digested
guvacine, and guvacoline. Sundqvist et al. demonstrated peptideanalysis,aCHCAmatrix(1mg/mLin50%acetoni-
that the aqueous extract and the above four alkaloids from trilecontaining0.1%TFA)wasmixedwithanequalvolume
the areca nut are highly cytotoxic and genotoxic to human ofpeptidesampleontheMALDIsampleplateandthenair-
cultured cells and have the potential to induce tumors [2]. dried.Thespectrawererecordedinreflectormodeusingan
Additionally, several reports have shown the association of accelerationvoltageof+20kVanda20nsdelaytime.Intense
chewing areca nuts with the development of various disor- MSpeakswereselectedforMS/MSanalysisandpeptidemass
ders,suchascardiovasculardisease[3],metabolicsyndrome fingerprinting (PMF). The precursor mass window was set
[4], and hypertension [5]. In spite of these various harmful at±0.45%oftheprecursormass.ThepeaklistfromtheMS
properties, the areca nut is still used in traditional Chinese spectrawasprocessedbyFlexAnalysisv.3.0software(Bruker
medicineandinIndia.Itwasfoundtobeahighlyeffective Daltonics, Inc.). The extracted peak list was compared
treatmentforhumantaeniasis[6]andincreasedmemoryand againsttheNCBInrdatabase(release20101220)usingMAS-
learninginaratmodel[7]. COTv.2.2.04(MatrixScience,UK).Searchparameterswere
Apart from the numerous studies on small-molecule selected as follows: taxonomy-Viridiplantae (green plants);
ingredientsinthearecanut,littleisknownabouttheprotein enzyme-trypsin; fixed modification-carbamidomethyl (C);
components,whichisthefocusofthisreport.Themajorpro- variablemodification-oxidation(M,H,W);peptidemasstol-
teincomponentinthearecanutwaspurifiedasanapparently erance±100ppm;MS/MStolerance±0.5Da;andsignificant
homogeneousproductandfoundtobeaperoxidase,whose threshold𝑃<0.05.
activitywassubsequentlyrevealed.Thenaturalsubstratesfor The molecular mass of the purified protein was deter-
thearecanut(AN)peroxidasewerealsodemonstratedinthis mined in linear mode. Equal volumes (0.5𝜇L) of protein
study. sample and SA matrix (saturated in 30% acetonitrile and
0.1% TFA) were mixed on a ground MALDI plate (Bruker
Daltonics, Inc.). Protein mass calibration was performed
2.MaterialsandMethods
using a protein mixture kit II consisting of protein A
2+ + +
2.1. Reagents. Arecanut was obtainedfroma local supplier [M+2H] ,trypsinogen[M+2H] ,andproteinA[M+2H]
in northern Taiwan. DEAE- (diethylaminoethyl-) cellulose (BrukerDaltonics,Inc.).
waspurchasedfromWhatman,Inc.(Clifton,NJ).Prestained
proteinmarkerwasobtainedfromFermentas(St.Leon-Rot,
2.4.ActivityAssayandZymogramofANPeroxidase. Activity
Germany).Guaiacol,(+)-catechin,and(−)-epicatechinwere
ofANperoxidasewasassayedbymeasuringtheabsorbanceat
obtainedfromSigma-AldrichCorp.(St.Louis,MO).PNGase
470nmin20mMguaiacoland5mMH2O2in0.1Mcitrate-
FwaspurchasedfromNewEnglandBioLabs(Ipswich,UK).
phosphate buffer (pH 5.5). For peroxidase zymography, a
Procyanidins B1 and B2 were obtained from Extrasynthese
native gel was used to analyze the purified protein without
(Genay, France), and sinapinic acid (SA) and 𝛼-cyano-4-
heatingandadditionofareducingagent.Afterelectrophore-
hydroxycinnamicacid(CHCA)werepurchasedfromBruker
sis, the gel was rinsed with water and immersed in 20mM
Daltonics,Inc.(Bremen,Germany).
guaiacoland5mMH2O2 inwater.Abrowncolorappeared
shortly after the addition and indicated the presence of
2.2.Single-ColumnPurificationoftheMajorProteinCompo- peroxidase.
nentintheArecaNut. Thefollowingprotocolwasperformed
at room temperature. Areca nut (330g) was homogenized
2.5.DigestionofGlycosylatedANPeroxidasewithPNGaseF.
with 1 liter of buffer A (20mM Tris/HCl, pH 7.0). The
The purified peroxidase (13𝜇L) was mixed with 2𝜇L of
homogenate was filtered and centrifuged at 13,000rpm for
∘ denaturation buffer (10X) and heated in boiling water for
20min at 4 C, and the clear supernatant was filtered again.
10min. The following components were then added to the
Proteins in the areca nut (AN) extract were fractionated
mixture: 2𝜇L of G7 reaction buffer (10X), 2𝜇L of NP-40
with 80% ammonium sulfate and centrifuged. The protein
(10%), and 1𝜇L of PNGase F (500U/𝜇L). The mixture was
precipitatewas redissolved in buffer A and dialyzed against
∘
incubatedat37 CovernightorlongerandanalyzedbySDS-
buffer A overnight. The dialysis step was repeated once,
PAGE. The bands of interest were excised and analyzed by
andthedialyzedsamplewasloadedontoaDEAE-cellulose
MALDI-TOF-TOFMSasmentionedabove.
column preequilibrated with buffer A. After loading, the
columnwaswashedwith100mLofbufferA,andtheproteins
inthecolumnwereelutedwithalineargradientof1MNaCl
2.6.DetectionofPeroxidaseSubstrate(s)inArecaNutExtract
buffer A. A brown-colored protein was eluted at a low
byLC-MS/MS. Wavelengthscanningwasinitiallyemployed
concentrationofNaCl.
todeterminewhetherperoxidasesubstrate(s)existedinareca
nut extract. In a 500𝜇L reaction vessel, 50𝜇L of areca
2.3.ProteinIdentificationbyMALDI-TOF-TOFMS. Thepro- nut extract was mixed with 5𝜇L of H2O, H2O2(0.5M), or
teinbandontheSDS-PAGEgelwasexcised,followedbyin- guaiacol (2M), in 0.1M citrate-phosphate buffer (pH 5.5).
geldigestionandpeptideextractionaccordingtotheprotocol Theabsorbanceofthewholemixturewasthenmeasuredat
described previously [8]. MS spectra were acquired with 380∼600nm.
Evidence-BasedComplementaryandAlternativeMedicine 3
AnAtlantisT3C18column(5𝜇m,2.1×150mm)(Waters 470nm using guaiacol as the substrate in the presence of
Corp.,Milford,MA)wasusedtoanalyzetheabovearecanut hydrogenperoxide(Figure2(d)).
extractusingalineargradientataflowrateof0.25mL/min.
The mobile phases consisted of ddH2O (solvent A) and 3.2. Dimerization and Glycosylation of AN Peroxidase.
acetonitrile(solventB),bothofwhichcontained0.1%formic
When performing peroxidase zymography, unexpectedly
acid (v/v). HPLC separation was performed on a Dionex
slow migration of active peroxidase was observed (Fig-
Ultimate 3000 HPLC system (Thermo Fisher Scientific,
ure 3(a)), but it showed much higher activity at a higher
Waltham,MA)equippedwithapump(DGP3600M)andan
pH(2inFigure3(a)).ComparedtotheCoomassiestaining
autosampler (WPS-3000T). An ion trap mass spectrometer
pattern,itclearlyshowedthatloweractivityatneutralpHwas
withanESIsource(HCTultraPTMDiscoverySystem,Bruker
the result of nearly complete dissociation of the peroxidase
Daltonics,Inc.)wasusedforLC-MSfullscanmeasurements
subunitsintoinactivemonomers(1inFigure3(b)),whereas
andLC-MS/MSexperiments.TheESIsourcewasoperatedin
theinteractionbetweensubunitswasstableathigherpH(2
positiveionmodewithnitrogenasanebulizing(50psi)and
in Figure 3(b)). Therefore, the two identical subunits were
∘
dryinggas(10L/min,350 C).MSandMS/MSweredonein
noncovalently associated to form an intact AN peroxidase
ultrascanmodeinthemassrangesm/z100–800andm/z50–
withfullactivity.
1000,respectively.
AN peroxidase was structurally similar to RPT peroxi-
dase,whichhasnineN-glycosylationsites[9].Thesmearing
patterninthegelofANperoxidasewasmostlikelythecon-
2.7. Oxidation of Catechin, Epicatechin, and Procyanidin B1
sequence of high glycosylation. To confirm this hypothesis,
by AN Peroxidase. The following oxidation reaction was
ANperoxidasewasdigestedtoformproductswithpartialor
performedin0.1Mcitrate-phosphatebuffer(pH5.5)atroom
complete removal of N-linked oligosaccharides through 24
temperature.Ina200𝜇Lreactionvessel,finalconcentrations
and48htreatmentswithPNGaseF(2and3inFigure3(c)).
of 2mM H2O2 and 0.5mM catechin or epicatechin were Two separate tryptic peptides in the intermediate and ung-
mixedwithANperoxidaseforwavelengthandtimescanning
lycosylated products were identified (Figure 3(d)) with the
andHPLCanalysismonitoredat280or475nm.ALiChro-
same sequences as those in Figures 2(a) and 2(b). Clearly,
CARTcolumn(4×250mm)(Merck&Co.,Inc.,Whitehouse
different extents ofAN peroxidase glycosylation resulted in
Station,NJ)wasusedforseparationwithalineargradientat
a broad spectrum of molecular masses, especially because
a flow rate of 1mL/min. The above mobile phases (ddH2O theglycanmoietywasestimatedtobemorethanhalfofthe
and acetonitrile) were used; however, no formic acid was
glycoprotein.
added.Foroxidationanalysisofthethirdsubstrate,0.1mM
procyanidinB1wasusedinthepresenceorabsenceof0.2mM
3.3.Catechin,Epicatechin,andProcyanidinB1AreArecaNut
catechinorepicatechin.
Peroxidase Substrates. When hydrogen peroxide was added
tothecolorlessextractofthearecanut,aslowcolorchange
from yellow to orange was observed, whereas addition of
3.Results
guaiacol or water had no such effect. Wavelength scanning
indicatedamaximumabsorbanceforthemixtureat440nm
3.1. Identification of Peroxidase as the Major Protein Com-
(Figure4(a)),andtheabsorbanceincreasesatthesamewave-
ponent in the Areca Nut. As illustrated in Figure 1(a), SDS-
length were similar to those observed in the time scanning
PAGE analysis revealed a major protein product with a
molecular mass of ∼55kD in areca nut extract. A brown- (Figure4(b)).ComparisonoftheLCchromatogramsclearly
indicated that the differences in the two components were
colored protein was eluted early at a low concentration
completely removed in the presence of hydrogen peroxide
ofNaClwhenperformingDEAE-cellulosechromatography.
(lowerpanelinFigure4(c)).Thesetwocandidatesubstrates
The colored eluent consisted of the major protein product
elutedat7.4and8.3minandshowedmolecularmassesof578
characterized by severe smearing in the SDS-PAGE gel
and290Da,respectively(upperpanelinFigure4(c)).
(Figure 1(b)). The homogeneity of the purified protein was
Furthermore,theMS/MSspectrumofthesubstratewitha
further confirmed using PMF to analyze the upper and
290Damolecularmass(Figure5(a))showedittobecatechin
lowerbandsinthegel(Figure1(b)).Thesamepatternswere
and epicatechin, both of which displayed indistinguishable
obtained for both bands (Figure 1(c)). The colored protein
spectra(Figures5(b)and5(c)).Ontheotherhand,fragment
exhibitedamaximumabsorbancenear405nm(Figure1(d)),
ionsintheMS/MSspectrumofthesubstratewitha578Da
andMALDI-TOFanalysisalsodisplayedasmearingpattern
molecular mass (Figure 5(d)) were very similar to those of
witha49kDmolecularmass(Figure1(e)).
procyanidinB1(Figure5(e)).Thesethreecomponentswere
MALDI-TOF/TOF mass spectrometric analysis led to
recognizedasthenaturalsubstratesforANperoxidase.The
the identification of two tryptic peptides with molecular
structuresofcatechin,epicatechin,andprocyanidinB1were
masses of 2308.131 and 1248.57Da (Figure 1(c)). Sequences
showninFigure5(f).
of bothtrypticpeptides (Figures2(a) and 2(b)) were found
to be identical with the amino terminus region of royal
palmtree(RPT)peroxidase,asshownbytheunderliningin 3.4.OxidationofCatechinandEpicatechinbyANPeroxidase.
Figure 2(c). Peroxidase activity of the purified protein was Whenperformingtheoxidationofcatechinandepicatechin
further illustrated by measuring an absorbance increase at with hydrogen peroxide, a color change was immediately
4 Evidence-BasedComplementaryandAlternativeMedicine
M AN M 3 4 5 6 7 8 9 10 11
170
170 130
130
100
100
70 70
55 55
40 40
35
35
25
25
(a) (b)
×104 PMF
2308.131 Upper band
y
nsit 1
nte 1248.558 1632.853 2878.388
I
2131.031
0
×104 Lower band
2308.147
y 1.0
nsit
e
nt 0.5
I
1248.570 1632.864 2878.413 3225.814
0.0
1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500
m/z
(c)
0.2
3000 [M+H]+
48997.1
e
banc nsity 2000
Absor Inte [M+H]2+
1000
24452.7
0 0
400 440 480 520 20000 30000 40000 50000 60000 70000 80000 90000
Wavelength (nm) m/z
(d) (e)
Figure1:Purificationofthemajorproteincomponentinthearecanut.(a)SDS-PAGEanalysisoftheconcentratedextract.M,prestained
protein marker with the molecular weights indicated on the left. AN, areca nut extract. (b) SDS-PAGE analysis of the collected brown-
coloredfractionsfromDEAE-cellulosechromatography.Thecollectedfractionnumberisindicatedatthetop.Theupperandlowerbands
werebracketedforidentificationpurposes.(c)PMFfortheupperandlowerbands.Themolecularmassofeachtrypticpeptideisindicated
atthetop.(d)Wavelengthscanningofthepurifiedbrown-coloredprotein.(e)Determinationofthemolecularmassforthepurifiedprotein
byMALDI-TOF.
Evidence-BasedComplementaryandAlternativeMedicine 5
×104
y20 y19 y18 y17 y16 y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
y7
2.0 761.421
S A V E A A C P Q T V S C A D I L A F A A R
b4 b5 b6 b7 b10 b11
1.5
u.) 457b.5981 b10
a. b7 1015.587
y ( 689.186 b11
ntensit 1.0 b4 y5 876y.4843 1114.501
I 386.934 535.121 y17 y20
y2 y4 y10 y12 1851.069 2149.983
0.5 246.072 y3 464.072 1107.587 1293.662
112.019705y.0174 317.034 529b.6004 648y.2634 947y.5928 119y41.6125 139y4.17335152y21.6431461y9.18569 178y01.0631 192y21.8056205y11.1927 2200.43523[M08+.1H3]1+
0.0
250 500 750 1000 1250 1500 1750 2000 2250
m/z
(a)
×104 y8 y7 y6 y5 y4 y3 y2 y1
y4
1.2
581.329
M H F H D C F V R
1.0 b6 b2 b3 b4 b5 b6 b7 b8
y3 828.380
y2 421.260
u.) 0.8 274.179
a.
nsity ( 0.6 b3 668b.3501 980y.5745
nte 416.183
I 0.4 b2 b4 b7
0.2 110.110 175y.1139 241.122679.113708.094 385.142 532.525336.277 696.y3566 833.y1649 975.402 b8117y78.549 1[2M48+.H57]0+
72.097 332.102 450.196 631.325 737.352 1074.499
0.0
200 400 600 800 1000 1200
m/z
(b)
0.6
m
n
0
7
4
at
e
c
1 DLQIGFYNTSCPTAESLVQQAVAAAFANNS an
b
31 GIAPGLIRMHFHDCFVRGCDASVLLDSTAN bsor
A
61 NTAEKDAIPNNPSLRGFEVITAAKSAVEAA
91 CPQTVSCADILAFAARDSANLAGNITYQVP 0
40 80 120 160 200
Time (s)
(c) (d)
Figure2:Identificationofperoxidaseasthemajorproteincomponent.(a)and(b)MS/MSanalysisoftwotrypticpeptideswiththesequences
atthetopofeachpanel.(c)SequencealignmentoftwotrypticpeptideswiththeN-terminal120residuesofRPTperoxidase.Thesequence
identityis underlined,and thenumbersonthe leftindicate theresiduenumberingofRPTperoxidase. (d)Spectrophotometric assayof
peroxidaseactivityforthepurifiedprotein.
6 Evidence-BasedComplementaryandAlternativeMedicine
1 2 1 2 1 2 3 4
130
170 100
170
70
130 130
55 Purified peroxidase
100
100
40
Intermediate
70
70
35 PNGase F
55
55
25
40
Unglycosylated
40
peroxidase
35
35 15
(a) (b) (c)
×104
Intermediate 2878.388
1.0
nsity 1194.615 1632.861 1856.901 2308.130
nte 0.5 VEAACPQTVSCADILAFAAR
I 1063.316 1994.001
1475.787 2705.177 3026.366
0.0
×104
1248.562 Unglycosylatedperoxidase
y MHFHDCFVR
nsit 1.0
Inte 0.5 1080.550 1994.000 2308.126
1475.788 1765.757 2705.177
0.0
1000 1250 1500 1750 2000 2250 2500 2750 3000
m/z
(d)
Figure3:DimerizationandglycosylationoftheintactANperoxidase.(a)Zymogramassayand(b)Coomassiestaining.Theenzymewas
desaltedwithTris/HClbuffer(20mM,pH7)(1)andHEPESbuffer(20mM,pH7.9)(2).(c)TreatmentofdenaturedANperoxidasewith
PNGaseFfor24(2)and48h(3).Thedigestedproducts,accompaniedbytheuntreatedcontrol(1)andPNGaseFitself(4),areindicated
byarrowheadsattheright.(d)PMFforthepartiallyglycosylatedintermediate(upperpanel)andunglycosylatedform(lowerpanel).Two
trypticpeptidessubjectedtoanalysisareindicatedbyarrowheadswiththesequencesshownineachpanel.
observed after AN peroxidase addition. The spectra of conditions,thesubstrateswerespontaneouslyoxidized,and
wavelength scanning for both reactions were similar to ANperoxidaseacceleratedtheoxidationprocess.
thatobservedforthearecanutextract(Figure4(a)).HPLC
examinationoftheoxidationreactionsrevealedthatcatechin
3.5. Occurrence of Procyanidin B1 Oxidation in the Presence
and epicatechin were oxidized to a major product with a
of Catechin or Epicatechin. Unlike catechin or epicatechin,
distinct retention time (Figures 6(a) and 6(b)). The major
therewasnocolorchangeorspectrophotometricabsorbance
oxidizedproducts,withthesamemolecularmassof576Da
increase when using procyanidin B1 alone as a substrate.
for both substrates, were undoubtedly the consequence
of condensation of two catechin or epicatechin molecules HPLC examination also indicated the lack of oxidation of
with the concomitant removal of four hydrogen atoms. procyanidin B1 alone (data not shown). The most striking
While chewing the areca nut along with the Piper betle featureofprocyanidinB1oxidationwasthatitoccurredonly
leaf in slaked lime in the oral cavity, the pH value of the in the presence of catechin or epicatechin. In contrast to
collected oral extracts usually fell between 8 and 9, in our spectra from conditions without enzyme (Figures 7(a) and
experience.ANperoxidasestillretainedtheabilitytooxidize 7(c)),theHPLCchromatogramsclearlyindicatedprocyani-
catechinandepicatechinatpH10,althoughtoalesserextent din B1 oxidation with the formation of additional products
than at neutral pH (Figures 6(c) and 6(d)). Under these (Figures7(b)and7(d)).Inadditiontotheabove-mentioned
Evidence-BasedComplementaryandAlternativeMedicine 7
0.4 0.45
H2O2 m
n
0
e 4
c 4
an at
Absorb Guaiacol orbance
H2O Abs
0 0
400 500 600 400 800 1200
Wavelength (nm) Time (s)
(a) (b)
×107
1.5 NoH2O2
nsity 1.0 579[M+H]+ 290.97[M+H]+
e
nt
I 0.5
×107
1.5 +H2O2
y
nsit1.0
e
nt
I
0.5
7.0 7.5 8.0 8.5 9.0 9.5 10.0
Time (min)
(c)
Figure4:Detectionofthenaturalsubstrate(s)inthearecanut.(a)Wavelengthscanningbetween380and600nmaftermixingtheAN
extractwithhydrogenperoxide,guaiacol,orwater.(b)Measurementofabsorbanceincreaseat440nmfortheANextractinthepresenceof
hydrogenperoxide.(c)LC/MSanalysisoftheANextractinthepresenceofwater(upperpanel)orhydrogenperoxide(lowerpanel).
catechin homodimer, a product with an 864Da molecular hemegroupwasshowntoexistinRPTperoxidase[10],which
masswasformedinthepresenceofsmallamountsofcatechin has a sequence similar to AN peroxidase (Figure 2(c)). The
(Figure7(b)).Thisproductresultedfromthecondensationof same prosthetic group, most likely resulting in the brown
procyanidinB1withcatechinwiththeconcomitantremoval colorwithamaximumabsorbancenear405nm(Figure1(d)),
offourhydrogenatoms. wasfoundinANperoxidaseandthereforeshouldbeclassi-
Inthepresenceofepicatechin,similarreactionsoccurred, fiedunderthefamilyofhemeperoxidases.
that is, production of an epicatechin homodimer and AN peroxidase existed mainly as an inactive monomer
an epicatechin-procyanidin B1 heterodimer (Figure 7(d)). at neutral pH (Figure 3(b)), while a small increase in pH,
Clearly, procyanidin B1 oxidation occurred through the to 7.9, led to a dramatic structural change in which two
condensationofonemoleculeofcatechinorepicatechinwith identical subunits associated to form a fully active, intact
procyanidin B1 in place of the second molecule of catechin protein(Figures3(a)and3(b)).Suchobservationssuggested
or epicatechin. However, a condensation reaction between thattheactivesiteofANperoxidasewascreatedonlywhen
twomoleculesofprocyanidinB1wasnotobservedineither two subunits were closely associated; that is, the active site
cases.Thus,thefollowingreactionswereindicated:catechin islocatedatthesubunitinterface.Itwasplausiblethatwhen
(or epicatechin) + procyanidin B1 + 2H2O2 → catechin- chewingarecanutintheoralcavity,ANperoxidaseisafully
procyanidinB1dimer(orepicatechin-procyanidinB1dimer) activedimerintheoralextractatalkalinepH.SimilartoRPT
+4H2O. peroxidase, secretory AN peroxidase belongs to the family
of class III plant peroxidases. However, AN peroxidase is
4.Discussion structurallydistinctfrommembersofthisclass,whichareall
knownasmonomericglycoproteins[9].
Mostproteinsinthearecanutwerefoundintraceamounts,as Peroxidasestabilityapparentlydependsonthepresence
determinedbySDS-PAGE,withtheexceptionofperoxidase, ofacarbohydratemoiety,asshowninstudiesoftherecombi-
whichwasidentifiedasthemajorproteincomponentinthe nantprotein[11,12].TheextremestabilityofANperoxidase
experiment. In addition to multiple N-glycosylation sites, a atroomtemperaturewasattributedtothesamefactorinthe
8 Evidence-BasedComplementaryandAlternativeMedicine
×106
3.0 123 139 291[M+H]+
2.5
ensity 21..05
Int 1.0 147 165
0.5 273
0
120 140 160 180 200 220 240 260 280
m/z
(a)
×107
2.0 139 Catechin
1.5
y
ensit 1.0 123 165
nt
I 147
0.5 273
169
0
120 140 160 180 200 220 240 260 280
m/z
(b)
×107
139 Epicatechin
2.0
nsity 1.5 123 165
nte 1.0
I 151
0.5 273
169 249
0
120 140 160 180 200 220 240 260 280
m/z
(c)
×105
8.0 409 579[M+H]+
427
291
6.0
y
ensit 4.0 301
nt 247
I 2.0 271
201 229 439453 543 561
0
200 250 300 350 400 450 500 550 600
m/z
(d)
×107
427 Procyanidin B1
409
1.5 291
y
nsit 1.0
e
Int 0.5 247 275 301
229 453 561
0
200 250 300 350 400 450 500 550 600
m/z
(e)
Figure5:Continued.
Evidence-BasedComplementaryandAlternativeMedicine 9
OH
OH
OH
OH
OH
OH
HO
O
OH
HO O OH
HO
O
OH
OH
OH O
OH HO
OH
OH
OH
OH
(+)-catechin (−)-epicatechin Procyanidin B1
(f)
Figure5:Identificationofthenaturalsubstratesinthearecanut.(a),(b),and(c)ComparisonoftheMS/MSspectrumofthe290Dasubstrate
withthoseofcatechinandepicatechin.(d)and(e)SimilarityoftheMS/MSspectrabetweenthe578DasubstrateandprocyanidinB1.(f)The
structuresofcatechin,epicatechin,andprocyanidinB1.
0.125 0.125
C-C EC-EC
MW576 MW576
S S
F F
U U
A A
0 0
0 15 0 15
Retention time (min) Retention time (min)
(a) (b)
0.15 0.15
Catechin, pH10 Epicatechin, pH10
m
m
n
n 0
0 4
4 4
4nt at ant at Peroxidase
a b
bsorb Peroxidase No enzyme Absor No enzyme
A
0 0
0 100 200 300 0 100 200 300
Time (s) Time (s)
(c) (d)
Figure 6: Oxidation of catechin and epicatechin. (a) and (b) HPLC analysis of AN peroxidase-mediated oxidation of catechin (a) and
epicatechin(b).ThedimerizedproductswiththesamemolecularmassareidentifiedasC-CandEC-EC.(c)and(d)Spectrophotometric
activityassayofANperoxidaseathigherpHusingcatechin(c)andepicatechin(d)assubstrates.Thespontaneousoxidationofbothsubstrates
intheabsenceofenzymeisalsoincluded.
10 Evidence-BasedComplementaryandAlternativeMedicine
0.125 0.125
NNoo eennzzyymmee PPeerrooxxiiddaassee
FS FS BB11--CC
U U MMWW886644
A A
CC--CC
MMWW557766
0 0
0 15 0 15
Retention time (min) Retention time (min)
(a) (b)
0.125 0.125
No enzyme PPeerrooxxiiddaassee
BB11--EECC
MMWW886644
EECC--EECC
FS FS MMMMMMMMMWW557766
U U
A A
0 0
0 15 0 15
Retention time (min) Retention time (min)
(c) (d)
Figure7:ProcyanidinB1oxidationinthepresenceofcatechinandepicatechin.HPLCchromatogramswereobtainedintheabsence((a)and
(c))orpresence((b)and(d))ofANperoxidase.ProductsderivedfromcondensationofprocyanidinB1withcatechinorepicatechinwere
indicatedasB1-C(b)andB1-EC(d),respectively.
PNGaseFdigestionexperiment.Theglycanmoietyalonehad through condensation of an A ring from one unit and a B
amolecularmassgreaterthantheunglycosylatedapoenzyme ringfromtheother[13].Twocatechindimerswerereported
itself(Figure3(c)),suggestingdistributionofglycansacross to be the main products: colored dehydrodicatechin A and
the surface of the whole holoenzyme molecule. The glycan colorlessdehydrodicatechinB4,withmolecularmassesof576
moiety affected not only the enzymatic activity of the AN and578Da,respectively[14,15].Comparisonofthefragment
peroxidasebutalsoitsconformationalrigidity.Withoutheat ions in positive mode revealed obvious differences in the
treatment,ANperoxidaseremainedintact,evenafterlonger MS/MS spectra between the observed catechin dimer (see
exposuretoPNGaseF(datanotshown). Figure S1A in supplementary Material available online at
Peroxidase-mediatedoxidationofcatechinsledtothefor- http://dx.doi.org/10.1155/2013/412851)anddehydrodicatechin
mationofproductswithdifferentdegreesofpolymerization A(FigureS1C).