Document vXv8yp4M7B0V9jyRG2Jz7bV6

art, - i UD 3k oT AG PERFLUOROOCTANOIC ACID INTERACTIONS win|e TIUMAN SERUM ALBUMIN Ill)MAN SERI.M ALBUMIN | llr GO1LDoN L. NORD]~Y* ^~ ,1. MURI{AY LUC~ ] (ro .~:" te Deri of Cerin, Stns Diet, Sing :;~ (From the D~partm~nl of Chemistry, Sla~tford University, Stanf~ California) civ for eblisiAovnge, , 00 ty (Received for publication, August I, I9~) eyFor svestyabmrpoeunoroantanci aed (1) (OFs-(OFe-GOOM) ha For sm,eral yeura Perflum'o(mt.anoie acid (1) (CP~-(CF~)~,COOH) }ms bas ben obse s protein prchint (5 chai s .:;~ ~en ,sed'in this laboratory as a proteiu precipitant (2). Q~alita~ively it rved 0 bin a campitly fo proto hak. ep .~Thas been observed ~,o bind so completely to proteins that little o~ the pre- cipitant remains in the tilt,rate; this is an obvious advantaga in chromatog- Sth nd in various anys posers ~phy s.nd in vm'ious analygleal procedures. B3 peeusorcctaonoi ceilpwilsl fecsti stn th conditions der wh The purpose of the present study is to define the conditions under which 20d 0 descr in detail 3 prociitation of ar es onic oic acid wil! effect a precipitation of hu~m ~erum albumin F Hunan se. l the niencion betmn ts e to describe in de~uil the in~raetion between these two reactam~s. amin ves ud ha of ty wm e Kuman serum albnmin was used because of its unusuaf properties in bindin vary of ion. peo ~g a variety of ions. 5 ates Mat~r~al~ 2 i1ysoluicn3of0hsumapnesocnu.r albumin eco! procedure) was lect A solut.ion of human ~erum albumin (decanol procedure) wa,s electrosare lcstion thouo sain sonducoly wet ca at 4~ vol~s ~r era. against conductivity water and clarified by isd a mgehrlin le pad. Ths pss filtra6on ~rough a sterilizing'filter pad. The product was lyoft ced dor sohrcedp1r5, snroprin moun of the pc bo ne stored at 3~; appropriate amounts o~ the powder were removed Eso epara of amis se for the preparation of albumin solutions. BE cncve0ti5ornrcciacncnei (25%) hin wee prepare by cat Sodium Per~uorooctanoate (PFt) aolubio~s were prepared by carefully Ce of sod hyirnb snes station of sh es Ctl a 0.5 per cent aqueous solution of the acid with a minimal l of sodium hydroxide. E Proeion Procedure BEPrcpiPtFiSn-v--rA mpurmbeo of bull 03 pe cnt lutions of seu Precipitalio~--A number of buffered 0.3 per cent. solutions of serum alJi oc d. Each ution see dan in PF8 were prepared. Each solution was distinctive with respect JB 0:1 + McPlES concnircion on 0 1 ps she Bt pg ns to i~s PF8 coneent~ration or to i~s pH, ~e later beir~g determined fo 5 ain buf 3. The pH of cach st eee 0,I M Mcllvaine buffers (3). The pH of each solution was between Fle 1d25(.PFS Ttioe aolFbcu onninofnatchitosni uwenerye eerred and 5.25. The PF8 concentration of each solution was su~ that ~he es thorugh mi min) ranged ben 0 and 3000 The sin ratio (i%'8 to MbumJu) ranged between 0 and 200? The solutions BE civic cous, alowed 0 stand To 13 mn, and r r thoroughly mixed, allowed ~o s~and for 15 minutes, and centrifuged BE ir ciopr room tempeforr20amti y a ehnica ce tr fuge at room temperature for 20 rninutes. For ~onditions JR ornostleaiinn.:siwvrees sstoamteitonimwesomsy rieialseBpeoerrt) gt.m protein in a given solution was only partially prec]pi~eed, Supernatant solu~ionu were sometimes formed; in all other c~es the Public l/ea~th Service Research ~ltow of the National Heart i AT 130 as Bel eh of 61 ,~ was .sod for ~hc molecular weight of human serum albumin i ope rn 399 -~ g~ "40? - 33001133..00000011 a es ae . Se E Euiea iind E C1a8 J bi 3A 3 ; 1 2 A3 E :A ai4 i3 3} 84 ErJ EEx3xh0hi1ibb3iitt 3013 State of Minnesota v. 3M Co., Court File No. 27-CV-10-28862 3MMAAO0E036131331100 iH 4 . Le be Eoa Ehe E= 4 sid Pe bs 2 , in FERFLTORS ALDMIN PREGHHTANT 4O0 supcrnat~nt solutions were clear. An nliquot of cach ~upernatant liquidI oTLro,~wswrorylyutieoft naalL.i~ mnmoovddpiifrteiiccc~aatptiiioto.nn~teooffexttihhseetedFF,oollxiiwnnvtt.eteisstt~an((55:))m.- MyPPzFFeS8d fdoureslmn~otcininbt~erf~er d~s no~ intcrfe~ | with t,his analysis. The percen~ag~ of albumin ~hat was precipitated fr0~ each solutio~ under ~he s~a~d condR.ions of pH and 1'1~ c~,ccntra%~ w~inciM oncBui~ndd.ingT--hAe rneusumbltesrapofpe0a.3r hp~eFicge.nt1. albumin solutions were p-'~3i Ani~ Bi~i~A number of 0.3 ~r cent albumin ~olu~ions were p~ Ee i sie woe pared in radioactive PFS. The ratio (PF8 to albumin) h~ each solu6~ w~ between 80 and 2O0. The solu~bns were tllea 6t.rated at 25 to abo~. pH 3 with hydrochloric acid. In the coume of the titration between prepared. ~cc-heli:tetahniintoheeded Half aamnnol'ee,'q~qrn:r BOUN 5O Ty Se te, T | N=ot A \ <X<OAS --~ JS~A J WE E 3 SSA Er SS 8g Fuo. 1. Thepercosfnhutmaansgereum albumin proiptated fromaqueces I $2, Curve 4 Fro 2. Curve A eR ~f the number of P To ~zlar ratio (album 4 4 aanndd 33,, tthhee pprroot[eeiiini precipitated precipitated completely; complexly; several severai 0.005 0.005 mi. ml. lique were removed from eacl~ supernatsant liquid, placed on aluminum and immediately dried under an infra-red tamp. The disks were ee to dik. Aer maxing able corectog | placed on an automatic sample changer which operated into a g~ counter (6). The time required for an arbitrary number of eomzb recorded for the sampb on each disk. M~r making sni~able for efficiency and background interfermme ht the cmm~ing, the relative: tizzies of the s~nlples were de~rmined and compared with the activigic~ of ~heir respective soNtions before the tit,ra~io~ was The molar ratio (bound PF8 to albumin) in eac~ sdufion w~ the~ N~d as a -fun0~ion d the molar ratio (PEg ~ albumin). The data: given in Fig. 2. Hydrog~ !o~ Bindin~A number of 0.3 per cent albumin wave Pecans l:m. 3, The ~f ~!~a pH of ~he s s~@t~m PF8: Cur~' was then tltrate The amount it, wa.~ calculate' "01ution and r~:~t~eted by :vas calcula~ 33001133..00000022 suaceezran 3,MA00631311 1 & pfeoeraproteinn bty hfdhf||| 1sTpFrSeccipointcaetendtfrraotdfos ii n in eachi woke tat.25* ha) in each tail| .-,.ted at 25 to 3 i& ~,ration between 5 2 a 1. von Ax 3M. TOK w G, lJ. NoRDBY AND J- M. LUCK 40l eretp.t, 1100 L re ivioe ns crivdwa ntonss xc ni . SEL prepared. Half of the soiu~ion~ cont~ained ~hat cottcenbraI,ion of PF8 for meni of scr. Exch 5 ~chicb the molar ratio (PF8 ~o ~lbumin) was 196; the remaining solutions contained an equivalent concentration of sodium chloride. Each solution aoe ore 7 sacosrofu ~oo .02 wo + wo ; a =To .,50 frie 200 ..?! .... ld ~01~-:" .... 7 wo150 Iil -A 3esgts1abun.moll.. Cor33e heerr Fro. 2. Curve A, t,he molar r~tia (PF8 ~e albumin) in a system determlne~ the I number of P~ pinions that bind ~o aach Mbumin molecule, Curve B, thv recip~ocM o~ the number of F)~ anlo~ bound ~o e~ch albumb~ molecule is plotted agaio~ the te 8 rs atedfrom aqueous scl ' a1 0.005 ml. sliquol al 0.005 mL aliq~( in 03 on aluminum The disks were Eonenoef acnonte e og] rated i,ato a gas umber of counts .g suit,sble nt,ing, the min, he gn] ,red with the ~i~ration was Ce ben cl] lation was ~hen esis 1 stain). The molar r~fio (Mbumia to [r~e PFS). / -5O 4+, 0 A I = 1 : IOO PpasB a Ltn or. vethe x stratod at 25 with cbr hydroctloi ak osFodium hydroxide nHanScdulLatm edi quiteo di. rectly e from theThtortd ale4droenoprs tbrhaeseiaaldnbduemdinFtiolagli3veen i 013.0003 3013.0003 I Ii i i| I i i I i i! i lL i ii | i i | i -- 3,MA00631312 i Ig | bi! 5 18:i PL Fy i f i 1 |i 402 Pr*clpita~iun--Alt~ough Fig. ! indicates a ce~'tain critical mng~ of ~ad PF8 concentraLi0~ which mus~ prevail for the precipitation of hu~na~ serum albumin, it. also indicates in a. qualitative serum albumin, ~t also indicates in a qualitative manner t~he manner the cond Additions under xvhic]l most other protein~ would be ~xpeeted to preclpit~e. Thr~. ratherstrikingfeaturesare noted for attention. rather striking features are noted for atMnticn. (i) The ccnditio~s DH and joni ctrongth under which complete (1) The cenit ma pH and ionic strength under wttich compleM albumin precipitation ocm albumin precipitation se 80 et are very ~nild. (2) The precipi~tion of albumin is completely bbweeithscoomrmepslppeleetcteetl,lyytoddpii~sHso.ollvveeAddn albumin precipita~ formed in a PF8 solution bbyy mmaakkiinngg tthhee ssoolluut6ioonn ssoommeewwhhaatt aallkkaalliinnee ~fpfH 3 ~ 7) to the pH at Which preclpi~agion occurred. The minimal pH which the solution mubesraitsed will depoef cnour,se, upon the pre BE hybound to Scwpohanicccehenntttrrhaaettiiosoonnluooftfiottnhheemssooullsuutttiibooenn..rais((3e3))d wilt depend, of course, upon the PF~ IInnaacciiddiicc ssoolluuttiioonnss,, tthhee pprreeccipiiittataitoino, | albumin is irreversible with r~pect to the PF8 concentration. Provided *ha~ an albumin precipitate i~ diabmed against an appropria#~iy acidic solution, it wil! not di~olve appreciably ~ the concentration of PF8 in cqulvium vith he procipitte desea. equ~fibrium with the precipitate decr~ses. However, the P~ precipitator can readily be dialyzed fromalbumia However, shoPRS recs arb can rcadiIy be dialyzed from albumin in neutra! or slightly alkaline sol,- in neutral o slightly alla By Stonhc dons. Ari dig Thosoufre i Fi. 1 Anion Bi~Min~The scope of Curve A in Fig. 2 ]s restrict~ed at the upper ricaat ee | orion limi~ by the ra~her low solubility of PF8 in aqueous solution and at the lower linait by the concentration of PF8 which wotHd adeqtmtcly precipi- ~ed, by virtue ~ apply to the conc.~.ntrati mo!ecu le Additions some less Curw number of P] be calcula" Ey acid ~36 ccta~mate isotof wigbound 5o ~ydrog~m. Ion a]bumln at app]ic~ble is readily appa~ melcu]~ by Stcir~ha~ 6-trinitroresor means nf tht t.a~ albumin under t,he conditions of the experiments. Rowever, it is carfrom the graphthat considerable number c]~r from t,!~e graph that a considerable m~mber of PF~ anions biud to each of PFS anions bsg sh albumin molecule. The molar ra6o (bound PF8 to albumins) i~crea~e~uite rapidlyasthePFS concentrisaintcrieaosend. qu~te rapidly ~ the PF8 concentration is increased. In contrast, ~he mola~ In contrast. the sor ratio (bound P~ to albumin) decre~es very slowly ~ the molar ratio tltt(ahaPhttee~Finn8mmggiitnna4oiimmggaiailavvblleeunnccmoocncincn~ocen)nencentiee~rtnnratdattrrteiaaicototirnonieora~noosffeoodPPffFFbaa8SellblboWuwuwhmmhiiiincacn,hhb, oititushshteeeeff1PPff2eFF0ecS8.tcitviiiessbiviiaanenllmsmoccooooIrssunttt~;piVolcneloetzmteelpctyyolenLptperarl.eeyi~oc:i?riapi~-g competes re. moved from the solution as par~ of the precipit,afh~g complex. tthheeTrhreesesuudlltattss, aaapopfppeeCaarurravasse CCAuruvearvrce tr@tcd by an BBooff FFii~g. 22.. exprcssion derived hv ~;lo~z (7~; of bound ~nm]3er agre~ Fe athe ~e albumin Rp of not dotted line BS~ fuTncetinon ooff - FpHa4a.2s5 :at in the prcse~ ~ extra hydro | Bboeunnsd tua~t,]w. ~ B~ XiIv;1 ITnin the mtthhaeelaaarbboorvvaeetioeeqq(uumaaattxiioiomnn,a,lr1 ghe to!!at ratio (maxima] ii.~s tthhee bboouunndd mmPPoFFolS8laarr~torraaaattliibooulm((bbbioonuu)un,nddcmiiPPsstiF~thhS8neepttom)aoioaa],nlnlbs.bruumrmmaHiihnno)), , (1 PF8 to albumin) in the solut.iou, and'K ~s a v~nstn.nt proportiomd to the Fei sive, Aeevvaiidncddne~Bn:ecte in alkalin E eqvi~ibrium vonst.ant for the PF8 n~ion-Hnd;ng reaction. The cevviiddce~nlccce lltthaatt itJhiec eeqquuaattiioonnddoocess aappppllyytto~Pl'FFS8 aanniioonn bbiinnddiinngg,, hit. ccaannbbee 25 Blow cuncentratic : 33001133..00000044 SuAo031313 3,MA00631313 G1. Nom avo5. 3. Lex 04O8 1 BJF3LaSp,ePpbbeylyysvvttiiiorortetutttihehoeoonffllirttnnheheaeevarerillinnsppeeooaratrtrhrtiatiitooty.nyn..ooffaaaTTsihpomeoirrtimtilion~o.dndniiccoooaftffteeCCd6duu3reervvxPxetetFrrSaBBa,pp,oaotlUnblaa~iatAtoiin,oosttnnhhbeetoioesqnuifunaatftiiotiniooiatt1nne Borcion~Acmmdcdonoikltterciacontunilloaenl ,brabeyytvieoraenacsa,t~acirtitoeohnnabs~souahhnaadmvviio~nngxgsimnnleeuaaumrrllmoyyf att6hth3eeH.iPssg~aah?mm8PeeaFneeSiqqouucnioisllniicbbberrininuitdumrmattocci.oooannnns- {BFtsioiomboeenAeslede,rsdssiCCtcoieauaof~r~snseaPlai,sleFylySaBodndae~ifonossficncorFsrFniiiiabbsg.ree,etdd22abssotbebeureerinccicad~oonomm~ofeopefsosansrrlesneebiaaeaubrccalmtltryiliyoiyonhnnivssvane.e~rdrthtiiAcAticagoLalth,la;vvnPee~tFrrahhyy8eerlle~hchfiloifgoognrhhriceetcPPnthmhY~Foe-erSl8~emmtccclaaoouoxxlnnn-~es- + BJBe usc:tcbvaeeaanccccicddauleoecaafuunnllPioiatoFontenen8eddbbabbinibinnyyidondiniettnhnshggee1te0hbbveyy~vividdoTTceeeatennr.5nrcecepesepseiioasavsaavasnoniiafblddiallyasbLiLbltubeluee.ciscn.kkdon((ITt88onnt))haccerrnooeonanvvatle~erldbabarl~sulustsLmm,i,ttnihhnaastmtmsttauuaoedldettyyoceout~aoolefIlf 30 o [8B.B5li5mse5oiieanrtrbbaaerosnonyouuannTibtdd.o~onsttpooiooBpentetihhsneeddbdiialiisutnl~nbidi.mgios~m--nnntToiinwssttoawmmauoooih~lelyesetcsdycurpulidoenleeig.ds.ceincoaafttfseeoitnetthhsb~ionndttihthnheegePPcaFFulbS8ruvogmesninnifoosm~r oh~sleurecmuarleen-. i 5 | [GiBn eslrteao`alsspbuppuiml]miii1caicnognpa~bpblaaaleBrreeeiLtn~ooddtiee~rsppfeeigivvocc~eettmrreeT~addwtbblhlioeenionstthFFiioy~citggdrr.ua.rartoti35vigo..oeennnsssTTohihatoveavetnseesrerPebRttihbbnShiiedonnsidndtppirigHHnonnggcg~urlccyarnuvnugregnvees~es[isuloltaarurrrseechtaruatccmtoeoetammddhl~e-. all| Bi~ir~iatdymmiloSyolteleeaccpiuulpnleaaerrecc:uhh~aa5 rrfgrseoomirointthh~aececilsddesGoisclrouualtruiivtooeinons.nto.hf awAtAaPlsspii~mrmioilltsaaterrrioneenfigfcenlcytt~hinehhfaalpssureebbnseceeeennnscetooohbbfe- 7vo% the JBJRysE5Amm4sbraeiiynan0niSnstisrteotnohorfciefibsot*taohhrrereedcsLiehesnyqqor(duu9lrca,)aotigtipfiieoooinoccrnlnrcfit,chddoeeenasasctccciidcrLdr,iob,rniaecbootdelrerondtnafarblaaooavbtvfovivieweiao,,nnoniottchhileen~padccrieeoiicxxddtat,~.teraeiansppooitlnlahat~ttiihooentnhper~oef saCCeuruilrr~vvleeossf i JBFEJEBsnruuBsmmoohtfefboriebbiroonsnuuf~iinngnrmddriocetelehhsesyyhcddwuywrirlodoietgtrgehhoneginnttehhneoaiaox~ntintossrfnooeuppucmenenolrrddyanalcblebabbycnyuuitmdmrTTiaasaintnicnnolfonummortarilioddneolendc(si1(ucf10i0la)ne).~.stissheT~T11hhp0h0u~r77uste,ssiit,nnehsntehehcceaeemchchoachfxhac~iatramharsgsgeaee.el. 2 FJoBeintailbonunomt ilc)oooffmnPoDdlFFeS8c2u5lrr,eeep7ipnr0reessPxeeFntnrStetemddsoeoblbysyy ara&cidemmosol~lbaalorurutirrno~adattiisowoien((ePPtrhFFe8Stthpe~soroesaealnlcbbocuunemmdoiiitfnni)t)ohnaoosftf oJ I exceed 28; 79 FF8 anions ~re bound under fl~eae col~ditions. ted in on Fig 3 represents the difference between Curves A snd dotMd line in Fig. 3 represents the diffsren~ ~twecn Our~c, A and bu function of pH. The mamxyimrayl difeence betweenSethe 60 curves a function of pH. The maximal difference be*wren the two curves +. JES enxintpHrfthlea~4pc.!r2d~5erresasoetcngowceehooicffihoPPnRFasSnS..aedxTtTrhthaheee6s4iGigg3hniysifdftcirrcaooanngngceclsney ooiobffnottshuheenadrcceooPiibnFnociSiuddenenadnnccieheoynbbseethtwtwehcieeacelnhn- rihocunnedxga1trnt0ao~hdhahthahtyLeedriitLoctsdingibtdiieesusnumbbanirironoonubuiJsgsghmhaontontotdtftv.pookKtinhnleasooe.wtt5w.ne.t6ent3uait~aotsTitnt(rm.othhh.niiiigslFFytmiihannenabcalsetoll;liuyyo,nhb,ndutaiLttlsP.,t~sFihhse8e gatccapphootppodiiaugnongrrcnehceidsnnie~dwnnecthtonhehh1cnaa'ih~etss of Benivec, Aixenviainddadeenn~icceceohthainstaoc.tlidutttehhiceaonbsPoF.FvSe8 completly pH 8~ This comph~ly disociate from the albumin is additional, although incon- di~ociaLes from the albumin in alkaline solutions. R bFErbcen onecnirsivonnsitaotf phearn,voiressstermeunmycibi5acn. caTnnitbohoonrfeoerrmsaihtileolnyrpoorfemsi )b . ~=~an 33001133..00000055 5 F 3= 3 53 Ff! -i1 73 xif 3i .H3y a3 -o1 f F# aE:5 i4 3i 3Go IMACDB31314, 3,MA00631314 4 !: : | ii i g. iE R?| a a aI] aI '|i { Hi a 1 [Er -- : `precipitate, the albumin tanto ion ~n mmoolleeccuultee bbiinnddss bbootthh hhyyddrrooggeenn iioonnss aanndd ppeerr~fiuuoor,~. ~ comple ha taosfomm,tucnhdaerrgtohf aconntdosser,ofthe epeimens sec oct~maff i~ ~h~orsmn,c~ucnhdaerr~~hocf calomnodsitti.o'znesroo.~ the ezperimen~ |8i THoE DRATEASLOFIN1x ftihaneinAtgoniacrd EMnaerrgfyacCtoamrmiingsiCoonmpfrary tfhoer M+insneisoet of pertucramrers | seid, Carboxy labeled (C*) prfuorooctanoi seid wascaine trey. | (Received FraAAccctkkinnoonowwlVleedd(gg11mm)e,enntdteciissanmmoaladdheeuttmooantthhseeorCCuuutm~tteearrlbLLuamabibono,rraattao,onrdriie~stoffootrrheaa Fraction V (lt), dccanol human serum Mbumin, and to the ssaammppleloef Minne on Mining and Manufacturing Company for a ~mple of ~rflu0roovtanoi~ acid. CarboxyMabeled (O0 perSuorooc~noic acid w~ obtained cing Company a Miri a the Atomic Energy Commission from the Minn~o{,a Mining and JRE BUALR(LF,rMom.thSSeTTEEFVuVlEEmNNeSrS,,OBrL (Received Er onto of Arms tacturing Company, fi.db~gs of Arnstein joident od Bsbvcasp i rr re JTREory (0-5) have esa toy dex Waals association of ovineserumalbumin in the presenocef perfuom acd Addc~dura--l~levens and E[lenbogen have recently published '~heir research on lk~ van dee WaM~ association of bovine serum s]bumin in the presence of perflcoro seid: BE Ccivly incorporate (12). 3 OB ad Diet, smzoomamy JBEcvciia, viheiothi ce BIBLIOGRAPHY ond ar Chr 8,55 cs. BB pte hae ue sfic 2 Lack 3. M. ifn, A.C ovGr.a,od Win, MJ. i. Chr 6,75 I. Kauck, E. A., and Diesslin, A. R., Ind. a~d Rn~. Chem., d3, 2~2 (1951). 2. Luck, J. M., Gri~n, A. C., Boer, G, and Wil~on, M., J. Biol. Ch,~., 2 Mh, at do TC.. ik.Lk oChr, A tn (1954). Cwohea iden Si113Gaim) BE:l ensdof pseiretnviel wbayt a| &~. LCLohowawrrrhyvy,oO,0o..dI,H'.IP.,,R,PoAs~e.s,beBrbo~rouovcghghc,hm,.NN.,t..,JJ,.5,F1Fa,rairrie,~, A(A.i,9L5L.2.,,).aan~ddR~a.avoddlal],l, IR~.. 1$.,,.J/.. BBi.ill Cien, Tr 1, ne. Ch, 5,0 3 FE ine 193, 26~ (t95I). 2. Riau rk , 6. Ny~, w. N+, ~nd Teresi, J. D+, Arml. Ch~m., ~, ~3 (!951). Te 50 akDh50 k8 } 7. Klo~z, I. M., Arch. Biochem_, 9, l~ (1946)~ 0 Bor, C G, 0h, 3 . : 899... STStteeerieinsnhih,aaJrrd.dqDtJ,.J.,.,,a nAAdnnnnL..uNcNkee,wwJ.]YorMrekr.,kAJcA.acdBid.i~. lSS.c.cC,.,he~4m1I,;., !94, g23 (I952). 228577 ((11998~11).). I Cn 5, GCrd . oc 5 Eo niin or !0. T&nford, C., J. Am. Chem. Sac., 7~, ~1 Derutun GeiJrn0,ne,as,, bs Dn,3. ons, 1.5. JE dermaof 11. Cobs, E. J., Gurd, F. R. N., gurg0nor, D. M., B~rnes~ B. A., Browa~ R. ein, A G2.me.ve.7 is 6 si [ on Deroua.ux, G., Giltespio, J. M., Xabnt, F. W., Lever, W. F., Liu, C. H., Mittd- th on Sn. EenePienn vofedrioon man, D., Mouton, R. F.~ Sehmid, K., and Uroma, E., ,L Am. Chem. Soc.,?L (3-5) have estab] [ig 0 addededdttoo ppeenniicciillli~ eate~sively incorporate that the entire cy.~ htilized, while the [88 ~wproer,tedcithsavie efaielendt' amino acids ioto seemed po~ibtc tha~ 1 of penicillin by JQEBA vvaalliinsee uunnddeerc ddiiffffeerreenntt , oa the rel~tion~ Spa pnroacetdeuress fooer oeuhll,m and the methods for of radios the present experimm at. varying times after 2 Tn. sd lt Dicia ns Fri, oc,1, co. Ritoegnnsiopapicilcin vas 12. Klevens, H. B., and Ellenbogen, E., Discusion, Fara~ay Sot., 1~, 277 penicillin was vas irlocultllT~ wR~ ser somnhas hier were somewhat higher fo icc omie o J~-cysfine was o Illinoiz, and ------ Company, Inc., Ec ne re~olved hy the genarM BI chino i ~esolution of labeled BB c dinne ~e and 350 mg c ndeye af ho~ water and crys vk ve ore work wau support,ed in Bnd Joes ~ndianaoolis, ~odlana, fEle 0 Wanton of Washington Init ~cp;trLinew J 33001133..00000066 sMaGoBa1315 3MA00631315