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(1)博士学位. 曇△,. 面冊. StudiesonAmylaseandProteinase`Inhibitors inPlantFoodstuff. 吉. 川. 秀. 樹. 文.

(2) DoctoralDissertation. StudiesonAmylaseandProteinaseInhibitors inPlantFoodstuffs. HidekiYoshikawa. September,20flO. KyotoKokaWomen'sUniversity CollegeofHumanEcology (Advisor:Prof.ToshioMitsunaga). SubmittedtotheGraduateSchool,KinkiUniversity,tofulfilltherequirement fortheDoctorateDegree..

(3) (英文題目) StudiesonAmyiaseandProteinaseInhibitors inPlantFoodstuffs. HidekiYoshikawa. September,2000. KyotoKokaWomen'sUniversity CollegeofHumanEcology (Advisor:Prof.ToshioMitsunaga). (和文題目) 植物性食品中のアミラーゼおよびプロテイナーゼインヒビターに関する研究. 光華女子大学短期大学部吉川秀樹 (主査:光永俊郎教授). SubmittedtotheGraduateSchool,KinkiUniversity,tofulfilltherequirement fortheDoctorateDegree..

(4) ABBREVIATIONS. BAPA. α 一ハTbenzoyl-D,L-arginineづ. BTPA. a-N-benzoyl-r.-tyrosinep-nitroanilideHCl. BTI. trypsininhibitorfrombroccoli. Bz-L-ArgpNA. α 一N-benzoyl一. Bz-Pro-Phe-ArgpNA. α 一N-benzoyl-L-prolyl-L-phenylalanyl-L-argininep-nitroanilide. ρ一nitloanilideHCl. レargininep-nitroanilideHC1. HCl. CBAI. cx-amylaseinhibitorfromkidneybeancultivarcranberry. CHD. 1,2-cyclohexanedione. HPLC. highperfomanceliquidchromatography. HSA. humansalivarya-amylase. KAI. cx-amylaseinhibitorfromkidneybeancultivarkintoki. NBS. N-bromosuccinimide. NPGB. p-nitrophenylp'-guanidinobenzoateHCl. PAGE. polyacrylamidegelelectrophoresis. PAS PIPES. periodicacid-Schiffsreagent sodiumpiperazine-N,N-bis(2-ethanesulfonicacid). PPA. porcinepancreatica-amylase. SDS. sodiumdodecylsulfate. TAI. a-amylaseinhibitorfromkidneybeancultivartora. TNBS. 2,4,6-trinitrobenzenesulfonicacid.

(5) CONTENTS. INTRODUCTION. CHAPTERI. 1. a-AMYLASEINHIBITORFROMLEGUMES. Sectionl. Distribu#ionofa-AmylaseIrihibi#orinLegumes. Section2. Purificationofα. Section3. InhibitoryPropertiesofKidneyBeana-AmylaseInhibitor. Section4. EffectsofVariousAmylaseSpecies,ChemicalModification. 一AmylaseInhibitor丘omKidneyBeanSeed. 3 10 19. andMetalIonsonInhibitoryActivityofKidneyBean. Section5. CHAPTERII. a-Amylaselnhibitor. 30. StabilityandDigestibilityofKidneyBeana-AmylaseInhibitor. 35. ANTINUTRIENTSINLEGUMES. Sectionl. InvestigationsofAntinutrientsinLegumesImportedforFood. Section2. PurificationandSomePropertiesofTrypsinInhibitorfrom BroadBean. CHAPTERIII. 42. 49. TRYPSININHIBITORFROMBROCCOLI. Sectionl. PurificationofMajorTrypsinInhibitorfromBroccoli. Section2. InhibitoryProperties,StabilityandDigestibilityofBroccoli TrypsinInhibitor. 55. 64. CHAPTERIVCONCLUSION. 73. ACKNOWLEDGMENTS. 77. REFERENCES. 78. PUBLICATIONS. 88.

(6) INTRODUCTION. Humanfoodsourcederivespredominantlyfromplants,fromwhichonlya. relativelysmallproportioncanbeutilizednutritionally.Oftheseplants,maymustbe. processedinsomemeanstoimprovethedigestibilityorremovenaturallyoccurring toxicants.Thegreateffortshasbeenmadeinelucidatingtheseantinutritionalfactorsin. fbodthatmay・constituteahazardtomanorthedomesticatedanimalswhenconsumed.. Onegroupofconstituentsthathasreceivedmuchattentionarethedigestiveenzyme. inhibitors.Theseinhibitorsarewidelydistributedinplants,andincludeintheedible. portionofmanyagronomiccrops(LienerandKakade,1984).Amongtheseenzyme inhibitors,theinhibitorsagainstproteolyticenzymes,particularlyserineproteinaseare. studiedintensively.. SincethesoybeantrypsininhibitorwasisolatedbyKunitzin1946,proteinase. inhibitorsinthevariousplantfoodstuffs,particularlyinseedsofgraminaceaeand. leguminosaehavebeenstudiedbymanyinvestigatorsfromthevariousviewpoints includingdistribution,inhibitorymechanism,physiologicalfunction,structure,. nutritionalsignificance,andclinicalapplication(LienerandKakade,1980;Laskowski. andKato,1980;RackisandGumbm㎜,1981;Ryan,1983;Laskowskiα01.,1983;. GallaherandSchneeman,1986;IkenakaandNorioka,1986;Limtrakuletal.,1993).. TheinhibitorswerealsoisolatedfromsomeseedsofCruciferae(Brassicaceae),i_e.,. radish(Ogawaetal.,1968),rapeseed(FukuzawaandKuroda,1980),kale. (Wilimowslca-Pelc,1985.),andwhitemustard(Menegattietal.,1985),butonlyafew. papersweredealtWltllthoseinvegetativetissues(ChenandMitchell,197). Recently,manyvegetableshavebeenfrequentlyconsumedraworhalf-cookedasa. saladcomponent.°Thereis,however,littleinformationonnature,intake,andnutritional. significanceofproteinaseinhibitorsinthesevegetables.. Ontheotherhand,theexamplesofamylaseinhibitorsarefew,butpotentially. 一1一.

(7) important.Takingintoaccounttheuseofstaplefarmcropsascaloriesourcerather. thanasproteinsource,theeffectofamylaseinhibitoronamylolysisshouldbeclarified. toutilizemoreeffectivelythecropsortoimprovehyperglycemiaorcorpulence.With. respecttonaturallyoccurringProteinaceousα. 一amylaseir血ibitors,Kneenand. Sandstedtreportedin1943thatwater-solubleamylaseinhibitorsoccurredinwheat,. rye,andsomeofsorghumcultivarsandin1945Bowmanreferredtoleguminous. a-amylaseinhibitor.Sincethen,proteinaceousa-amylaseinhibitorshavebeenfound. inavarietyofcerealsandotherplants(Buonocoreetal.,1986).Someofleguminous. cx-amylaseinhibitorshavebeenisolatedfromkidneybean(MarshallandRauda, 1975;PowersandWhitaker,1977a;FrelsandRupnow,1984),andinvestigatedthe. aminoacidsequence(Kasaharaetal.,1996;Nagaguchietal.,1997),butlittleis. knownabouttheirinhibitorymechanismandstructuralfeaturesaswellas. bioavailability.. Thisdoctoraldissertation,dealswiththedistribution,purification,and. characterizationofleguminousa-amylaseinhibitors(ChapterI)andwiththe. investigationofantinutritionalcompounds;tannin,proteinaseinhibitor,andlectinin. legumes(ChapterII).Furthermore,thepresentthesisdealswiththepurificationand. inhibitorypropertyoftrypsininhibitor丘ombroccoli(β70∬ICGIoleracea)ofaleading. Cruciferaevegetable(ChapterIII).. 一2一.

(8) CHAPTERI. Section1. Thedistributionofα. a-AMYLASEINHIBITORFROMLEGUMES. Distributionofa-AmylaseInhibitorinLegumes. 一amylaseir血ibitorinleguminousseedsand止echangesof. a-amylaseinhibitoryactivityinkidneybeansduringgerminationaredescribedinthis. section.. MaterialsandMethods. Materials Theleguminousseedswereobtainedfromamarketandafoodimporter.Porcine pancreatica-amylase,bovinetrypsin,andBAPAwerepurchasedfromSigma ChemicalCo.PIPESwaspurchasedfromWakoPureChemicalIndustries.An a-amylaseinhibitor(CBAI)waspurifiedfromthekidneybean(Phaseolusvulgaris cttltivarcranberry)accordingtothepurificationmethodasdescribedinsection2.. Extractionofa-amylaseinhibitorandassayofitsactivity. Seedswexegroundtopassthrougha60-meshsieve.Theflourwasextractedwith. fivevolumes(V/W)ofwaterfor2handcentrifugedat10,000xgfor30min.The. supernatantwasadjustedtopH4andheatedat70°Cfor15min.Thesolutionwas. clarifiedbycentrifugationandneutralized,andthenusedfora-amylaseinhibitory. assay. Theactivitiesofa-amylaseanditsinhibitorweremeasuredaccordingtothe. iodine-starchmethod.Thereactionmixture(1.5ml)containingadefinedamountof. theenzyme,0.5%solublestarch,50mMNaCI,andsmMCaC12ina20mMPIPES. 一3一.

(9) buffer(pH6.9)wasincubatedat37°Cfor10min.Thereactionwasstoppedby. addingamixture(2.5ml)ofO.08MHCIandO.4Maceticacid,andthenanaliquot. ofthereactionmixturewasaddedtoaniodinesolution(0.005%iodineandO.05%. potassiumiodine)tomeasuretheabsorbanceat660nm.Oneunitofenzymeis definedastheamountcapableofproducingaO.8decreaseintheabsorbanceat660. nmover10min.Theinhibitoryactivitytowarda-amylasewasevaluatedfromthe residualenzymeactivityinthepresenceofthelrlhlbltor.Oneinhibitorunit(IU)is. definedastheamountoftheinhibitorrequiredfor50%inhibitionoflunitof. a-amylase.. ImmunizationofarabbitwithCBAIandImmunoassay. ANewZealandmalerabbitweighingabout3kgwasimmunizedbysubcutaneous. injectionwithawater-in-oilemulsionofCBAI(5mg},pH7.2phosphatebuffer. (1ml)andFreund`scompleteadjuvant(1ml).Aboostershotof2mgofCBAIwas. given2weeksafterthefirstinjection,andaweeklater,theanimalwasbledtocollect theserum.. Doublesimpleimmunodiffusionforqualitativeanalysiswascarriedoutinglat. agarosegelaccordingtothemethodofDuchterlony{1958,1962).Singleradial. immunodiffusiontechnicquewasemployedforquantificationoftheantigen(Mancini. etal.,1965}.Inbrief,6mlofprewarmed1%agaroseinphosphatebuffercontaining. O.5mloftheantiserumwaspouredontoaglassplate(3x10.5cm}andinalittle. while,2mmwideholeswerepunchedinthegelontheplateandfilledwith4ｵlof. the _antigensolutions.Theplatewasthenincubatedinahumidatmosphereforawhole day,andtheareaofholewasdeterminedbydiametermeasurementasafunctionof. theantigenconcentration.Quantificationisbasedontheobservationthat.theareaof. theholehasalinearrelationshiptotheinitial. _concentrationoftheantigenfora. definiteconcentrationoftheantiserum.Precipitinlines.inagarose_gelafter. immunodiffusionweremadevisiblebystainingwithCoomassiebrilliantblue. -4一.

(10) Assayoftrypsininhibitoryactivity TrypsininhibitoryactivitywasdeterminedbythemethoddescribedbyIbukietal (1983)usingBAPAasasul)strate.Theinhibitoryactivitywasevaluatedfromthe residualtrypsinactivityinthepresenceofaninhibitor.. Measurementofproteinconcentration. ProteinwasmeasuredbythemethodofLowryetal.(1951),usingbovineserum albLUninasastandard.. Germination. Thekidneybeans(Phaseolusvzclgariscultivarcranberry)weresoakedinfour. volumesofwaterfor15hatroomtemperature.Theseedssoakedasstatedabovewere. germinatedinthedarkat20-25°Cfor7dayswhilesinglelayeredincontainerslined withseverallayersofwetcheesecloth.Samplesfora-amylaseinhibitoryactivity. measurementswerepreparedfromtheseedsofdifferentstagesofgerminationbythe. samemethodasabove.. ResultsandDiscussion. Distributionofa-Amylaseinhibitorinleguminousseeds. Tablelshowstheoccurrenceofa-amylaseinhibitoryactivityinvariouslegumes.. Thea-amylaseinhibitoryactivitieswerefoundinallkidneybeancultivarsexamined. butnotinotherleguminousseeds.Theseresultsarecontrarytothosefortrypsin. inhibitoryactivitywhichhavebeenfoundinailofthesameastestedhere(Lienerand. Kakade,1980}.. Aqueousextractsfromgkindsofbeancultivars(Table2);3cuitivarsof. Phaseolusvulgaris,2cultivarsofPhaseoluslunatus,2cultivarsofVignaanglaris,and. cultivarsofPsophocarpustetragonolobusandViciafaba,wereexaminedfor -5一. ..

(11) T油. ユ藷1. a-AmylaseinhibitoryactivityinLegumeseeds. .. Inhibitoryactivity. Scientificname. 工egumes. (unit/1皿9-protein). Kidneybeans{13samples). Phaseolicsvzclgaris. White(SouthAfrica)a. 16.4. Greatnorthern(USA}. 31..9. Smallwhite(Thailand). 5.3. Daifultu(Japan). 19.i. Lightred(USA). 1. 33.6. Darkrecl(USA). 28.9. Smallred(USA). 23.9. Pink(USA). 35.5. Kintoki(Japan). 35.3. Pinto{Thailand). 33.0. Cranberry(USA). 56.0. Tora(Japan). 38.5. Uzura(Japan). 55.2. Limabeans(5samples). Plcaseolusluraatus. NDb. Runnerbean. Phaseoluscoccineics. ND. Mungbean. 1)ん α3θ01πSaureus. ND. Adzukibeans(6samples). Vignczangrtilayis. ND. Cowpeas(2samples). Vaga￠. ND. Wingedbegin. P30助0αZ,'加Stet,'α90剛0∂ass. NU. Broadbea;i. 7`磁faba. ND. Gardenpea. 1ゼs初〃zsα'ゴ〃2〃12. ND. Lentil. Lensescu?esata. ND. Chiclepia. Cげ08782ゴ8'ゴ. Soybean. Glycinemax. aKidneybeansweregrowninthe bND二Notdetectable. α3ゴフ2θ72sげ3. countriesgiveninparentheses. .. 一6一. 〃該〃ra. ND ND.

(12) Table2 cttitivar. Samplelegumes Habitat. scientificname. a. cranberrybean. USA. Phaseolusviclgayis. b.. whitekidneybean. SouthAfrica. Phaseolzasviclgaris. C,. piny:bean. USA. d. largeIimabean. USA. Phaseolc`Slc〃2α. e. redbutterbean. Burma. PIzaseolus〃. 'has召olx`S〃. f. kafaebean. SouthAfrica. Vigntzangul'aris. g. redbean. Thailand. Vignaangzclaris. h.. wingedbean. Indonesia. P3ρ. i,. broadbean. China. Viciafaba. π忽 α7ゴ∫ ま鋸S ∫"α∫π∫. ρ乃OCα ゆ π3∫6'/:1,'0如. 西πS. Allleguminousseedswereobtainedfromafoodimporter.. 、⑤ ,. す州. "離. 雛 .. ,、ヤく夕. '1 謙轍④ 聾鱗騰嚢騨"'蟻難講講蕪驚難'雛ワ♪' 、、. が・、;, 、,,陣 ,,fト. ちう. ぎ. 弊`ヨ. F且9.1. とヘウロへもノメマ. 雛撫轟1癒撫ll蹴瓢難懸. 、媛。rr〃㍉冠. Irnrnuno-precipitinreactioninagarosegelbydoublesimplediffusion betweenrabbitanti-CBAIserumandaqueousextractsfromIeguminousseeds. A,antiserum;a∼i,therespectivecultivarscorrespondingtothose listedinTable2。. 一7一.

(13) i㎜. 皿o-reactivityagainstrabbitanti-CBAIse㎜.Amongvariousbeancultivars,only. thoseofPhaseolusvulgariswerefoundtobeimmuno-reactivewith止eantiserumas well(Fig.1).Asinglelineiscompletelyfused,indicatingtheoccurrenceofthe. a-amylaseinhibitoridenticalinantigenicityin3cultivarsofPhaseolusvulgaris.On. theotherhand,theantiserumgavenoprecipitinreactionwithaqueousextractsfrom. anyotherbeans.Kotaruetal.(1992)have. _alsoreportedthattheeachsingleprecipitin. linefbrtheaqueousextractsfrom7kindsof1)haseolus槻. 忽or'5cultivarswas. completelyfusedintothattowardCBAIinirnmuno-reactivity.. Theseresultsindicatethata-amylaseinhibitorisexclusivelyoccurringincultivars. ofPhaseolusvulgaris.PickandWOber(1978)havereportedthatVicia/abaseeds,. nothavinganyinhibitoreffectsonmammaliana-amylase,areinactivein. immuno-reactivitywithrabbitantiserumagainstwhite.kidneybeana-amylase. inhibitor.. Changesina-amylaseandtrypsininhibitoryactivitiesduringgermination. Changesincx-amylaseandtrypsininhibitoryactivitiesduringgerminationupto7. daysareshowninFig.2.Thelevelofa-amylaseinhibitoryactivityincranberrybean. initiallydecreasedandthengraduallyincreased.Itreachedamaximum4daysafter. germination.Asimilarchangeswasobservedforthetrypsininhibitoryactivity.Many otherlegumeshave..been-. _examinedforchangeintrypsininhibitoryactivityduring. germination.Resultssimilartothiswereobtainedfrom-otherplantseeds;wheat (Mitsunagaetal.,1983>andblackmatpe(Tsu7rushiinetcrl.,1979)。Mitsunagaα. (1983)indicatedtheplumulesandradiciesofwheatcontainedtrypsini曲ibitory. activity.Contrary,Nagahiro(198.1)reportedthata-amylaseinhibitoryactivityin. kidneybeansoccurredinseedsbut-notinthegerminatedtissues.. ・. α1,.

(14) . 5 か150 ・r. .二ぢ鵡. ≧0 +」100 三・r .r.. 雲お痒ゆ. 412345G7 Germination(day) 】F萱9.2ChangesofAIandTIactivitiesinkidneybean(cranberry)duringgermination. O,ATactivity;・,TIactivity. Relativeinhibitoryactivitywasexpressedasa percenttothespecificactivity(unit/nユ9-protein) incontrol{Odayofgermination).. 一9一.

(15) Section2. Purificationofa-AmylaseInhibitorfrom KidneyBeanSeed. Intheprevioussection,Kidneybeans(Phaseolusvulgaris)containedlarge amountofa-amylaseinhibitors.Thissectiondealswiththepurifcationofa-amylase inhibitorsfromthreekidneybeanseeds;Phaseolusvulgariscultivars,kintoki,foraand cranberry.. MaterialsandMethods. Materials. KidneybeancultivarkintokiandtorawereproducedinHokkaido,Japan.Kidney. beancultivarcranberIywasproducedinUSAandobtained丘omafbodimporter.. Porcinepancreatica-amylasewaspurchasedfromSigmaChemicalCo.PIPESwas. purchasedfromWakoPureChemicalIndustries.Sephacryl5-200,PBE94,and. polybuffer74wereobtained丘omPharmaciaFineChemicals.Allotherreagentswere ofanalyticalgrade.. Measurementofa-amylaseinhibitoryactivityandproteinconcentration a-Amylaseinhibitoryactivityandpxoteinweremeasuredbytheiodine-starch methodandthemethodofLowryetal.(1951)describedinsection1.. Electrophoreticanalysis PAGEwasdonebythemethodofDavis(1964},using7,5%acrylamidegelatpH. 9.4.SDS-PAGEwasdonebythemethodofWeberandOsborn(1969),using12.5%. acrylamidegel.Allthesamplesforelectrophoresiswereheatedat100°Cfor3minin. 2%SDS.ThegelswerestainedforproteinwithCoomassieBrilliantBlueR-250and. 一10一.

(16) forcarbohydratewithPAS(Fairbanksetal.,1971).. Evaluationofthemolecularweightand-carbohydratecontent ThemolecularweightoftheinhibitorwasevaluatedbySDS-PAGE.andgel filtrationinaSephacryl5=200column(2.6x95cm}.The.-markerproteinsusedwere bovineserumalbumin(MW67,000),ovalbumin{MW43,000),bovine chymotrypsinogenA(MW25,000),andribonucleaseA(MW13,700).The carbohydratecontentwasdeterminedaccordingtothephenol-sulfuricacidmethodof Duboisetal.(1956),glucose-beingusedasthestandard.. Chromatofocusing α 一Amylaseinhibitorwasdissolvedina25mMpiperadine-HCIbuffer(pH5.6),. andputintoacolumn(1.Ox30cm)ofPBE94thathadbeenequilibratedwiththe. samebuffer.TheinhibitorwaselutedbyaPolybuffer74solution(diluted10times,. pH4.0).Theactivefractionswerecollected,dialysedagainstH20,andlyophilized.To removethePolybuffer74,thislyophilizedmaterialwaspassedthroughacolumnof. SephadexG-75(2.6x40cm)thathadbeenequilibratedwitha1%NaCIsolution,. dialysedagainstHZO,andlyophilized.. Aminoacidanalysis. Theproteinwashydrolyzedinsealedevacuatedtubesat110°Cfor24hwith6N. HC1.TheaminoacidanalysiswasconductedwithaHitachi835autoanalyzer.Cystine. andmethioninewereassayedinasampleoxidizedwithperformicacidbythemethod. ofMoore(1963).. ResultsandDiscussion. 一11一.

(17) Purificationofα. 一amylaseinhibitor. FinelygroundbeanwassuspendedinH20(1:5,W八T),andthesuspensionstirred. for2hatroomtemperature.Thesupernatantobtainedbycentrifugation{10,000xg. forlh)wasadjustedtopH4with1MHCI,heatedat70°Cfor15min,andrapidly cooled.Aftercentxifugation,thesupernatantwasneutralizedwith1MNaOHand. madeupto40%ethanolbyaddingof95%ethanolat4°Coveraperiodof30min.. After3hofstirringat4°C,themixturewascentrifugedat10,000xgforlh.The supernatantwasadjustedtoan84%ethanolconcentrationandstirredfor3hat4°C.. Aftercentrifugation(10,000xgforlh),theprecipitatewasdissolvedinHzO. ,. dialyzedagainstH20,andlyophilized.Thelyophilizedmaterialwasdissolvedina50. mMphosphatebuffer(pH7.0),andputintoacolumn(2.6x40cm}of. DEAE-cellulosethathadbeenequilibratedwiththesamebuffer.Afterelutingofthe. unadsorbedmaterialwiththestartingbuffer,theinhibitorwaselutedwithalinear. gradientofOtoO.4MNaClinthestartingbuffer(Fig.3).Theactivefractionswere collected,dialyzedagainstH20,andlyophilized.Thislyophilizedmaterialwas. dissolvedina50mMTris-HCIbuffer(pH7.5)containingO.1MNaCIand. chromatographedinaSephacryl5-200column(2.6x95cm}thatwaselutedwiththe. samebuffer{Fig.4).Theinhibitorfractionswere.collected,dialyzedagainstHzO,and. lyophilized. Thepuri丘cationofα. 一amylaseinhibitor丘omkidneybeancultivarkintoki.is. summarizedinTable3.A110mgamountofthepurifiedinhibitorwasobtainedfrom. fihebeanflour(100_g).Thespecificacfiivitywas. .increased11-fold..Theyieldofthe. inhibitorfromkidneybeancultivarcranberry. .wasabout2-foldthatfrom.kidneybean. cultivarkintokiandcultivartors.Thefinalpreparationswereexaminedfor. homogeneitybyusingelectrophoreticanalysis,andeachinhibitorwasfoundtogivea. singleproteinbandaspresentedinFig.5.. 一12一.

(18) (E[ 虞0 0DC℃} ω Uロー0ρh︻0 鋤∩︻. G501001500 FractionNo.(7m1/tube}. F且9・31・n・x・h・agech・. ・m・t・9・aphy。f40-80%. ethanolfractiononaDEAE-cellulose column(2.6x40cm) Thefractionsrepresented.bythebarwerecollected,dialyzedandlyophilized.pAbsorbanceat 280anm,・inhibition,一. 一一一NaCIconcentration.. lGO. (卵 V. 宥 qO。o巴. 2.0. ⊆0回紗 4 '勲引4口 H. Φo鶴偲漁﹂o㎝. 1.0. 09050607080900 E'ractiortNo.(4.3m1/tube) F且9・4G・1員lt・ati・n・fth・inhibit…ep・,at・d byDEAE.cellulosechmmatographyona Sephacryl5-200column(2.6x95cm} Thefractionsrepresentedbythebax・werecollected,pAbsorbanceat280nm,rinhibition.. 'Table3 Step. Purificationofcr-amylaseinhibitorfromTiintokibean. Totalactivity (TU). Protein (mg). Speci丑cactivity. Y三cld. (IU/mg). c≫. Ratio. Heattreatment. 74,800. 量00. 1.0. Ethε 亀nolfユ・action. G5,900. 370. 17B. 88. 7.0. D);AE-cellulose. 39,600. 186. 213. 53. Sephacryi5-200. 31,400. 110. 2B5. 42. 2,952. *OneinhibitorunitisdefinedIStheamountofinhibitor・requiredfor50夕6三nhibitiollofa portionofcr-amylasethatproducedaO.$decreaseintheabsorbanceat6GOnmforIOmin at37°C.. 一13一. 15.3. !1.4 11.

(19) B. A. A. B. 翻糊. C i. 、f. (5-2)PAGEandSDS-PAGEofTAI.. (5-1)PAGEandSDS-PAGEofKAI.. AB. II. :. o==1∼ ＼c、!. .,l. 幽 i・. (5-3)PAGEandSDS-PAGEofCBAI.. Fig.5PAGEandSDS-PAGEofa-amylaseinhibitorsfromPhaseolusvulgaris. (5-1),KAI;(5-2),TAI;(5-3),CBAI. 一14一. a 1 0. 礁磯と∠b 嚇rへ＼. . 灘. 麟髄. a.

(20) Molecularweightandcarbohydratecontent ByitselutionvolumefromtheSephacryl5-200column,theapparentmolecuar. weightsoftheinhibitorswerecalculatedtobe45,000forCBAI,45,000forKAIand. 40,000forTAI,respectively.UponSDS-PAGE(Fig.S),CBAIandKAIdissociated. threekindsofsubunitscorrespondingtomolecularweightsof16,000-18,000(a),. 14,000-16,000(b)and11,000-13,000(c}inthepresenceorabsenceof. 2-mercaptoethanol.Althoughthesubunit(a)and(b)ofKAIwaspositivetoSchiff stainingwithPAS,thecarbohydratechainofCBAIwasexclusivelylocalizedinone. (a)ofthesubunits.Ontheotherhand,TAIdissociatedtwokindsofsubunits correspondingtomolecularweightsof16,000(a)and14,000(b}.Bothsubunitswere. positivetosugar-stainingwi血PAS。Theseα. 一㎜ylaseinhibitorshadnocysteinyl. residuesTable4),soitcanbeconcludedthateachsubunitwasnotlinkedby. disulfidebonds.. Thea-amylaseinhibitorfromwhitekidneybeancomprisesfouridenticalsubunits. withamolecularweightof11,000(PickandWober,1978),whiletheredkidneybean. inhibitoriscomposedoffoursubunitsofthreedifferenttypeswithmolecularweights. of11,000-12,000,12,000-15,000and15,000-17,000(PowersandWhitaker,1977a).. Kasaharaetal.(1996)observedthatthewhitekidneybeana-amylaseinhibitor. (heat-stabletype)wascomposedoftwokindsofsubunitswithmolecularweightsof 10,000(a-subunit)and14,600(f3-subunit),andthenativeinhibitorhadthe tetramericstructure,az,QZ.Theseresultssuggestthatthekidneybeana-amylase. inhibitorsinthisstudyprobablywerecomposedoftwosubunits,{a}and{b),and. hadatetramericcomplex.However,thecalculatedmolecularweightofthenative. inhibitor(TAI)wasabout60,000,beinggreatlydifferentfromtheresult(MW. 40,000}obtainedbygelfiltration.Thisdifferenceinmolecularweightrequiresfurther. investigation,becauseglycopolypeptide-SDScomplexescanyieldabnormallyhigh. molecularweightestimates(Yamaguchi,1991). Ananalysisofthecarbohydratecontentbythephenol-sulfuricacidmethod -15一.

(21) indicatedthatCBAI,KAIandTAIcontained14%,15%and9.1%carbohydrate,. respectively.a-Amylaseinhibitorsisolatedfromotherbeanshavemolecular. _weights. ranging丘om20,000to60,000,andthesecontained7.5-15%carbohydrate(Hoand Whitaker,1993).. IsoelectricseparationofTAI InthechromatofocusingofTAI(Fig.6),twoinhibitorpeakswereelutedatpH 4.70and4.60,thesebeingdesignatedasTAI-IandTAI-II,respectively.Thistechnique resultedinarecoveredactivityof47%forTAI-Iand30%forTAI-IIcomparedwith theoriginalactivity.Thisrelativelyhighrecoveryofactivityleadsustobelievethat theseiso-inhibitorsdidnotresultfromanyartificialproductinthepurificationprocess. BothinhibitorswereexaminedforhomogeneitybyusingPAGE,andeachinhibitor showedasingleproteinbandaspresentedinFig.7.Themolecularweightofboth inhibitorswasthesame,beingcalculatedas40,000bySephacryl5-200gelfiltration.. o.s. 5.0 0.4 100. 50. S 月醤. 0.2. 45. 40. 0 080120160200 Elutionvolume(ml) F量9。6ChromatofocusingpatternforTAI. ●,abs{}rbanceat2801m1;△,α 一一 … …. 一amylaseinhibitoryad,重vil'y;. ,pH.TAIwasapphedl;oa`ヱdllmn(1.〔. 〕x30cm)of. PBE94tllathadbeenequilibraLedwlthLL25mMpipera(lille-HCIbuffer(pH5.6),Theinhibitorwaselutedbya Polybuffer74sohltioll((liluted10・ fractiollsI℃. しimes,pH4.0).The. 」>rc}se1U,edbythel)arswerecollected.. -16一. 賦.

(22) 9難. 願. abc Fig.7PAGEofTAI-IandTAI-II. Lanea,TAI-1;laneb,TAI-II;lanec,TAI beforechromatofocusing.. Ontheotherhand,thechromatofocusingpatternsofCBAIandKAIshowedthesingle proteinpeakcorrespondingtotheisoelectricpointsof4.68and4.55,respectively. a-Amylaseinhibitorsisolatedfromotherbeanshavetheisoelectricpointsof4.65for redkidneybean(PowersandWhitaker,1977a)and4.35forblackkidneybean (LajoloandFilho,1985).. Aminoacidanalysis. TheaminoacidcompositionsofCBAI,KAI,TAI-1,andTAI-IIaregiveninTable. 4.Theseinhibitorscontainedlargeamountsofasparticacid,serine,valine,threonine,. andglutamicacid,butitdidnotcontaincystine.Theaminoacidcompositionsofthese. inhibitorsaresimilartothatreportedforthewhitekidneybeaninhibitor(Hoand. Whitaker,1993).Theyhadnocystine,inspiteofitspresenceinredandblackkidney. beaninhibitors,whiletheyhadprolinewhichwasnotcontainedintheredkidneybean. inhibitor(PowersandWhitaker,1977a;LajoloandFilho,1985).FrelsandRupnow. (1984)havereportedthattwoiso-inhibitorsinblackbeanhadasimilaraminoacid compositionandwererichinasparticacid,serine,glutamicacid,valine,andthreonine. -17一.

(23) NonoticeabledifferenceintheaminoacidcompositionbetweenTAI-IandTAI-IIwas. alsoapparent.Thedifferenceinaminoacidcontentbetweentheinhibitorswas. estimatedtobeabout4%ofthetotalaminoacidresidues,sothedifferenceofpI. betweentheinhibitorswouldhavebeensmall.. Table4Aminoacidcomposition,molecularweight,carbohydrate content,andpIofα. aminoacid. 一amylaseinhibitorfromPhaseolusvulgaris. CBAI. KAI. Asx. 17.5. 16.6. 16.5. 16.i. Thr. s.a. 9.0. 8.7. 8.9. Ser. 14.1. 14.6. 13.4. 14.1. Glx. 7.6. 8.5. 7,8. 8.2. Pro. 2.3. 2.3. 2.9. z.s. Gly. 4.8. 4.5. 4.7. 4.7. Ala. 5.9. 5.9. 5.2. 5.5. 1/2Cys. 0. 0. 0. 0. Val. Q.. s.s. 9.9. 9.4. Met. o.8. o.s. 1.0. 0.9. 口e. 4.5. 4.5. 4.9. 5.1. Leu. 5.1. 5.1. 5.9. 5.7. Tyr. 3.7. 3.7. 5.0. 4.$. Phe. 5.6. 5.6. 5.7. 5.5. Lys. 4.8. 5.1. 4.8. 4.7. His. 1.4. 1.4. 1.3. 1.3. Arg. 2.8. 2.8. 2.9. 2.8. Trp. nd*. nd. nd. nd. 丁Al-1. TA1一!1. t%ofresidues/mo1). molecularweight. carbohydratecontent(%). 45,000. 45,000. 40,000. 40,000. ー P. i4. 15. 9.4. 8.9. 4.7. 4.5. 4.7. 4.6. CBAI,cultivarcranberry;KAI,cultivarkintoki;TAI,cultivartoga. *notdetermined. -18一.

(24) Section3. InhibitoryPropertiesofKidneyBean a-AmylaseInhibifior. Thea-amylaseinhibitorsisolatedfromkidneybeansinhibitedpancreaticand. sali町. α 一㎜ylaseactivities。Thissectiondealswiththei血ibitoIyactivitiesofkidney. beana-amylaseinhibitoragainstporcinepancreatica-amylaseaswellasamylases frommammalian,plant,andmicrobe.. MaterialsandMethods. Materials Thea-amylaseinhibitors,KAI,TAI,andCBAI,werepurifiedfromkidneybean cultivarkintoki,Loraandcranberry,respectively,accordingtothemethoddescribedin section2.Porcinepancreatica-amylase(typeI-A,DFPtreated},humansalivary a-amylase(typeIX-A),andamylasesfromAspergillusoryzae,Bacillussubtilis, barleymalt,andsweetpotatowerepurchasedfromSigmaChemicalCo.Allother reagentswereofanalyticalgrade.. Assayofa-amylaseinhibitoryactivity a-Amylaseinhibitoryactivitywasmeasuredbytheiodine-starchmethoddescribed. insection1.Forthepurposeofstoichiometricinvestigationontheformationofa. complexbetweena-amylaseandinhibitor,afixedconcentrationofenzyme,and inhibitorwasincubatedtogetherin20mMacetatebuffercontaining50mMNaCIand. smMCaC12(pH5.0)at37°C.Theresidualamylaseactivitywasmeasuredbythe. iodine-starchmethodusing3.0%solublestarchsolutionasasubstrate.. 一19一.

(25) Measurementofinhibitionconstant. Porcinepancreatica-amylaseconcentrationwasheldconstantat1.1x10.8M. whiletheinhibitorconcentrationwasvariedfrom1.7x10-9to1.4x10-$M,andthe enzymeandinhibitorwereincubatedatpH5.Oand37°Cforlh.Aftercomplex. formation,theresidualamylaseactivitywasmeasuredbytheiodine-starchmethod. using3.4%solublestarchsolutionasasubstrate.Theinhibitionconstantwas. determinedfromtheamylaseactivityleftatthevariousconcentrationsoftheinhibitor. asdescribedbyBieth{1974).. Measurementofproteinconcentration ProteinwasmeasuredbythemethodofLowryetal.(1951)describedinsection 1.Themolarconcentrationoftheporcinepancreatica-amylasesolutionwas determinedspectrophotometricallywithanextinctioncoefficientA=24.0(1%,280 nm)andamolecularweightof52,000(Elodietal.,1972).. EffectsofpHandpreincubationtimeonamylaseinhibition. a-Amylase(1unit)wasincubatedat37°Cfor30minwiththeinhibitorin. variousbufferscomposedof20mMcitrate(pH4.0-5.5}or20mMphosphate(pH. b.0-7.0),40mMNaCIand2m1VICaClz.Afterincubation,theresiduala-amylase. activitywasmeasured.Acontrolexperimentwasrunwiththeenzymealoneateach. pHvalue,andthepercentageinhibitionwascalculatedfromthecontrolvs. enzyme-inhibitorreactionateachpHvalue.Formcasurementoftherateofamylase. inhibition,amixtureoftheinhibitoranda-amylasewasincubatedatpHS.Dand. 37°C,andthepercentageinhibitionofaliquotswasmeasuredatdifferenttimes.. Effectsoftemperatureandionicstrengthonamylaseinhibition. Totesttheeffectoftemperature,theinhibitorwasincubatedwithanrx-amylase. (lunit>atatemperature丘om25℃to50℃.Afterpreincubationfbr30minateach -20一.

(26) temperature,residuala-amylaseactivitywasmeasured.Ateachtemperature,the extentofinhibitionwascalculatedbysettingenzymeactivityinthecontrolwithout. inhibitorat144%.Theionicstrengthwasvariedbyadditionofdifferentamountsof. NaCItothereactionmixturein50mMPIPESbuffer(pH6.9}containingsmM. CaCIZ.Followingpreincubationofana-amylase(1unit}andinhibitorfor30minat. 37°C,theresiduala-amylaseactivitywasdeterminedintheusualmanner.Control. wasrunwithenzymealoneateachionicstrengthandpercentinhibitionwascalculated fromthecontrolvs.enzyme-inhibitorreaction.. ResultsandDiscussion. Inhibitoryactivity. TablessummarizestheinhibitoryactivitiesofKAItowardamylasesfromvarious. sources.Theinhibitorwasactiveagainstporcinepancreaticandhumansalivary. amylases,butdidnotinhibitplant,fungalorbacterialamylases.Inrespectofthis. specifcity,thesameresultshavebeenfoundforTAI,CBAIaswellaswhite,red,and. blackkidneybeana-amylaseinhibitors(MarshallandLauda,1975;Powersand. Whitaker,1977a;FrelsandRupnow,1984).Thus,itispresumedthattheα. 一amylase. inhibitorsinPhaseolusvulgarisbeansactneitherasaregulatorofa-amylasefor. starchmetabolismintheplantnorasaprotectoragainstmicrobialattacksontheplant.. Table51nhibitoryactivityofIiintokibcanaamylaseinhibitoronvariousamylases. Amylase. InhiUition(%). a-Aznylase. 0 0 ー. PorcinePancreas. 8 0 Qぜ. Humansaliva Barleymalt. 0. ∠霊sfier'gi〃usO7フzac. 0. Bacillussubtilis -amylase. 0. Barleymalt. 0. Sweetpotato. 一21一.

(27) Figure8showsthetitrationpatternofporcinepancreatica-anriyiasewithKAI. Porcinepancreatica-amylasewasstronglyinhibitedbyKAIandtheinhibitionwas. showntobelinearupto95%inhibition.Basedonthemolecularweightsof52,000for. theenzymeand45,000fortheinhibitor,andassumingthattheenzymewasiOO%. activeaswellas100%activeinhibitor,thestoichiometryoftheenzyme-inhibitor. complexwasconcludedtobea1:1molarratio.Eacha-amylaseinhibitorisolated 丘omredkidneybean(PowersandWhitaker,1977b)and丘omwhitekidneybean. (MarshallandLauda,1975)alsoformedastoichiometriccomplexof1:1with a-amylase,whiletheinhibitor(1-i)fromblackkidneybeanformeda2:1complex. withtheenzy皿e(FrelsandRupnow,1985).. TheinhibitionconstantoftheKAI-porcinepancreatica-amylasecomplexwas. 1.1x10'1°M(Fig.9).Theinhibitionconstantsfbrredkidneybeanα. 一amylase. inhibitor{PowersandWhitaker,1977b),blackkidneybeana-amylaseinhibitor. {FretsandRupnow,1985),andwhitekidneybeana-amylaseinhibitor{Hoand Whitaker,1993),respectively,withporcinepancreatica-amylasewere3.5x10-"M. (pH6.9,30°C),2.2-3.8x10-9M(pH6.9,30°C),and1.Ox10""M(pH5.4,30°C}.. ThemodeofinhibitiontestedbyLineweaver-Burkplotwasfoundtobe. noncompetitive(Fig.10).ThisresultisagreementwiththoseofMarshallandLauda. (1975)andFrelsandRupnow(1985)whoreportedthatanoncompetitiveinhibition ofporcinepancreaticα. 一amylasebykidneybeanα. contrary,theinhibitorsfromwheat(SaundersandLang,1973},corn{Blanco-Labra. andIturbe-Chinas,1981),andyam(SharmaandPatterbiraman,1982)havebeen. reportedtoinhibita-amylasethroughuncompetitiveinteractions.Itisgenerally. knownthatinanoncompetitiveinhibition,thesubstrateandthelnhlbitoroccupy. differentpointsoftheenzyme.. 22. 一amylaseh血ibitors.Onthe.

(28) Molarratio(」{AI/PPA) 1Fig。S. Stoichiornetryofinhibitionofporcinepancreaticcc-amylase (PPA)byKAI.PPA(1ユ. ×10噌8M}wasincubatedatpH5.Oand. 37°Cfar60minwithKATatvariedconcentrationsof1.7×10-9 Mto1.4×10-8M,andther1,セheresidualα. 一amylaseactivity. wasmeasured.. 1/a Fig。9[. InhibitionconstantofKAI-porcinepancreatica-arnylase complex.ExperimentaldetailsweregiveninFig.8..Thegraph showeddatafromFig.$replottedbythemethodofBieth. `l o'istheinitialKAIconcentrationand`a'is.relativeresidual enzymeactivity.Theslopeofthelinegavetheinhibition constantKi=1.1×10-10M.. 一23一.

(29) 一〇 .5-0.4-0.3-0.2-0.100.iO.20.3 1/S(mg/ml). F且9。10. Lineweave卜Burkplotsforhydrolysisofstarchbyporcinepancreaticu-amylaseinthepresenceandabsenceofKAI.Enzyme thathacibeenpreincubatedfor60minwithorwithoutKAI was-addedtotheassaymediumcontainingdifferentconcentrationsofsolublestarch,50mMNaCIand5mMCaChin20mM acetatebuffer(pH5.0),followedbyreactionat37°Cfor10 min.'S'istheconcentrationofsolublestarch(mg/ml)and `V'istheinitialreactionvelocityexpressedasmilligramsof solublestarchhydrolyzed/miforlminbyporcinepancreatic α一amy至ase(5.0×10-yNI).●,intheabsenceofinhibitor;▲, inthepresenceofKAI(3.5×10-9M).. EffectsofpHandpreincubationtimeonamylaseinhibition. FigurellshowstheeffectsofpHontheinhibitionofporcinepancreatic. a-amylasebyKAI.TheoptimumpHforinhibitionoftheenzymewas5.0.The. optimumpHforinhibitionagainstporcinepancreatica-amylasehasbeenreportedto. beintherangeof4.5to5.5forinhibitorsfrommanybeans(MarshallandLauda, 1975;PowersandWhitaker,1977b;FrelsandRupnow,1985).However,wheat. a-amylaseinhibitorswerethemostinhibitoryatpH6.4-8.4andpH5.8-7.Oagainst humansalivaryandvariouspancreaticα. 一amylases,respectively(0'domeIland. lVlcGeeney,1976}.. Maximuminhibitionofporcinepancreatica-amylasebyKAIrequiredabout30. minofincubationatpH5.0(Fig.12-1).However,themaximumlevelofinhibition. couldnotbeachievedevenafterincubatingfor3hatpH6.9(datanotshown).The. rateofinhibitionofporcinepancreaticcx-amylasebykidneybeana-amylase. inhibitorsatpH6.9wasslowerthanthatatpH5.0.PowersandWhitaker(1977b). 24一.

(30) havereportedthatthemaximuminhibitionbytheredkidneybeaninhibitorrequired. over3.5hincubationatpH6.9,whileitoccurredwithinlhatpH5.5,thisbeingthe. optimumpHforthewhitekidneybeana-amylaseinhibitor(MarshallandLauda,. 1975;HoandWhitaker,1993).Ontheotherhand,theryeinhibitor(Gramm,1978). andtheStreptomycesinhibitor(Araietal.,1985)associatedwithporcinepancreatic a-amylasewithin20minandafteronlyafewminutesofincubation,respectively.. Theinhibitionof1.1x10"8Mporcinepancreatica--amylaseby6.2x10-9MKAI. approached56%(Fig.12-1),thisbeingthetheoreticalpercentageinhibitionwhenthe. enzymeandinhibitorreactwith1:1stoichiometry.AsshowninFig.12-2,Thedegree. ofamylaseinhibitionatpH5.Ooftheinhibitorapproachedamaximumafter30minof incubationforTAI-Iandafter20minofincubationforTAI-II.Thetwoinhibitors(1-Y -25一.

(31) 工00. 50. (ま )わΣ も邸ご￡量. 三 020406084100120. 1ncubationtime(min) 1F且9。丑2一丑Rateofinhibitionofpancreaticα. 一amylasebyKAI. Themixtureofa-amylaseandtheinhibitorwasincubated atpH5.Oand37°C.. 02040GO I血tbationtime(㎞) 圃9・. 且2・2Rateofinhibitionofpancreaticα TAI-IandTAI-II. Themixtureofa-amylaseandtheinhibitorwasincubated atpH5.Oand37°C.・,TAI-1;O,TAI-II.. 一26一. 一amylaseby.

(32) andI-2}fromblackbeanhadsimilaractivityagainstpancreatica-amylase,butthe. maximumamylaseinhibitionrequired12hofincubationforI-1and6hofincubation. forl-2at30°CafipH6.9(FrelsandRupnow,1985).Thecarbohydratecontentsof. theseinhibitorswere7.5%forI-1and9.0%forI-2.Theincubatingtimewhichwas requiredtoproducethemaximumamylaseinhibitioninthepresentworkshowed. TAI-II,thetypewithlowercarbohydratecontent,tobethefasterofthetwoinhibitors.. Thisresultis. ,,contrarytotheobservationbyFrelsandRupnowonthecorrelation. betweentheinhibitoryactivityandcarbohydratecontent,andsothecontributionofthe carbohydrateportiontotheinhibitoryactivitymaybesmallforTAI.. Effectsoftemperatureandionicstrengthonamylaseinhibition Figure13Ashowstheeffectsoftemperatureoninhibitionofhumansalivary a-amylaseandporcinepancreatica-amylasebyKAI.The.activitiesofboth. Temperature(°C)NaCI(M) 】Fig.13Effectsoftemperatureandionicstrengthoninhibitionofporcinepancreatic a-amylase(●)andhumansalivaryα. 一amylase(○)byKAI.A:Aftereachenzyme. (1unit)wasincubatedwithKAIfor30minatpH5.9andtemperaturesranging from?StoSO°C,theresidualinhibitoryactivitywasdetermined.Controlswererun withoutinhibitorateachtemperature.B:Theionicstrengthwasvariedbythe additionofdifferentamountsofNaCItothereactionmixtureofenzyme(]unit) andKAI.AfterthereactionmixturewasincubatedatpH6.9and37°Cfor30min, theresidualinhibitoryactivitywasdeterminedintheusualmanner.Acontrolwas runwithenzymealoneateachionicstrength.. 一27一.

(33) amylasesincreasedinproportiontotheriseintemperaturefrom25to50°C.Therate. ofinhibitionagainstporcinepancreatica-amylasebyKA.Iincreasedwitharisein temperature,buttheinhibitionofhumansalivarya-amylasereachedamaximum. above35°C.Theeffectsofionicstrengthoninhibitionagainsthumansalivary. a-amylaseandporcinepancreatica-amylasebyKAIareshowninFig.13B.An. increaseinNaClconcentration丘omOto15Mcausedanincreaseintheinhibitory activitiesagainstbothamylases.However,thesearnyiaseinhibitoryactivitiestendedto. decreaseabove1.5MNaClconcentration.Thelossofenzymeactivityincontroltube. whichcontainedonlyamylasewaswithin10%evenin2.OMNaClconcentration.. EffectofNaCIoniodine-starchreactionalsowasnotobservedinthisexperimental. COriltiOri.. PowersandWhitaker(1977b)reportedthattherateofcomplexationofporcine. pancreaticcx-amylaseandredkidneybeana-amylaseinhibitoroccurredmore rapidlyat37°Cthan25°CwhenreactionswereperformedatpH6.9.However,. LajoroandFilho(1985)reportedthattheincreaseinactivityoftheinhibitorfrom. blackbeanatpH5.4,beingtheoptimumpHforinhibitionwasnotsomarked,andthe. pHandtemperatureeffectsontheinhibitoryactivitywasrelatedtopH-induced conformationalchangesoftheinhibitor.Althoughtheeffectoftemperatureonthe humansalivaryα. 一amylaseinhibitionbyakidneybeanα. knownwell,Granum(1978}reportedthattherateofinhibitionbyryecx-amylase. inhibitordecreasedattemperatureabove30°C.Theinhibitionofhumansalivary. a-amylasebyKAIreachedamaximumabove35°C,butthedecreaseofinhibitory. activitywasnotobserved.. Ionicstrengthinthereactionmixturehadaprofoundeffectonthereactionofthe. kidneybeana-amylaseinhibitorswithporcine-pancreatica-amylaseorhuman. salivaryα. 一amylase.Inalowionicstrength(∼1.OM),similarresultswerereported. fortheeffectofincreasingionicstrengthontheratesofcomplexformationbetween. kidneybeana-amylaseinhibitoranda-amylase(HoandWhitaker,1993;Powers -28一. 一㎜ylaseinhibitorwasnot.

(34) andWhitaker,1977b;FrelsandRupnow,1985),butaneffectinhighionicstrength. hasnotbeenreported.Inourresults,thea-amylaseinhibitoryactivityofKAI decreasedinahighionicstrength(above1.5MNaC1).Thisindicatesthatacomplex. formationbetweentheinhibitorandenzymeneedssomeionicstrength,butthe. excessiveincreaseofioru.cstrengthperhapsinterferedwiththeirassociationandcaused aLOSSOfirihlbltoryaCtlVlty.PowersandWhitaker(1977b)suggestedthattheeffectof. ionicstrengthcouldbeduetoaneffectonconfbmlationalchangeofthe丘eeenzyme. and/orfreeinhibitorwhichrequiredtoformtheenzyme-inhibitorcomplex.However,a. conformationalchangeoftheinhibitortoformthecomplexwithenzymewillprobably. havegreaterinfluencebecausethelossofenzymeactivityincontroltubeiswithin. 10%eveninahighionicstrength.Ontheotherhand,PowersandWhitaker(1977b). suggestedthattherateofcomplexformationbetweenkidneybeanα. 一amylaseinhibitor. andporcinepancreaticcx-amylasewasveryslow,andsoaconformationalchange. requiredtoformtheenzyme-inhibitorcomplexwastherate-controllingstepinthe. complexation.Ariseoftemperatureinvolvedanactivationenergywillprobably. encouragethisstep.Actually,thesecondorderrateconstantsofbindingofredkidney. beana-amylaseinhibitorwithporcinepancreaticcx-amylasewerereported. 1.1xlOa/M,sec.at37°Cand9.77x102/M,sec.at25.7°C(PowersandWhitaker, 1977b).. _29_.

(35) Section4EffectsofVariousAmylaseSpecies,Chemical ModificationandMetalIonsonInhibitoryActivityof KidneyBeana-AmylaseInhibitor. Thekidneybeanα. 一㎜ylasei血bitorswereactiveonmammalianpancreaticand. salivarya-amylases.Accordingly,thespeciesspecificityoftheinhibitoragainst. pancreaticamylasefromvariousanimalswasdescribedinthissection.Theeffectsof chemicalmodificationandmetalionsoninhibitoryactivityoftheinhibitoralsowere. described.. MaterialsandMethods. Materials. Theα. 一amylaseinhibitor,CBAI,waspurified丘omPhaseolusvulgariscultivar. cranberryaccordingtothemethoddescribedinsection2.Porcinepancreatic. a-amylase(typeI-A,DFPtreated)waspurchasedfromSigmaChemicalCo.. Pancreasfrompig,dog,cat,horse,sheep,cow,rabbit,guineapig,rat,andmousewere alsoobtainedasacefionepowderfiromSigmaChemicalCo.,fromwhicha-amylases. werepurifiedinthisexperimentbyaffinitychromatographywithpotatostarch. accordingtothemethodofTsujisaka(1975).Ailotherreagentswereofanalytical. grade.. Assayofa-amylaseinhibitoryactivity a-Amylaseinhibitoryactivitywasmeasuredbytheiodine-starchmethoddescribed insection1.Forthepurposeofspeciesspecificinvestigationonamylaseinhibition,A fixedconcentrationofinhibitorwasincubatedwithlunitofamylasefromanimal, plant,fungalorbacterialsourcein20mMPIPESbuffer(pH6.4}or50mMacetate. 30.

(36) buffer(pH5.5)for30minat37°C.Theresidualamylaseactivitywasmeasuredby theiodine-starchmethodatpH6.9withα. 一amylase丘omanimalsourceandatpH5.5. withtheamylasesfromtheothersource.. Chemicalmodification TheinhibitorwastreatedwithTNBSaccordingtothemethodofHaynesetal. (1967),withCHDaccordingtothemethodofPatthyandSmith{1975),withNBS accordingtothemethodofSpandeandWitkop(1967)andwithNaBHQ-formaldehyde accordingtotheme#hodofRiceandMeans(1971).Thesemodifiedinhibitors,after beingwelldialyzed,wereassayedfortheirinhibitoryactivities.. ResultsandDiscussion. Inhibitionofvariousamylases. Table6comparestheconcentrationsofCBAItoyield50%inhibitionagainst. pancreatica-amylasesfrommammals.Althoughmammalianpancreatica-amylases correspondingtolunitwereusedateitherpH,theconcentrationofCBAItogive50%. inhibitionwasmuchloweratpH5.5thanatpHb.9formostoftheenzymes.Under. thephysiologicalpHconditions,a-amylasesfrompig,dog,cat,andhorseweremost. effectivelyinhibitedbyCBAI,towhichtheenzymesfromsheep,rabbit,guineapig,. andmousefollowed.Noteworthily,theenzymesfromcowandratwerelittleinhibited. byCBAI.Theseresultssuggestthata-amylaseinhibitorinPhaseolusvulgarisbean. wouldnotalwaysserveasaninhibitorintheintestinalenvironmentsoftheseanimals.. Inrelationtothispossibility,Savaianoetal.(1977)reportedthatadministrationof. redkidneybeancx-amylaseinhibitortoratsdidnotaffecttheirgrowth.Onthe. contrary,MarshallandLauda(1975)describedthathumanpancreatica-amylase wasasmuchinhibitedbywhitekidneybeana-amylaseinhibitorleadstoa. considerabledecreaseintheamylaseactivityremaininginthelumen. -31一.

(37) Table6ReactivityofCBAIwithmammalianpancreaticα. 一amylases. Sourceof. .. AmountofCBAI(nM) requiredfor50"/ . pancreatic a-amylase". inhibition. (pHG.9)(pH5.5). Pig Dog cat Horse Sheep Cow Rabbit Guineapig Rat Mouse. 40 28 22 30 25Q >3,000 160 180 >3,000 350. `Preparationsfrompancreaticacetonepowderwereusedasamyl. 7.3 7.'7 8.9 8.0 6.7 100 9.S 12 27 8.4 ases.. EffectofChemicalmodification. Theinhibitoryactivityofα. 一㎜ylaseir止ibitor丘om、Phaseolusvulgarisbeanwas. affectedbychemicalmodificationwithvariousreagents(Fig.14).Treatmentsofthe. inhibitorwithTNBS,CHD,andNBScausedmoreorlessremarkeddiminutioninthe. activity,whichwasexhaustivelylostwithin3h,whilereductivealkylationoflysyl. s-aminogroupswithformaldehydeandNaBH4wasnotresponsibleforinactivation. duringincubationfor3h.Inaddition,thistreatmenthadnoeffectontheinhibitory. activityevenaftertheincubationperiodofmorethan24h(datanotshown).Wilcox. andWhitaker(1984}reportedthattheoxidationoftryptophanresidueofredkidney. beana-amylaseinhibitorwithNBSledtolossofinhibitoryactivity.Similarresult. wasfoundforwhitekidneybeaninhibitor.However,itisimpropertoemphasizethat. arginineaswellastryptophanaresituatedinthereactivesiteoftheinhibitororits. vicinitybecauseinsomecasesconformationalchangesmayarisefrommodificationof. guanidogroupsorcleavageofindolering.. _32_.

(38) 100. 50. 隅. (a) 0 00 1. (b}. ¶iIl. コ ●. 9⊥. ●. .,. 一一. 一. (d}. (c) 盧. 置1璽. 1量II魯1. Oi230123 hlodification(h) Fig.14EffectsofchemicalmodificationoninhibitoryactivityofCBAI.CBAIwas treatedwithTNBS(a),CHD(b),NBS(c)、andNaBH4十formaldehyde(d), accordingtotheprocedurespreviouslydescribed.Inhibitoryactivitywas measuredagainstPPA(○)andHSA(●).. Effectsofmetalionsandreagents. Afterrespectiveincubationsoftheinhibitor(4.4x10-5MofCBAI)inmetalsalt. solutionsorreagentsolutionsat37°Cfor2and24h,eachmixturewasdiluted. 100-foldwithwaterandtheremaininginhibitoryactivitywasmeasured.Theeffectsof. metalionsandreagentsona-amylaseinhibitorareshowninTable7.Theinhibitor. wasscarcelyinactivatedby2hincubationwiththemetalionsotherthanHgion.. Respective24hincubationswith20mMCuSO4andHgClzresultedin40%and89%. lossoftheoriginalactivity.IncubationoftheinhibitorwithH202asanoxidizing. reagentcaused24%lossoftheinitialactivityafter2hand56%lossafter24h.. However,. ■1 …. 50. (訳) 台 ; コ u価さ B ヨ三 ⊆H. 闇. .reducingreagents,chelatingreagentsandtheothercompoundstestedhadno. effectontheactivityofinhibitor. -33一.

(39) Tab艮e7. Effectsofmetalionsacidreagents ontheactionofCBAI. %remaininginhibitoryactivity 2h. 24h. None. iaa. ユ00. MnCIZ. IOG. 103. CoC12. 92. 94. BaC12. ioz. 98. ZnSO4. 128. 120. CtxSO4. 14G. GO. HgC12. 39. 11. H202. 76. 44. NaNO2. 92. 102. NaZS203. 99. 112. 2-Mercaptoethanol. 1Q4. 92. EDTA. 102. 104. o-Phenanthroline. Sao. 101. CBAIwasincubatedwith20mMmetalionsin waterandwith20mMreagentsin20mMPIPES Duffer,pH6.9at37°Cfor2and24h.. 一34一.

(40) Section5. StabilityandDigestibilityofKidneyBean a-AmylaseInhibitor. ThereisnotmuchinformationonpHorthermalstabilityofkidneybean a-amylaseinhibitorsorontheirdigestibilityandchangeofactivityduringcooking, exceptforaseriesofinvestigationontheinhibitorsfromgrains.Accordingly,pH stability,thermalstabilityanddigestibilityofkidneybeana-amylaseinhibitorwere describedinthissection.. MaterialsandMethods. Materials. Thekidneybeanseedstestedherewereobtainedfromamarket.Thea-amylase. inhibitors,KAI,TAI-1,TAI-II,andCBAI,werepurified丘omkidneybeancultivar. kintoki,tora,andcranberry,respectively,accordingtothemethoddescribedinsection. 2.Porcinepancreaticα. 一amylase(typeI-A,DFPtreated)waspurchased丘omSigma. ChemicalCo.Allotherreagentswereofanalyticalgrade.. Measurementofa-amylaseinhibitoryactivityandproteinconcentration cx-Amylaseinhibitoryactivityandproteinweremeasuredbytheiodine-starch methodandthemethodofLowryetal.{1951}describedinsection1.. pHandthermalstability TotestthepHstability,theinhibitor(1.5x10遁M)ina10mMbufferwas. incubatedat37°Cfor24h.Thebuffersusedinthisexperimentwereasfollows:. Na-citrate-HCIforpH2.Oand3.0;acetateforpH4.Oand5.0;phosphateforpH. 6.0-8.0;H3BO3・KC1-NaOHforpH9.Oand10.0.Afterthedesiredperiod,analiquot. 一35一.

(41) waswithdrawn,adjustedtopH6.9withthePIPESbufferandassayedforitsresidual inhibitoryactivity.Twomethodswereusedtothetestofthermalstability.Inthefirst. method,their血ibitor(1.5x10崎M)ina20mMphosphatebuffer(pH7.0)was. incubatedatvarioustemperatures.After20minofheating,analiquotwaswithdrawn, cooled,andassayedforresidualinhibitoryactivity.Inthesecondmethod,theinhibitor. washeatedat80°Cand90°CforO-20mininthe20mMabovebuffersofpH3.0,. 5.Oand7.0.-Afterheating,theremaininginhibitoryactivitywasmeasured.. Heattreatmentofbeans. Kidneybeans(Phaseolusvulgariscalutivartora)weresoakedinfourvolumesof. distilledwaterfor15hat20°C.Hydratedseedsalongthesoakwaterwereheatedand. cooledinanicebathaftervaryingtimesofheating.Theseedswerehomogenizedina. mortarandhomogenateobtainedwasstirredwithfivevolumeofwaterfor2h.After. centrifugation,thesupernatantsobtainedwereusedforassayoftheiramylase. inhibitoryactivitiesandmeasurementofsolubleproteincontents.. Inヅ1かodigestion. Afixedconcentrationoftheinhibitorwasincubatedat37°Ctogetherwithtwo fixedconcentrationsofpepsin(pH2.4),trypsin{pH7.5),orchymotrypsin(pH7.5),. respectively.Aliquotswithdrawnathalf-hourintervalswereeitheradjustedtoneutral. pHwithPIPESbufferinthecaseofpepsindigestionorheatedat70°Cfor10minin thecaseoftrypsinorchymotrypsindigestioninordertoinactivatetheproteinases.. Afterthisheattreatment,theeffectoninhibitoryactivityoftheinhibitorwasnot. observedandtheproteaseactivitieswasnotdetectedbytheusualmethodusing. chromogenicsubstrate.. Resultsand.Discussion. 36一.

(42) pHandthermalstability TAIwastestedforitsstabilityunderdifferentpHandheattreatmentconditions. WhenTAIwasallowedtostandatvariouspHvaluesfor24hat37°C,itsinhibitory. activitydecreasedunderalkalineconditions,althoughitwasalmostunchangedinthe rangepH3.Oto7.0{Fig.15A).Thecx-amylaseinhibitorsfromblackkidneybean. werestablefrompH3.Oto4.0(FrelsandRupnow,1985);TAIwasstableovera. widerpHrangecomparedwiththose.. TAIretainedSO%and65%ofitsinitialactivityafter20minofheatingat70°C. and80°C,respectively(Fig.15B}.Cincoetal.(1985)havereportedthattheactivity. oftheheat-labileα. 一amylaseir血ibitorfromwhitekidneybeanwasreducedby70%. after20minofheatingat70°C.Fromtheseresults,itwasconcludedthatLorabean,as wellasredkidneyl)ean,hadasingleα. 一amylaseir血1)itoroftheheat。stabletype.. TAI-IandTAI-IIhadsimilarthermalstability.Theactivityofeachinhibitorwas. reducedbyabout40%after20minofheatingat80°C,andtherewashardlyany. changebefbre丘actionationbychromatofbcusing.. 一37一. ..

(43) TheinhibitoryactivityofTAIwhichhadbeenheatedfor20minatpH7.Oand80 °C ,wasretainedbymorethanhalfcomparedtoitsalmostcompletelossatpH3.Oor 5.0(Fig.16).Thus,a-amylaseinhibitorinkidneybeanseemedtoundergolittle. inactivationsolongasphysiologicalconditionsareconsidered. Onthestability,thesimilarresultswereobtainedforKAIandCBAI.. Activitychangeofa-amylaseinhibitorduringcooking. Figure17representsthechangeinamylaseinhibitoryactivityinkidneybeans. duringheating.Soakingfor15hdidnotcauseanylossoftheinhibitoryactivity,while. itledto15%lossinthesolubleproteincontent.Noamylaseinhibitoryactivitywas. detectedintllesoakingmedium.Afterheattreatmentfor15min,theinhibitoryactivity. reducedbyabout10%oftheinitialactivity,anditdisappearedcompletelyfollowed15. minheating.Theseresultssuggestthatkidneybeanseedsifcookedexhibitedno -38一.

(44) amylaseinhibitoryactivity.Whenthesametreatmentwithoutpresoakingwas performed,theactivitywasremained50%oftheinitialactivityevenafter15min heating(datanotshown).Manyworkers(Kadam,S.S.etal.,1986;AbdusSattar, S.K.etal.,1989)reportedthatthedecreaseoftrypsininhibitoryactivityinbeanseeds duringsoaking.However,thedecreaseofa-amylaseinhibitoryactivityinbeanseeds wasnotobserved.. Invitrodigestionbyproteolyticenzymes. ChangesintheinhibitoryactivityofTAIduringtheincubationintheabsenceor. thepresenceofpepsinareshowninFig.18A.IntheincubationofonlyTAIatpH2.0,. theinhibitoryactivityslowlydecreasedto90%oftheoriginalactivityafter2h.Inthe. presenceofpepsin,TAIwashardlyinactivatedwhenitiscomparedwiththelossof inhibitoryactivityofTAIintheabsenceofpepsin.AsshowninFig.18B,theinhibitor. wasquiteresistanttothe. _proteolysisbytrypsinfor2hdigestion.Qntheotherhand, -39一.

(45) TAIwasrelativelysusceptibletotheactionofchymotrypsinandlostabout90%ofthe originalactivityduring2hincubation(Fig.18C).Andrioloetal.(1984)have demonstratedthatthea-amylaseinhibitoryactivityofawhitekidneybeanextractis susceptibletochymotrypsindigestionincontrasttotheresistancetopepsinortrypsin. (a). 100. 50. 0 00 1. (渓 ) 倉﹀唱蕊. Cb}. 50 0. 昏。董澄 q一. 0 0 1. (c). 50. 0123 Time(h) Fig.18Effectsofdigestionby(a)pepsin,(b) trypsinand(c)chymotrypsinonthe a-amylaseinhibitoryactivityofTAI ThereactionmixturecontainingTAIand pepsin,trypsinorchymotrypsinina10to1 (●>andlto1(△)molarratiowereincubated at37°Cforindicatedperiods. Opencircle(○)in(a)indicatestheinhibitoryactivityintheabsenceofpepsin.. 一40一.

(46) digestion.OuxobservationonthedigestibilityofTAIisinagreementwiththeirdata. Despiteahigherconcentrationofchymotrypsininthisexperiment,however,theloss. ofinhibitoryactivitywithin2hwasalmostequal,suggestingthatTAIismorestable. tothechymotrypsindigestionthanwhitekidneybeana-amylaseinhibitor.Asforthe trypsin-resistanceofa-amylaseinhibitor,itmaybeduetothepresenceofcertain. factorssuchassteriehindranceoraminoacidsequenceaffectingthetrypsindigestion. However,Andrioloetal.(1984)andOldeneto1.(1982)suggestedthatthe. trypsin-resistanceofa-amylaseinhibitorhadrelevancetothecarbohydratemoietyof. inhibitor.Infact,thecarbohydratecontentsoftwoα. 一amylaseinhibitors(1-1andI-2). 丘omblackbeanwere7.5%and9%,respectively,andI-lwasmoresusceptiblethan. I-2totheactionoftrypsin{FrelsandRupnow,1985).Thecarbohydratecontentof. TAI(9.1%)isalmostthesameasI-2fromblackbean,andacontributionofits. carbohydratemoietytothetrypsin-resistancecouldbesupposed.. 一41一.

(47) CHAPTERIIANTINUTRIENTSINLEGUMES. SectionlInvestigationsofAntinutrientsin LegumesImportedforFood. Thissectiondealswiththeresultsofaninvestigationontheantinutrientsin legumesimportedforfood.Proteinaseinhibitoryactivity,hemagglutinatingactivity, tannin,andphyticacidwereanalyzed.. MaterialsandMethods. Materials. Eightdriedlegumesamplescomprisedofadzukibean(Phaseolusangularis. (Willd.}Wight),mungbean(PhaseolusaureusRoxb.),twovarietiesoflimabeans. (Phaseoluslunatus),twovarietiesofkidneybeans(PhaseolusvulgarisL.),andtwo varietiesofcowpeas(ifignasinensisEndel.),wereimported丘omthecountriesshown. inTable8.Theseedswerepulverized.. WingedbeanseedsoffourPapuaNewGuinea,fourIndonesiaandfourOkinawa. (Japan)avarietieswereused.TheOkinawavarietieswereexperimentallycultivated. inOkinawaprefectureofJapanandsuppliedbytheOkinawaBranchoftheTropical. AgricultureResearchCenter.Theseedsweregroundintofinepowderanddefatted. withn-hexane.Thedefattedpowderwasusedasasampleforanalysesexceptforthe. measurementoftanninandphyticacidcontents.. Measurementofchemicalcompositionandproteinconcentration Thechemicalcompositionoftheseedflourwasdeterminedbythemethodsof AssociationofOfficialAnalyticalChemists(1975).Proteinwasmeasuredbythe. 一42一.

(48) methodofLowryetal.(1951),usingbovineserumalbumuzasastandard.. Assayofproteinaseinhibitoryactivity Theseedflour(1.5g)wasextractedwith20mlof2%NaClat4°Covernight. andcentrifuged.Thesupernatantwasusedforproteinaseinhibitorassay.Thetrypsin andchymotrypsininhibitorassayswerecarriedoutusingBAPAassubstrateforbovine. trypsinandBTPAforbovinea-chymotrypsinaccordingtothemethoddescribedby Ibukietal.(1983).. Assayofhemagglutinatingactivity. Theseedflour(2.Og)wasextractedwith20mlof10mMphosphatebuffer. containingO.15MNaCI{pH7.0)at4°Covernightandcentrifuged.Thesupernatant wasusedforhemagglutinatingactivityassay.Thehemaggiutinatingactivitywas. measuredbyaserialtwo-folddilutionmethodonmicrotiterplateusinga4%. suspensionoftrypsinizedhumanerythrocytes(LisandSharon,1972).Isolationof. hemagglutinatingmaterialbyaffinitychromatographyonConcanavalinAbound. Sepharose4Bwasperformedaccordingtotheme止odofJlmqueiraandSgarbieri. (1981).. Measurementoftanninandphyticacidcontents. Tannincontentwasdeterminedbyavanillin-HCImethodofBurns(1971).Phytic acidwasisolatedbythemethod-ofOgawaetal.(1979).Pytate-phosphorusandtotal. phosphoruscontentsweredeterminedbyAllen'smethod(1940)afterdigestionofthe samplewith2NHCIandperchloricacid,respectively.Phyticacidcontentwas. estimatedbymultiplyingtheamountofpytate-phosphorusby3.55basedonthe. empiricalformulaC6P602aH且8.. 43一.

(49) ResultsandDiscussion. Antinutrientsinseverallegumesimpartedforfood. TheproximatecompositionoflegumeseedisshowninTable8.Theprotein. contentrangedfrom18.4to2b.4%.Trypsinandchymotrypsininhibitoryactivitiesof leguminousseedsareshowninTable9.Bothactivitieswerefoundinthevarietiesof. limabeansandkidneybeans.Inthesescases,inhibitoryactivitiesagainsttiypsinwere. higher・thanthoseagainstchymotrypsin.ElkowiczandSousulski(1982)reportedthe. similarresultswithrespecttotrypsininhibitoryactivity.. Table8. 、部% A . Protein' (OiQ). Testacolor. L し・ ♪ 恥伽. Le;ume. Proximatecompositionoftropicallegumeseeds.. Moisture tia3. Adzukibean(Burma)b♪. Yellow-brown. 18.4. 3.3. 4.0. 12.3. Mungbean(Thailand). Peagreet. 23.4. 1.8. 3.3. 11.3. Babylimabean(U.S.A.}. White. 21.0. 1.8. 3.9. 13.6. Licnabean(SouthAfrica). White. 24.1. 4.4. 4.4. 13.8. 工ightredkidneybean(U.S.A。). Lightred. 26.4. 1..2. 3.7. 12.fi. Whitei:idneybean{SouthArica). Wh1te. 19.8. 1.2. 4.1. 10.8. Rosepea{Thailand). PiElgE. 22.6. 2.3. 3.$. 12.1. Blackpea(Thailand). BIack. 23.8. z.5. 3.1. 12-0. a)ハ1x6. .25.. b)Thelegumesweregrowninthecountriesgiveninparenthese. .. Tablegalsoshowshemagglutinatingactivityintheseeds.Twolimabeansandtwo. kidneybeanshadtheactivitywithhumantypeAerythrocytes.Lectins. (hemagglutinatingmaterials)wereisolatedfromtheserespectiveseedsbyaffinity cluomatographyonConcanavalinA-Sepharose4B.Relativehemagglutinatingactivities. withfourtypesofhumanerythrocytes(A,B,OandAB)ofisolatedlectinsaregiven. inTable10.Lectinsfromkidneybeanswerenon-specific,whilethosefromlimabeans. werespecificfortypeAerythrocyte. -44一.

(50) Table9. Trypsin,chymotrypsininhibitoryandhennaggiutinatingactivitiesoftropical legumeseeds.. TIactivityA)CIactivitya) (unit/mg-protein}. Legume. Hemagglutinatingactivi亡yb) (titer/mg-protein). Adzukibean Mungbean BabyIimabean. 330.O. 131.1. 40G. %,imabean. iao.7. 70.8. 340. 73.6. 57.6. 353. 151.0. 8?.1. 764. Lijhtredkidneybean Whitekidneybean Rosepea Blacklea a)TIactivity. 76.7 ,trypsininhibitoryactivity;CIactivity,chymotrypsininhibitoryactivity.. Oneunitwas. theamountofinhibitorrequiredforcompleteinhibitionof1,ugoftheenzyme. b)HemagglutinatingacfivitywithhumantypeAerythrocytes.. Table10. Relativehemagglutinatingactivitywith variouserythrocytesoftropicallegume seed夏ectins.丘). Erythrocytes source. :.. lima bean. Lima bean. Light red kidney bean. White kidney bean. Human TypeA. zoo. TypzB. TypeO TypeAB. goo. 100 100100 (12,000)b) (5,000)b' 100100 (1,250) 100ioo (1,250} 100 100100 (1,250) (3,000}. Rat. 200200. Mouse. 400200 {3,125). a)TheactivitywithhumantypeAergthrocytes wastakenas100. b)Thevalueswererelafiiveactivitiesofpartial purifiedlectinsbyaffinitychromatographyon concanavalinAboundSepharose4B.. 一45一. (625).

(51) Antinutrientsinwingedbeanseeds Thetannincontentsoftheseedsoftwelvevarietiesofwingedbeansareshownin Table11.A5-foldvariationintannincontent(1.35-6.75mg/gofbean}wasobserved, whileTanetal.(1983)reporteda25-foldvariation(0.3-7.5mg/gofbean).No correlationbetweenseedcoatcolorandtannincontentwasobserved. ThephyticacidandtotalphosphoruscontentsarealsoshowninTable11.Except forthevalues-obtainedfromIndonesiavarieties,theseresultsarecomparablewiththat ofsoybeanandhigherthanmanyotherlegumes(ElkowiczandSousulski,1982).The phytate-phosphoruscontributedasubstantialportionofthetotalphosphorus (44.3-54.8%}. TrypsinandchymotrypsininhibitoryactivitiesareshowninTable12.Inallwinged beanspecimens,inhibitoryactivityagainstchymotrypsinareabout2-foldhigherthan thatagainsttrypsin.Thisobservationmaybeaccountedforbythepresenceofa trypSlnlnhlbltor,whichalsoinhibitschymotrypsin,inwingedbeanseed(Korn,1979}. Itisknownthattanninsarenonspecificinhibitorsofenzymes.However,deLuman. andSalamat{1980)reportedthattrypsininhibitoryactivity,causedbythetannin, occupiedonly1%ofthetotalinwingedbeans.Inthissection,nocorrelationwas. foundbetweentannincontentandtheproteinaseinhibitoryactivity. Table13showshemagglutinatir..gactivityinwingedbeanseeds.Wingedbean. lectinagglutinatedalltypesoftrypsinizedhumanerythrocytes(A,BandO).Inthis. section,theagglutinationspecificityforhumanerythrocytetypeswasclassi丘edinto. threepatterns.`TiTingedbeanvarietiesOOIand2826showedthatagglutination. specificityforhumanerythrocytesdecreasedintheorder,bloodgroupA>B=O;. varieties3and4,A=B>O;others,A=B=O.TurnerandLiener(1975)reported thatsoybeanlectinshavenoapparentdeleteriouseffectsonanimal,whileHiguchiet. al.(1983)showedthatwingedbeanlectinsaretoxictorats. Theeffectofheatingonhemagglutinatingactivitywasalsoinvestigatedto. inactivatewinged-beanlectin.Afterheatingthetestsolutioninboilingwaterbathfor r46...

(52) り・肩. 6う寺い. ◎◎6 い. 箇寺寸. 寸りい. 寸りい. NO・い. α寸寸. ゜oり寺. 6りΦ 寸. 寸ひ寺. ひoQ・ N. ◎oや 04. ooO出. 9 ・ N. 寸い内. ON・ N. 巳内. ︻hO・日. ゆN・臼. い一・ひ. 。、α・ 9. めN・◎。. OO・ひ. 一Q。・卜. 卜卜・ト. 凶り・祠. ゜ooQ高. ゜◎6ら山. °oり山. O寸∴. O寸∴. いトb. ◎。・寸い. 一寺い. ① Qo寸. ひ︹出. いoQ・い. αOめ. ゜。りい. 〇一・ひ. いひ∴. Q。一・ N. 心卜N・ α. b 。＼・。ミ ). 、℃ q. 9 ミミ. o= 6銘﹄. 剛毎。↑ ℃器. ミミ緊葭. 跳ひ∴. ooN出. トリ9. Nひ・ N nO出一や 6ー. 賄ミミ薯。貫. 0卜・ 9. ◎◎い・ N. 鴇∴. 目也. o。・ O寸. oo・胡. hミミ魯。ミ. ( § 避も. 、ミミ承旨 hミミ寓。貫. ℃ミ ≧ 霞. へ. 、ミ￡. 自出. 翻. ℃⑪・D三 ≧ ωε ㎏。・・巳。謂q・、。歪. 忌ミミ店. hミミ昏。ミ. り o。寸. ひ・卜寸. 諾・い. り寸い. 、. 衷。り環8 ℃日. m. 寸. ・いい出 × ・・ヨ。琶・・。且器一盈山 . 切↑昼邸﹀唇自ヨ 8 罵 9 。. 滋。り窪 o℃属. 一. N. o剛 dq 淵≧ o﹂ロq. 酒旨 8 ℃￡. 信≧ oお. 図8 国. 寸い一の. 凪。,⑪口O℃口同. 邸貫お O ≧. 。Z 邸薮￡. OdQQd. ・富黛§. 一〇〇. NOO. 6㌦OO. 寸OO. 一αoON. 器口眉 O 器 Z 耐鼠￡舘巷 o 器 z "a ￡. 寸一 9. ミ・ミ oり. ( 器忌じ窪。コ三謂O 宕巴琶邸詫口逗O. 省巴・3h)雲邸三図○ ( 口巴琶窪實薫 ○. 器 ε うO 詰 Z 讐似￡. 口弦 e ρq 自≧ 2 的自≧ 9 自自￠≧ 2 β自 9≧ o﹄国￠≧ O﹂ρ自 q≧ O﹄ロ自≧ O﹂βQ 蝦≧ 2 山. kQ s 、魅◎b. 、電恥. ℃唱 Q< Qヨ畷氏、三目俘. 一47一.

(53) 10min,AssayforhemagglutinatingactivitywascarriedoutusingtypeAerythrocyte. TheboilingresultedinthedisapPearanceoftheactivities丘omallwingedbean. varietiesusedhere.Lectinsinwingedbeanseedcouldbeinactivatedeasilybysmin. ofautocleavetreatment{Tanetal.,1983).Theseresultssuggestthatwingedbean seedmaybeacceptableforafoodsourceifcooked.. Table12, TrypsinandChymotrypsinInhibitoryActivitiesofkheWingedBean Yrrriety. TrtIpsin. C1り〃η0'リノf15%II. inhibitory. 加乃'ゐ'〃〃・y. αc'∫り`'ア. rtctEVrty. (ε〃1〃ぶ!'η9〃. °0ごぞ加)n. 001. 0・12. 0.24. 002. 0・10. fl・22. 003 004. 0・14. 0・28. 11'. 0・21. iaia 2826. 0・12. 0.22. 0・]夏. 0.20. 2891. 0・12. 0.23. 3154. 0・14. 0.26. 0・11. 0Zo. 2 3. 0.12. O・21. 0.13. 0.23. 4. ato. 0zs. 1. Oneunit;theamountofinhibitorrequiredforcompleteinhibitionoflmgofenzyme.. Table13 HemagglutinatingActivityoftheWingedBean{titer/mgprotein} Vtrriety. Tンneofery,〃. 〃・0のノ'C'S. A. B. 001. 2aO. ioo. 10(?. 00z. 110. 110. 110. OO3 1ii. 94. 94. り4. 94. 94. 94. 1014. 97. 97. 97. 2826 2891. 240 490. 124. Sao. 490. 490. 3154. 130. t3a. 1. 130 210. 210. 21U. 2. z20. 27Q. 220. 3. 220. 220. 110. 4. 480. 480. 240. 一48一. 0.

(54) Section2. PurificationandSomePropertiesofTrypsin InhibitorfromBroadBean. Ithasbeenknownthatbroadbeans(Viciafaba),likemostotherlegumes. containedtrypsininhibitors.However,trypsininhibitorsinViciafababeanshavenot. beeninvestigatedasmuch,probablybecauseofthelowerconcentrationoftheinhibitor comparedwiththatinsoybeans.Thepurificationandsomepropertiesoftrypsin. inhibitor丘ombroadbeansaredescribedinthissection... MaterialsandMethods. Materials. Theseedsofbroadbea血s(Vicia/abaL.,var.major)werepurchasedfromalocal. market.Bovinetrypsin{typeIII)andBAPAwereobtainedfromSigmachemicalCo.. CM-SephadexC-25andCNBr-activatedSepharose4BwereproductsofPharmacia FineChemicals.Anhydrotrypsin-Sepharose4Bwaspreparedaccordingtothegeneral. method.. Measurementoftrypsininhibitoryactivityandproteinconcentration. TrypsininhibitoryactivitywasdeterminedbythemethodofIbukietal.(1983). describedinsectionl.Oneir雌bitor㎜it(11U)wasde丘nedastheamountof inhibitorrequiredforcompleteinhibitionof1ｵgofenzyme.Proteinwasmeasuredby. Cowry'smethoddescribedinsection1.. Aminoacidanalysis. Theproteinwashydrolyzedinsealedevacuatedtubesat110°C-for24hwith6N HCI.TheaminoacidanalysiswasconductedwithaHitachi835autoanalyzer.Cystine. 一49一. ,.

(55) andmethioninewereassayedinasampleoxidizedwithperformicacidbythemethod ofMoore(1963}.Tryptophancontentwasdeterminedspectrophotometrically.. ResultsandDiscussion. Purificationoftrypsininhibitor. Theseedflour{30g)wasextractedwith300mlof2%NaClat4°Covernight, andtheresiduewasextractedagainfor2hwith150mlofthesaltsolution.The. extractswerecombinedandcentrifuged.Solidammoniumsulfatewasaddedtothe. supernatantto90%saturation,andtheprecipitatewascollected,dialyzedagainstwater. andlyophilized.ThispreparationwasdissolvedinO.1MNaHCO3containingO.5M. NaCIandchromatographedonaSephadexG-75column{2.6x90cm)withtheuse. ofthesamesolutionaseluent.Thetrypsininhibitorfractionwasappliedtoa anhydrotrypsin-Sepharose4Bcolumn(1.1x18cm),andelutedfirstwithO.1M. NaHCO3containingO.5MNaClandthenwithO.01MHCI(Fig.19-1}.Trypsin. inhibitorelutedinasinglepeakwascollected,desalted,andlyophilized.The. lyophilizedproteinwasdissolvedin50mMphosphatebuffer(pH6.5),putona. colu㎜(1.6x25cm)ofCM-SephadexC-25equilibratedwiththebuffer,andeluted. firstwiththisbufferandthenwithalineargradientofNaCI(0-0.5Minthesame. buffer).AsshowninFig.19-2,threetrypsininhibitorpeakswereobservedanda. majorinhibitor,thesecondpeakintheorderofelution,wascollected,desalted,and Lyophilized.ThepurificationofamajortrypsininhibitorissummarizedinTable14.. Thefnalpreparationwasshowntobeahomogeneousproteinbyelectrophoretic analysis(datanotshown).. の. Somepropertiesofmajortrypsininhibitor. Isoelectricfocusingoftheir血ibitorindicatedthattherewasasinglepeakat8.66.. Table15showstheaminoacidcompositionoftheinhibitor,whichconsistedof99 -50一.

(56) 3。0. 100. (邑 ⊆￡三三⊆= 奮さト. ∈ 広 OooN }⑩ Φo⊆o￡ ogうOく. 1.5. Q. Fractionnumber(3.5mQ/tube) Fig.19-1. Anhydrotrypsin-Sepharose4Baffinitychromatographyofbroad beantrypsininhibitor, TheinhibitorsolutionobtainedbySephadexG-75gelfiltrationwas appliedtoacolumn(1.t×18cm)o正anhydrol:rypsin-Sepharose4B equiliUratedwithO.1M:Na.IICO3containingO.5MNaCJ. ..The. columnwaswashedwiththissolutionandthentheinhibitorwas elutedwithO.01MliCI.Thearrowindicateswhereusinghydrocliloricacidasaneluentwasstarted.Thefractionsrepresented l)yabarweエecollected・. ○ 一〇,absorbanceat280nm;0-●,. trypsininhibitoryactivity.. 100. a.5. 50. 岳 qさト. Q. 0 130. 0 Fractionm」mber(2.5m2/tube>. Fig.19-2. ( や")董 ↑ 至葦. E C OoQN 幽 o Φo⊆㎝￡﹂ooPく. 1.4. CNI-SepliadexC-25ion-exchangechromatographyofbroaddean txypsininhibitor. Theinhibitorobtainedbyanhydrotrypsin-Sepharose4Baffinity chromatographywasappliedtoacolumn(1,6x25cm)ofCMSephadexC-25equilibratedwith50mMsodiumphosphatebuf正er (pH6.5).TheI)roteinwaselutedfirst;wi士hthis1)ufferandthen tuithalinearsalt:concentrationgradient(OtoO.5MNaClinfile samebu丑er).Thefrac七ionsrepresentedbyabarwerecoUected. ○ 一〇,at)sorbanceat280nm:●-0,i:xypsiitinhibitoryactivity: … …,NaClconcen七ration.. 一51一.

(57) Table14. Surnrna,ryof七hepuri且cationofbroadbeantrypsininhiUitor. Total activity (IU). Procedure. Total protein (mg}. Extract. 72,500. 3,720 754. Specificyi eld. activityt°r. i9.5. Ratio o)(IU/mgprotein). 100. Salting-out. 59,200. SephadexG-75. 51,300. 79.8. 643. 70.s. AIITa-Sepharose4B. 46,300. 18.9. 2450. 63.9. 旦26. CM-SephadexC-25. 37,000. 12.3. 3020. 51.0. 1SS. uAFITstandsforanliydrotrypsin. Table15. 78.5. 1.00. S1.7. .. Aminoacidcompositionofthebroad beantrypsininhibitor. .Aminoacid. Residuespermole*1. IntegezI. Lysine. s.s. 9. H三sddinc. 4.8. 5. Argln三ne. 4.6. 5. 14.1. 14. flsparticacid Threonine. 6'.4. s. Serine. 9.1. 9. Glulamicacid. 5.1. 5. Prolillc. 5.9. 6. Glycine. 3.3. 3. 3.3. 3. 21.2. 21. Val三nc. 5.8. 6. Methianine. 4. 0. Isoleucine. 0. 0. Leucine. 1.7. 2. Tyrosine. 2.4. 2. Phenylalanine. 3.1. 3. Tryptophan.. d. 0. Alanine Half-cystine*2. Total. 99. *1Basedonamolccularwcightof. 11,00U.*2Deter一. minedascysieicacid.. 一52一. 4.03 33.0.

(58) aminoacidresidues.Thiswascomputedonthebasisofthemolecularweightof. 11,000measuredbySDS-PAGE.Theinhibitorcontainedahighlevelofhalf-cystine, andlackedmethionine,isoleucine,andtryptophan.Asaoθ'α1.(1991)showed. isoelectricpointsof7.7,8.7,and9.1forthreeinhibitorsfromfababeans,andreported thattheinhibitorwithpi8.7accountedformostoftheinhibitoryactivity.Although. thetrypsininhibitorfromfababeanshasbeenreportedtocontainlowlevelsof. isoleucineandtryptophan,itsaminoacidcompositionisgenerallysimilartothat. obtainedinthisstudy.. Stability. Whentheinhibitorwasallowedtostandinvariousbuffersfor24hat37°C,its. inhibitoryactivitydecreasedunderalkalineconditionsabovepH10,although. remainingalmostunchangedoverawidepHrangelessthanthis(Fig.20-1).The. effectofheattreatmentontheinhibitoralsoisshowninFig.20-2.Theinhibitory. activitywhichhadbeenheatedfor20minat700r100°Cwascompletelyretainedat. pH3and7.AtpH10,however,theinhibitorwasrapidlyinactivatedat70°Cand above,andallofitsactivitywaslostbyheatingat100°Cfor10min.Thesestability. oftheinhibitorforpHandheattreatmentaxecontrarytothoseofWilsonetal.(1972) reportedthatapurifiedinhibitorfromfeldbeansdissolvedin2.5x10-3MHClwas. inactivatedbyautoclavingat120°Cfor30min,andWarsyetal.(1974)described. thattwopurifiedinhibitorsfrombroadbeanswerethermallylabileatneutralpH. values.. 一53一.

(59) ハ訳 "zoa あ .ピ .ヒ u. 御. ゜. s?50 ヨ .ヨ需づ v'あ v a 24681012 plI Fig.20-1EffectofpHonthcstabilityofthcbroadbean trypsininhibitor TheinhibitorwasincubatedatvariouspHvaluesfor24 hat37°C.Afterincubation,aliquotswcrcwithdrawn andtbcrcsidualinhibitorアactivitywasmcasurcd.. d102dd102d Heati11只time{min) Flg.2-2EfT'ectofheattreatmentonthestabilityofthe broadbeantrypsininhibitor Thelnllibitorwashcatedat70°C(a>and蔓00°G(b)in variousbul洗rs:O,pH31●,pH7;▲,pH10.Aliquots werewithdrawnatvari・usL三mes,c・. ・1edinaniccbath,. alldtheresidualixahibitoryactiv五tywasmcasurcd.. 一54一.

(60) CHAPTERIIITRYPSININHIBITORFROMBROCCOLI. Section1. PurificationofMajorTrypsinInhibitorfromBroccoli. OccurrenceoftheproteinaseinhibitoryactivitiesinthevegetablesofCruciferae andpurifcationofamajortrypsininhibitorfrombroccoliweredescribedinthis section.. MaterialsandMethods. Materials Broccoliandcauliflowerusedherewereobtainedfromamarket.Bovinetrypsin (typeXIII},bovinea-chymotrypsin(typeII),BAPA,andBTPAwereobtained fromSigmachemicalCo.DEAE-SephadexA-25,SephacrylS-100HR,PBE94,and Polybuffer74wereproductsofPharmaciaFineChemicals.. Assayofenzymeandinhibitoryactivities TrypsinactivitywasmeasuredbythemethodofErlangeretal.(1961)witha. siightmodification.Thereactionmixturecontaininganappropriateconcentrationof. trypsin,1%BAPA,and10mMCaClzinaO.1MTris-HCIbuffer{pH8.0}was. incubatedat37°Cfor10min.Thereaction-wasstoppedbyadding10%aceticacid,. andtheabsorbanceofthemixturewasmeasuredat410nm.a-Chymotrypsinactivity. wasmeasuredspectrophotometricallywithBTPAasthesubstrate.Theinhibitory. activitywasevaluatedfromtheresidualtrypsinactivityinthepresenceofaninhibitor.. Oneinhibitorunit(111」)wasdefinedastheamountofinhibitorrequiredfor. completeinhibitionof1ｵgofenzyme.. 一55一.

(61) Measurementofproteinconcentration. ProteinwasmeasuredbythemethodofLowryetal.(1951),usingbovineserum. albuminasastandard.Theenzymeconcentrationsweremeasured spectrophotometricallywiththeabsorptioncoefficientsEα1%=154(at280㎜)for. bovinetrypsinandE°'t%=2.05(at282㎜)forbovinea-chymotrypsin.. Eiectrophoreticanalysis PAGEwasdonebythemethodofDavis(1964),using7.5%acrylamidegelatpH 9.4.SDS-PAGEwasdonebythemethodofWeberandOsborn{19d9),using12.5% acrylainidegel.ThegelswerestainedforproteinwithCoomassieBrilliantBlueR-250.. Aminoacidanalysis. Theproteinwashydrolyzedinsealedevacuatedtubesat110°Cfor24hwith6N. HCi.TheaminoacidanalysiswasconductedwithaHitachi835autoanalyzer.The reductionandS-carboxylationofcystineinthesamplewasdonebythemethodof. Crestfieldetal.(1963}.. ResultsandDiscussion. Proteinaseinhibitoryactivitiesinbroccoliandcauliflower. Thecutvegetableswashomogenizedinahigh-speedmixerwithwater(1:2,Wハ1). forsmin.Thehomogenatewascentrifugedat8,000xgforlh,andthesupernatant. wasusedforinhibitoryassay.Trypsinandchymotrypsininhibitoryactivitiesinthe. vegetables.areshowninTablel6.Bothactivitieswerefoundinbroccoliand. cauliflower,andbroccolihadhigherinhibitoryactivitiesagainstbothproteinasesthan. cauliflower(Table16A).Thechymotrypsininhibitoryactivitiesinthesevegetables. wereNigerthanthetrypsininhibitoryactivities.Thespecificactivitiesoftheinhibitors. instemofbroccoliwerehigherthanthatinheadsofbroccoli(Table16B).About -56一.

(62) 70%ofthetrypsininhibitoryactivitiesintwovegetableswerelostafterheatingfor. lOminat80°C.. Table16A. Pro七easeinhibitoryactivitiesofvegetables. Protein. Vegetable. Txypsininhibitox. Chymotrypsininhibitor. S.A.**. (mg). T.U.芸. Cauli$otiver. lbl.1. 746. 4,6. 862. 5.4. Eroccoli. 229.3. 1692. 7.4. 1988. 5.7. T.U.. S.A。. *TotalinhibitoryLinits. **Specificactivity(T.U./mg,protein).. Table16B Par七. PxoteaseinhibitorvactivitiesofUxoccoli.. Protein ,(mg). Trypsinanllibi七. 〇r. Chymo廿ypsininhibitor. T.U。*. S.A.**. T.U.. S.A.. Heads. 156.2. 950. 6.1. 995. 6.4. S七em. 73.1. 742. 10.2. 993. 13.E. *Totalinhibitoryunits **Specificactivity(T. . .U./mg,protein).. Broccoliwashomogenizedinamixerandextractedwiththe50mMTris-HCl. buffer(pHS.0)forIh.Theextractwasfiltratedwithcloth,andthenammonium. sulfatewasaddedtothecombinedsupernatantsto85%saturation.Theprecipitatewas collectedbyfiltration,suspendedindistilledwateranddialyzed(tubeofMWcutoff. 3,500)againstwaterat5°C.Aftertheprecipitatewasremovedbycentrifugation,the. dialyzatewaslyophilized.Thelyophilizedsamplewasdissolvedin50mMTris-HCl. buffer(pH8.0}containingO.1MNaCI,putonaSephacrylS-100HRcolumn(2.6x. 94crn)equilibratedwiththebuffer,andelutedwiththesamebuffer.Ainhibitor. fractionwascollected,dialyzed,andlyophilized.Thelyophilizedsamplewasdissolved in25mMimidazole-HCIbuffer(pH7.4),putonaPBE94column(1.Ox20cm). equilibratedwiththebuffer,andelutedwithaPolybuffer74--HCl(pH4:0).Asshown --57一.

(63) inFig.21,severaltrypsininhibitorswerefoundinbroccoliwiththesimilarmolecular. weights,about10,000andthedifferentisoelectricpoints,4.2-6.9.Theseplvalueswere. similartothoseofrapeseed(4.6-6.8}andleafmustardseed(4-7}{Ogawaetal., 1971a).Thetrypsininhibitors丘omrapeseed(FukuzawaandKuroda,1980),radish. seed(Ogawaetal.,1971b),andcabbageseed{FukuzawaandKuroda,1982)hadthe. molecularweightof7,000-8,000,7,000-12,000,and16,500,respectively.Amajor. trypsininhibitor,whichhadabout60%oftotaltrypsininhibitoryactivityinbroccoli. hadtheisoelectricpointofaboutsandthemolecularweightofabout10,000.The. inhibitorstronglyinhibitedbovinetrypsinandweaklyinhibitedbovine. a-chymotrypsin.. 臼昌 O ooN. Q.4. 口qJ ΦO 目耐 ρ醤 O uDρ<. 0.2. Fig.21. Purificationofamajortrypsininhibitor(BTI-1)frombroccoli. Broccoliheadsandstem(2.5kg)werehomogenizedinamixerandextracted -58一.

(64) withthe50m1VITris-HCIbuffer(pH8.0}forlh.Theextractwasfiltratedwithcloth andtheresiduewasreextracted.Ammoniumsulfatewasaddedtothecombined. supernatantsto85%saturation.Theprecipitatewascollectedbyfltration,suspended indistilledwateranddialyzedagainstwaterat5°C.Aftertheprecipitatewasremoved. bycentrifugation,thedialyzatewaslyophilized.Thelyophilizedsamplewasdissolved. inSOmMTris-HClbuffer(pH7.5),putonaDEAF-SephadexA-25column(2.bx30 cm)equilibratedwiththebuffer,andelutedwithalineargradientofNaCIfromOto. O.4Mintheequilibratingbuffer.Thepeaksoftwotrypsininhibitors(1-Aandl-B). wereobservedinunadsorbedfractionsandathirdtrypsininhibtor(1-C}wasobserved inadsorbedfractions(Fig.22}.TheI-Bfractionwhichoccupiedabout45%ofthe. totalinhibitoryactivityrecoveredinthischromatographywasdialyzedagainstwater. andlyophilized.ThelyophilizedsamplewasdissolvedinSOmMTris-HClbuffer{pH. 7.8),putonaDEAE-SephadexA-25column(2.6x30cm)equilibratedwiththe. 2.0. O oo N .O .O. 1.0. goo と゜・r. 50±'. ・r. 0,4. ε o・2毫. .,_. z. ξ 050gooX500 FractionNo.(15m1/tube). Flee22. Ion-exchangechromatographyolthecrudeinhibitorpreparati DEAE-SephadexA-25column(2.6×30cm)equilibzated Tris-HCIbuffer(pH7.5).. with50mM. EIutionwasdoneataflowrateof50zn1/hr.The. #ractionsrepresentedbythebarwerecollected.●,Absorbanceat280nm;○, trypsininhibitoryactivity;一. preparationona. 一一,NaCIconcentraion.. 一59一.