Fundamental studies on the utilization of olive fruits. II. Identification of the amino acids in the protein hydrolyzate of ripe olive flesh by paper chromatogrphy-香川大学学術情報リポジトリ

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Tech.Bull∴KagaWa Agr.Coll. 194

FUNL)AMENTAL STUDIES ON’THE UT王LIZÅTION OF

OL‡VE FRUITS

∬“Identifieation of血eAm五noÅ¢idsin仏¢Protein Ⅲydrolyza七e ofRip¢01iv¢椚esh by‡>a騨r(漁roma七og・rphy

By TeiichiNARASAKIand KenjiKATAKURA

(Laborator・y Of AgT・icultur・alProducts Technology) (ReceivedJuly28.,1954)

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Inthepreviouspaperithasbeenshown that oliveflesh contains onlyl−3%of

ptoteinonfreshbasisandthelevelofitwasmaintained almost constant during the

wholeperiodoftheexperiment・Thelowcontentandconstantleveloftheproteinseem

toindicatethat theproteinexsistsinolivefr・uitsnot as accumulativebut asconstit− utive component.

Onthepracticalpointofviewtheproteincontentissosma11,thatit maybeof

noimportancetoconsiderthesignificanceofit.However,th占protein should rightly

participatesomewhatin the nutr・itionalvalu占ofpickledolives withthe oilandthe

carbohydrates

Thus the authors studied thepr・Otein components by paper chromatogrIaPhy and therIeSults ar・egivenin the pr’eSent Paper・

EXPERIi沌ENTA‡。

Material:RipefrIuits of theMiisionvariety wereusedinthepresentstudies・ Pr坤W・atio?lqf’Sa〝ゆIs:Fleshwassepar’atedfrom seeds,Cutinto smallsections,

andthendefattedwith ether・ina SoxHLETapParatuSfor30hrs.Thedr’iedanddefatted flesh wastakenforproteinextr・aCtion.Thepr−OteinextractswerIepr’eparedbyextracting from about2grいOf the defatted flesh(ca巾50mg巾Of pr’Otein)with90%formic acid

accordingto thedirectionofR.J”BLOCK&Dl、BoLLING(2)小Afterremoving the carbo− hydr・ateby precipitation with two volumes of ethanoithe extracts were evaporated to drynessin vacuo,aud then hydrolyzed under・reflux with20ml.of6N hydrochlolic acid for20hours..The excesshydrochlolicacid wasremoved byevaporationtodryness i?ZVaCuO On a Water bathand theresultingthinfilm of thehydrolyzate wa$takenup

in5ml.ofl%acetic acid.The mixtur’e,afrer filtr’ation,WaS paSSed slowly througha COlumn of sulfonated polystylene resin(AmberiteIR−120,in the H十cycle,100×10

mm.)(3)(4)(5),WaShed with20ml,Of disti11ed waterI,and the amino acid mixtur・e waS eluted with a sufficient volume of4N aqueous ammonia.Theeluate was againevap・ Orated to dryness,and finally taken upin about2ml..oflO%isopr・OpanOl

CircularP郎er Chromatograクわ′‥(8)This methodis ver・y Simple and speedy,but glVeS Only unsufficient separation.Inspite ofits poor・Separ・ationit was conveniently used preliminarily toinspect theapplicabilityofsamplesand to determinethequantity

to beapplied。Filter・paper・T6y6Not6(dia.,11cm‖)was employed,and the chr・OmatO− grams were r・un With n−butanol:aCetic acid:Water(4:1:5,Ⅴ/v)小

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Vol一.6,No、2(1954) 195

0チ7e−Di毎β77Si.07;al.物er Cゐ㌢OmatOgr(砂hy:One−Way Chr・OmatOgramS Were run aSCe− ndinglywith n−butanol:aCeticacid:WaterOrCOllidinesatur・ated withboratebuffer ofpH

9.3.prepar・ed from200ml.,OfO巾1Mboric acid andl13小5ml..ofo.1Msodiumhydroxide, Onfilterpaper・Str・ipsT6y6No。50(2×40cm..)usingChromatographicappar・atuST6y6−B

(T6y6 Filter・Paper Co。,Japan).The exp6rimentaldetailes were essentialy the same as that describedbyE..FいMcFARREN(6)and K”OTOZAI(7)..Itisrepor・ted(S)that elabor・ate

thermalcontrIOldoes notimpr・0Ve the resolution of each amino acid,and thus the

Chromatogr・amS Were run at rOOm temper・ature(ca.at 250 C.)without special thermalcontr・01.

7bo・Di加eプ:Si.0;:alEbi,er Chromatogrqi,勿′:AscendingtWO−Way method described

by M.MuTO(S)was employed,and theimproved methed of A..L.LEVY&D..CHUNG(!)〉 WaS also r’efer’edin precise techniques”Chromatography was carr・ied out by using

ク2−butanol:aCetic acid:Water,preViously described,aS thefirstsolvent,and m−Cresol: phenol(1:1,Ⅴ/V)saturated withboratebuffer of pH9.3for thesecond runaccording to A∴L LEVY&D.CHUNG,.Collidine of pH9い3,mentioned above,WaS also employed for the first run,but,in this case,double development was necessary,for・this solvent gives toolowRf values forIeaCh amino acidい Filter・Paper・SheetsT6y6No、50(20×20

Cm.)werIe uSed

Color Deuel(沙me;12iqf’ihe Chromatogram:Thedried paper・WaS SPrayedwithO.2% ninhydrinin water’−Satur’ated n”butanoIsolutior).and developed aboutlOOO C”for fewminutes as usually.Iodine,(】0)alkalinepermanganate,(11)diazotized sulfanilic acid,(12) p−dimethylaminobenzaidehyde,(13)andisatin(14)were also employed to detectindividual spots on paper

R貰≡SU‡」TS AⅣD DISCtTSS‡ON

The one−Way Chr・OmatOgramS Obtained are shownin Fig.1,and the two−Way Chromatogr・amS are givenin Fig.,2(by BuOH:AcOH:H20剛血mCresol:PhOH

SyStem)and Fig・.3(by Collidine・・・−‖コロじ−・m−Cr.esol:PhOH system).

Theidentificationofeachspot onthechr・OmatOgramSWaSCarr・iedout by a.ddition test with standard amino acids,deter・mination ofIU values,detection with color r’eaC,

tions,Or the relative positions on the chromatograms..The r・esults are summarizedin TableI ・−・Jヨ噂 ⊥∠

亘二重垂二妻 lノ

番長壷壷

壷 ● I l 1 090 =)0 010 020 0、30 040 Rチ 050 0.60 Fig.10ne−dimensionalpaperchromatogramsofaminoacids王ntheolivehydrolyzate

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Tech Bull.Kagawa Agr..Coll. 196 双︰一骨−正月pヨOU −一 2 ○飼H︰HOU可︰HOぷ甲良

紗9 8⑳ 去重 6 義■ 5♂ 6

重電牒 i

⑳′2 軌 ⑳ ⑬ I 1 15 ウ , ‡

」 鵬_._

__..腑_ −ゝ ∽−CfeSOl:PhOH,pH9.3 称CTeSOl:PhO‡す,pH9.3

Fig小2lTwo−dimensionalpaper chromatog− Fig”3」Two−dimensionalpaper chromato− ram of amino acidsintheolivehydrolyzateby gram o土amino acidsin七heolivehydrolyzate the BuOH:AcOH:Ⅰ‡20M m−Cresol:PhOH, by theColiidine,P‡‡9.3−ml・Cresol‥PhO軋

pH9.3,SyStem p‡i9.3,SySte〉‡n

Tablel,Identification ofEach Spot on Chromai:OgramS

Rf Color Reaction

Others Correspond Ninllydrin

No.Of OneLWay Two−Way

Spots Butanol− Collidine Butanol−(

Acetic acid Acetic acid

l ( ) (.) 0い14 2 0.16 0‖14 0.21 3 ( ) ( ) 0“16 4 0..13 ( ) 0‖16 5 ( ) 0.01 0..10 6 0り20 0.17 0い22 フ 0.23 0.21 0り29 8 0.32 ( ) 0。39 9 0..40 0い33 0.44

二dine m・・Cresol− ColorIntensity

Phenol O小18 0‖04−0..06 B.P ≠ 0い20 0…05“○い14 Pl ・什 0‖18 0一.19一っ…22 Y..R.0Ⅰ・R.P..十 0..15 0‖26−0‖29 BI・…R 十I Oい01 0ハ36−0..45 R..0工Pけ +・ 0.21 0.45−0.46 P‖0工R.P..1け 0‖25 0.8フ−0い91 Y‖ ・什 to aspar’tic acid glutamic acid SeIlne glycine lyslne alanine isatin proline +,B tyr■OSine L P 0.、59 0.55−一刀.58 0..39 0.フ2ぺ■フ6 十 ≠ valine ぐ O iodine A恩A..tryptophan phenylalanine isoleucine leucine D.S.A. histidine 十,RりYu aTglnlne 10 0.51 0.5フ 0い53 0一67 0.66・−0一.71 L. 士 11 0い53 (0.5フ) 0.59 0‥85−−−0一B9 BPいOr且 十 12 (0。56) 0..46 (0.59) 0り52 0..84 P 廿 13 (0..59) 0..50 (0..66) 0..5フ 0.82 P tti ユ4 0.、08 ( ) 0−12 0.、13 0ぷ]−づ.64 L 十 15 ( ) ( ) 0..14 − 0.7フ L. 士

(‘):OVerlap of spots,D,S・A.:diazoti2;ed sulfanilic acid,A・BAい:P−dimethylaminobenzalde− hyde;B.:blue,P..:pink,Y…:yellow,R:red,Brl‥ brown,L・:1avender; 士‥nOt Su・ re,+:$mal1,廿:middle,≠:1arge

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Volご6iNo.2(1954) 197

As showninTableIabout15 a血iiio atidil:weredetectediIlthe hケdrolyzate of

Oliveflesh,butserine,tyrOSifle,histidi壷,・andphenylalaninewereverysmallinamount, and the presence oftryptophanand arginine wasthotsure..Methioninewas notdetected

either with theiodine or with the alkalineperrhanganate tegt.Anothet essentialamino

acid,threonine,WaS also undetectable..From the畠etesultsit willbe easilすConcluded that olives areinferior asproteinfood’bothintheconteht and quality.

AsshowninfigureSleucine,isoleucine,andpheilylalanine☆汝emoredearlysep左− rated withthecollidinesolventthanwiththebutanolandthephenoIsorlvents∴Thespot No.10was determined as tryptophan according toits positions on the chromato− grams(inFigs.2and3),butcouldnotbeascertainedwithp・dimethylaminobenzaldehyde・

Proline,intwo・・WaythromatQgtamS,Wa占easilydetectedbyblu占colorwithisatinfrIOm browncoloratinat the soIvent front,butisatin was not so usefulforphenylalanine, tyrosine,and tryptophanLaSdescribed byA..SAIFER&Ⅰ.ORESKES(14)

StJM】撼ARY

(1)01ivefleshwas studied fof aminb acidsbypr畠patingone−and two−dimensional

paperchromatograms.

(b)Thepr・Oteinhydio】yzatewaspreparedbythe∵forImicacidekttactionasprbvided by R”J:BLOCK&D”BoiLING(2),and deioniz6d by cation−eXChange resin,and th6h used forchromatography..

㈱ The amino acids foundate∵as fo1lows:aSpar・tit、atid,gluta血ic acid,Setine, glycine,1ysine,alanine,pr・01ine,tyrOSine,Valine,tr・yptOphan,phenylalanine,・′leucine, isoleucine,histidine,ノandarg・inine;thepresenceoftryptophanand arlglnlneWaSuilCert−

ain小

㈲ The two essentialamino acids,methionineand threonine,WerenO阜found (5)Isatinwaseffectiveonly forprolineasacolorreagent.

AC且Ⅳ0Ⅵr互一EDG】ⅦRⅣT

Theauthors wish to thankProf.S小 KAWAMURA,Labor・atOrIyOfBiologicalChem− istry,thisCollege,forhisconstantinterestandhelpfulsuggestions,andMr.M..MINO, Labor’atOry OfChemistry,、thisCollege,for agift ofstandard amino acid samples.

Rl∃FER王壬ⅣCES

(1)K.KATAKURA and T.NARASAKI:7セch.Bull.BbgawaAgr.Collり6,1−7(1954) (2)R,J”BLOCK and D。BoLLING:TheAmino Acid CompositionofPr・Oteins andFoods,

Charles C..Thomas,Springfield,Illu,576p,1951

@)R.J..BLOCK,R..LESTRAND,and G.ZwEIG:Paper ChrIOmatOgraphy,AcademicPress Inc=,N.Yり195p1952.

㈲ P.BouLANGER and G.BISERTE:Bull.soc.chiれ biol.,33,1930・−9(1951),C..A..46, 7610c(1951)

(句 A..P.,PRtOR and T.P.WHITEHEAD:NdtuYle,172,358−9(1953) (6)E.McFARREN:A循扇.C加綱.,23,168−74(1951)

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Tech.Bull..Ka畠a☆a、、A岳r:Coll.

198

の K“OTOZAI:ム.わ少の2.Cゐβ仰.,′4,23ニー31(1950)

㈱ M.MuT百:′√4gグ・.・C如沼.草紙Jt砂αクプ,24,321・ニ4(1951) (9)A.L.LEVY and D.CHU甲G:A??al.Chem.,25.396−・9(1953) 叫 G.BRANTEr:■A厄≠〝γ♂,163,.651(1949) (11)G・E・pALGLIESH:NatuY■e,166,1076(ユ950) (1功 Aい行SuKA:ムJ%α吼Cゐe〝‰,、8,′181・−3(1954) q⑳ L,FoⅥ7D由N:肋わ‘γβ,r167,1030(1951) (14)′A.’SAIFER andI.oRESKES:Scieク3Ce,119,124l−5(1954)

オリーブ果実利用に関する基礎的研究 −

2.完熟果実果肉蛋白中のアミノ酸のペーーノべr−

クワ・マトグラアイ一による検出 檎崎丁市・・片・倉健→二 前報に於て,筆者等埠,凝然オリ∧rブが乾燥果肉のい−・3%の粗蛋白な食有し,〉且この含量は笑晩 期間中殆′ど・−・定である審を報告した.この熟ま.,、オリーブ蛋白ほ構成蛋白であ・り貯蔵蛋白ではない番 を示している小 本報に於てほ.蛋白加水分解物申のアミノ酸のぺ−∴パークロマトグラフイ・−・による検出実験の結果 に】就いて報告した. 1) リジン ,アラニン,プロリン,チロシソ,バリン.トリブーファン,フエニーリレアラニン,ロイシ ン,イソロイシソ,ヒスチジン,アルギニ・ンであるがこの申トリプトファン及びアルギエソほ明確で なかった.

2) 必須アミノ酸の中,メチオニン及びスレオ・=ンは仝然捻出されなかった.

3) 以上の結果より,オリーブ蛋白は栄養約にすぐれているものでない事が結論された. 猶クロマトグラフ実施上の問題についても二,三考察を加えた,特にクロマトグラフ用試料の調製 港に・ついては詳細町野草した 終り笹.臨み本研究を行うに当り引続き有益な助言を争えられつ一ゝある本学川村教授,並びに.アミノ 酸標品を供与された≡野講師に感謝する.

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