TRE UBERA'HON OF PHOSPHORUS FROM CASHN DURENG PROTEOLYSES BY TAKA-DlASTASE Thuhforthobogmoffiks. MiCHEGAN STATE UNIVERSH‘Y Bobbie Jean Nicholson 1955’ LIBRARY Michigan State I University THEILIBERNTIOI 0F PflDSPIDRUSIIRflM CASEII DURING PROIEOEISISJII'TAKA~DIASIASE By Bobbi. Jun 11mm A THESIS Submitted to tho 601103: of Science and Arts otilinhig:n S‘ato'filtvvruity*ot Agriculture Ind AppliOd Science in pnr‘ill fulfill-nut of tho roqnirunonto tor the aggro. of MASTER OF SCIENCE napam of Chantry 1955 acm‘mmr flu «the: fish” to apron her gratitude to Dr. H. A. mum: and other when or m funny at the Chaim Depart-mt at Michigan sun Hunt-it: for sundae. And monument during the period of thin van-k. W W W m G. ‘I m1 Tho ocular m born October 3, 1931 at Pomona, lcrth Carolin. Sb inducted tro- Brcurd High School, Bronx-d, Ion): Carolin, in 1939. In 1951 we received on Auodato of m. Dog-cc tro- Hm m1 Junior 0011.“, Hart m1, North Coronal. ' m 1953 ch- Mtod troll Mon Unimoity, Granville, South leinondth c Bachelor of Science Dogroc. Sh. no cdnittod to tho School of mu Studio. ct 11101313“ State College in September 1953 and ' hll boon to Itmec thorn line. that tile. Tho unborn oal: clplcyncnt, cthcr tho a account. fucking I lad-cm m1. ct moms“ out. 0011.30 , on during Mn manor of 1953 in tenth rocou'ch ct Dcoflng-Hfllikcn Banach Tract, tendon“, South Comm. l’hc outba- u a Junior nubcr o: the mica Clinical Socioty, labor 9! Chi flcto Phi and Sign Bolts Epsilon. THE LIBEREIIOI OF PHDSPHORUS PROM CASEIH DURING PRO'IEOL! SIS BI rmansusa: By Babble Joan Nichol-on “ABM Bub-lttod to tho college of Scions. and Arts cthlchigmn State Univoroity or Agricultcrc cud Appliod Science to partial fulfillment of the ”quit-cumin for tho cop-cc of mm C? coma Dopartnoct ct'Ohonictry Iear 1955 Apprond 1v LBW? Protoolytio onoynoo fro- nicrohiologicol oourooo no): no I. subtiluo or A. m bring about the digestion of coooin with occcnpenyinz too-notion of o ling oppoaring oolution. Such io observod when Ton-0W io eddcd to o clcor, control oclnticn of min. This otudy no concerned dth tho noturo of phcophomo liberotod during ceoein protoolyoio by tokaiootooo. Tho quontitioo of inorganic and 1H trichlmoootio ocid oolublo phoUphcc'uo rolooood more dotcrlinod . Proteolytic octivity no oincltonocuoly followd by increooo in option donoity ot 280 m o! the ocid ooluhle hydroly- ooto out by chongo in its epecitic conductivity. Both roto of opooii‘ic conductivity incrcooo ond inorganic phosphoruo lihcretion indicotod o tn etop reaction. The til-It etop prccodcd onoot of nilldncos vhorooe tho eeoond tollovod. Incroaeco in optical dcnoity and totol ecid eoluhlo phoophcc'uo whoa compared with digooticn tine ooro linoor. Protoolyo’io end on tone of phcophcroo liberotod ohm-Id identicol dependency upon ccncontrotiono of cum , ouhotreto , ond hydrcgon inn. Thornton thou voriobho ore boliovod to be directly oooooiotod with one ond the lane proteinooo. Theeo experimental ”onto ouzgeotod thot tho initial roocticn(o) preceding mun»- involvod the diooppooronoo of :5 -coooin. Further- Ioro thot thio no eloc cocood by e protoolytic cnoyno cetolyoed hydroiyoil of the phoophodiooter end ocmo peptido bondo of thio (notion. Aloo, of. o much clover rote thoro no ocno hydrolyoio of nonophoophoto groups to produce inorganic phosphoto ions. The reactionh) following nilkimco no regarded oo protoc- my cotolynod hydrolyoio at tho phosphorus ond nitrogen bondo o! the d-coocin fraction. the on or o opociric phoophodiootorooo , i.o. choke voncn tron tho diamond book rattler (Ontoluo odamntouo) , on coooin brought about tho roactionh) of otop one. Tho diooppoor- once of 6 min only, following incubation, no danonotrotod by eloctrcphorotic molyoio. TiBLB OF coma-ms Pogo IISTOBICAL. 2 A.EquipmentUIed........................................... 15 materialismSolutionl.........................‘......... 16 0. Emermontal fiethoda...“................................ 18 D.1'ab1u ofResult-“u“.................................. 23 E.F1gm'ea.. 28 Discussml. 36 WIOOOOOOOOOOOOOOOOIICC'QIOCOOCOO'OIOOOOOOOOOOOOOOOOOOCOOOCOI 1‘6 BnumRAPMQQeOOOOII...-OOQOOCOOOOIOOOOO0.000000000000...-.O..O. 1‘9 711 I. INTRGWCTIW rho protoolytio hydrolylie or cacoin hoe boon invcotigotod with voriouo my. proporotiono for tho purpooo of gaining interaction obout protein otrnotnre, "cheni- ct rooction, or tho rclo of tho protein in nutrition. Solo «once of onoyno ouch no tron cultureo of Bucilluo Ebtilu (38) or W 33133; (35) bring obout tho tornoticn of . nillq oppoorinc dizcot otter addition to o. clear control oolnticn o: omin. Itudioo on tho protoolytie changoe with roopoct to nitrcgon producto during thio taut-notion hovo boon nodo by new (35) end tho r... enlto eeon to indicote thot o two otop reaction ie involved. moctro- phcretio onalyoio during tho reaction ohm that 64mm: diooppcorl prior to nilkinooo end oubooquontly d -ooooin io ottockcd (37). Since coooin io ono of the noot provolont phoophoprotoino and due to the ocorcity of proviouo study, it named logicol to invootio goto the noturo ct phoophcruo liberoticn during proholyeio . II. HISTORICAL is. The Occurrence of {110322931335 Phoophoprctoino ore found in obnndonco in embryonic tioouoo end in the food for tho young. The phoephcprotoino of milk and one hove been the noot extensively otudied. Vitollin, ovolbunin, end phcevitin are the outotonding phoephoproteino at age. The noturo end proportieo of these proteins hove been merited in oovcrol booko (62), (8h), (28). Coooin, found in am, hoe received the moot ottenticn of ell the phoephoprotoino. Ravine hove been written by Sutorneiotor end em (18), bananas}, end oloo by Moflocldn end Polio (ts). WW - 0mi- N W W by moral Meant nothode. hereton (2h) aplmd oeotic ooid tor ioolectrio pneipitotion end hie purified product oentoinod 0.85 percent phothreo. Il‘he ooooin propmd by Von Slylco and Boucrth (as) by coin; unaut- hydroxide tot- autumn. end main euloto to room cold. contoinod only 0.71 percent phoephoroe. toner (J) repertodthotoholocooeinpreporedbyuoingonixturoottive percent mun-it end nitrio oeido contoinod 0.16 mm phoophom. Geooiovoolcngoenoideredepuropretoin. 31927 Moms-#- in. end no. (39) by ontroetion end procipitotion nothodo decon- etrotod thot ooooio in o nixture of oovorol proteino. Lotor W1 “0) “coded in eoporeting cooein into three ”MW “3thth cm. The pore- eontopo were 0.96, 0.52, end 0.10 respectively. Cherbnliez end oo- "bro (h). (S), (6) principolly with the oid of five percent ”in chloride on! ooetone precipitotiono preporod o nunbor ct oooeintx'octionoondouhfroctionootphoophorueocntentrouingtru 0.55 to 2.3: mt. cub (at) by the one e: mi, phenol, end oleeholio o-enio oncoooded in oeporoting ton coeoin tactical o: phoophcrle content. rousing tron 0.65-0.90 percent. mfl' (51) found three mu when he electrophorotieony min-c m ooooin o: 0.86 percent pea-phone. It nod nu- do, 61nd Y‘ cooein booed on their do‘ereooiug nobilitieo. Werner (85) no the tint to tone! the {rootionotien ct coooin by electrophoretio onolyoio. Iron untrocticootod cooein of 0.86 percent phoephcu-oo, he preporedtho dw oniéwrrootionooithophoophonoomtcntctlfl end 0.61 percent reopoctively. Bordon end coworker. (20) offinod the one phoophcnl content in o(- and daemon. on 1950 app on! coma-lore (29) deter-nod the We content othooein oo 0.11 percent. ‘l‘h‘oyoloo pl‘oplredd- ondé-oooeinbyo mdcohol Iothod tilt onolynod 0.98 percent end 0.55 percent phoephoruo reepeetivoly. the phoophorno content of Y-coooin eloeely race-bled thot of tho oleohol eolublo protein or coooin «puma by Ochoa-no «a M(57)131918. Iydryig‘rinding o: «sitcom (a) in 19k} produced o voter eolnblo froction o! 0.68 percent pboophcrno end on Mable (motion of 0.85 percent. Who» of main and rroctiggg - Poeternok (66) in 1916 ioclotod o phoephcpeptone from o too to three doy tryptio digoet thot onolyeod 5.86 percent phcephorno, About the one tine Ellington oodloyflfl) ioelotodophoophopeptcoointhoouononnerthot con- taboo 3.8peroent phoophcrco. um? 9mm (66) mime the phuphcpoptcnootrnctnrothothehodieolotodthcprovioueyoorou ototod thot it contoined three Ioloo oi' ioolcuoino, three noleo of urine, three noloo or glutonio ocid, ond oix noloo of ooportie ocid. zips-nun anizwtatbtrinttisstbstpbospm rooottochodtothohydroulpoupoteorineinooeoin. Biophoopho- poptono propored by tryptio Ivdrclyoio of ooeein contoined mt P01" eentphoephorue. Mono mango) were thetirotnerkere to ehov not amino with night he ottoohed to oerino phoophoto. They ioolotod o dipcptide cupoood of urine phoophoto ondglutanic ocid ond pro- poeed .w otrncbcre to be either phosphooeryl-glutonic ocid or Murine phoophoto. Three grown o2 writer-e published further findings during the yoor c! 19141. Landon, lioeoro, end Plinor (£2) ieoloted on octo- poptido tr. eooein tryptio hydrolyoote containing too nice o: clutuic ooid , too who of phoephooerino (S .67 percent phoophcru) as ssis or dicorbcxyiio ocid (probohly ”pol-tic), end three Ioleo 0: turns- einplo nuns oeid (probably iooloucine). mington (68), (69). (to). (m. tru- bi- work an ”rpm momma-s. mega-m thot tho oeoentiol phoophcruo lining in cooein cooled to be between phoophcrio ocid on conne. Serino oppoorod to be united in peptide none. tith othor enino ocido, prodcninotely nuts-is end poeoihly ioolonoino. He found the phoophopeptone lith Lb percent phoophurouo to be composed of five noleo of glutmic ooid, four noloo of ocrine end three moles of phoephcric ocid. Danodaran ond Rmhmdran (9) prepared o phoephopeptono by peptic followed by tryptic hydrolynie. The boriun oolt of the phospbopoptons icoloted snsiysed 1;.) percent rho-phone. . Hollander (h?) icoloted o trypsin reeietant phoophopeptono on the biz-1m oolt :rsn mun silk coeoin. no later (he) ototed thot o lorgo [port ot the phoophcruo in human coeein io converted by inteetinol protoclytic enzyme to phoephopeptonee of h.9 percent phoophcruo content. Hollondcr (is?) also determined thot hunch milk .tdiole moin cont-inn Ira 0.25-0.1t2 percent phoophcrue. moist snd Shine (510 by coooin ti-yptio digestions snd froctionol precipitation obtoined whet they believed to be e relotively pure octopeptido, The wine ocido preocnt were one mole of urine, too ssiss of phcophcoorino, tss noloo s: iecleucine, tso ncloo o: glutoIic edd,ondenelclenetidentii‘iod. monthopeptmomtreoteduth o phoophotooo, eoryl-tlotuio ocid no liberotod. Thio no the tint indicotion of the phoophooeryleglutonic ooid redone in «coins ci-yotollino phoophooorino c: 15.80 percent phoopherco content rootirot obtoinodndeoqoredoithooynthetio oolplobylgron,0o Vordier one disosst (1) in 1951. The tone-wing you- Do tsrdisr (10), warm, isolated end compared phoephothreonine tron caeein digee‘te. Re eleo publiehed the one year (11) the finding that bovine casein conteine 6.) percent phosphoeerine end h.9 percent phoephotlu'eonine. _ The only our): reported on phoephopeptonee of eepareted oaeein traction- hee been that of Peter-on, fierrington, and Meekin (6b) in 1951:. They ieeleted e phoephopeptone from the tryptic digs-t of B omein that contained 3.0 percent phoephorue. Perlnonn (59) reported this relatively chart polypeptides core team then 5-eeeein no digeeted with e phoephodieetereee followed by phoephalonoeetereee. ot-ouein (63) me einiler pm“ when it an: pretreeted vith l phcephcdieeteraee renewed by e combustion of phoepimonoeeterue end e pyrophosphetue . B, flame Studies Releted to PhosEhoruo Linkage in Cuein We and rhoephorue Liberetion . In 1895 Sebelien found ceeein to be completer eolubilized by trypein but mode no investigation- with regard to the nature of the phosphorus liberated. However, in 11398 81121, e etudent of Salkowld, extended thin egoti- nent by precipitetin; about 27 percent of the eolubilised phoephu'ne uthugnemumeendpruuedthe reettobeergeniepheephoree (65). Bali-e in 190k eteted thet eerily tryptic eotien on ceeein pre- dneed e met inoreeee ct electricd conductivity end Iith Eliminer (65), e your inter, reported on the retee of eepmtien o! phoephcne tr. ends by tmein, pepein, pepein, and new. by utilising tunic ecid as protein precipitant, they concluded thet the tot-J. eeideoluhlepheepheteaeeplitoflineuyeinilerte thetofthe ecu eoluble mtrogen end cal-equaled in the eerly etegee to the elects-ice]. conductivity increeee . Poplin m Inch clover end never coupletely mobilised the min phoephol'ul. Pepein renting in metro]. nedie produced rceulte oiniler to trypein. Ellington end to: (72) extended their etodiee to dietinguieh crude Ira inorganic phoephorce eelubilieed during preteelyeie. Io incrgenio phosphoric one found being pepsin, but with high mun“ ottrypeineunemntuenyccmrted. Duringthreetotivehmr dimtiom ct pn 8.h end 37°c. totel phoephorue ea. libereted et e rote coupes-able to chino-nitrogen production. The organic pheephme roleeeed eppi-oeehed e um elm: end then dininiehed. Inca-genie ‘pheophemeproduoeddm'ingtm tineeeeceneidereblyeloverthen on: e: the other for”. Hettenheiler, neon-nu, end Zflllor (ht) teeted the pouible phcophe-nnkece eplitting power of rennin. Gryetelline rennin did let chow en: pheephorce liberetien ectivity, unleee it eee activated eith therneeteble ultra-tutu“ of crude retain or milk. The Erect of mutton on mole 0% .. Phoepheteeee ere cape thct cetdyee the hydrolyeie o: phoephcrue oompounde (31;). With reepect to wgenic phoephorue compolmde (73) the phoepheteeee Ieybe epecitic co to whether the linhgc ie in phoepmnoeetereee, phoophedieeter, ptneprieenide, pyrophoephete , or ecyl phosphate cabinetion. Further eubclueiricetion it booed upon optimu- pa, end/er need or netel ion ectivetien. Rom, other pheepheteeee ere quite epecitic for one or lore Inhetr'etee only. Phoephoprotein pheepheteeee (not chained by any authority) ere timely epecitie tor “home M'h u chain, Vitenin, end phoevitin. They do not teen te be entire en eingl: bucked cigemphoepherue eonpmnde. the amino phoephononoeetereeee (optim pa 6.6.9.13) trcn bone, kidney, on epleen, end inteetine here not eheen pohepheteee ectivity en mutated omin (23), (1s). ' out in 19% mt ieoleted tn eneyne tron bt‘in frog egge (gag; flebiane) end leeperd (reg one (a 241129,) thet liberated tt pa 5 inorganic phoephce'ne m- min, Vitellin, or heeted egg yolk. Glycerophceplnte end nonophenyl phoephete treeted in the cane nemer geve no each reaction. Since thie eneyne bed not been pmiouely deecribed, he celled it pheephOprotein phoe- pheteee. In 19h? mired (a) discovered en 0118er in the ninetioetttt init- at oitrue Mite (W, 1m, end grapefruit) thet releeeed inergenie phcephete tron ceeei‘n ct e pi! e: 6.0. In en ettapt to m the cane, he trheted d aglyoerophoephnte, ’5 -glycerophegu... putt, end fv-nitre phenylphoephcte in the out me, the first three enbetretee yielded eene inorgedc phosphate , but the leet one did at. Hamlin 10! M erretic utifltion. He decided that theeneyleldtichprcdncedinergenicphoepheteeeeenecidphcepho-ene— «term. he elee found ribonucleeee, eeyl phoepheteee, end epyreee ectivity in thcee trnit juioee. It not reported in me by hinttein and won: (13) that to onlyle in ret epleen eplit inorganic phoephete tron cecein. Megneeim ion counted the eneyne vhoee main. activity one et pH 6.0. The enzy- mnld not produce inorgenic pMephete rm phocphceerine under the one condition. Inhibition nith eodim fluoride produced the re~ auction of inorganic phoephete but the appearance of eoid-ocluhle nitrogen nu Inflected, end there to: no ecmmuleticn of organic Whom. They concluded that there no on encyne noting upon protein phoephorue not requiring proteelytio activity. Newer; (56) toiled to oenfire the reeulte of reinetein end Yolk. In 1950 he eet out to dononotrote the cxietenoe of on independent phoephoprotein phcephetue eneyne end to find ite dietribntion in the different out-gene. le ieoleted en eneyne tron rot eplm that et pH 5.8 releeoed theta-do phoephete tro- cceein end phonitin. He found high activity eleo in halo, edrenele, eon alende, end kidney. Lev eetivity no found in heert, eleeletel noeclee, red bone terror, end erythncyteo. However. he one unable to dencnetrete thie activity to either nun-pmmpepteoo or phenyl phoophlto under the em condition. at tapered heat lthiiity, pr sensitivity, optimal pH, end netel ectivetien beheeicr e! thie eneyne with thet e: ecid phoe- ph-atceetereee , mutt-cl pyrcphoepheteee , edewl phoepheteee end dune-oophcepheteee. lo found theee propel-nee were not coupenble ' with try of the other may.» and concluded that ho had ieoltted t phosphoprotein phoephetue . 10 loote end Kind (16) isolated an enzyme from chick embryo in 1953 thlt no motive at pH 5.8 in releaeihg inorganic phalphate fro- oeeein and phoevitin. Ewen, it one inective against 3 ~glyeero- phoephote, fructose diphoophate, phoephoglycerio acid, adonoeine tri- pheephate, urine phosphate, and eodiu pyrophoophete. Hence they regarded the emyue u o pholphoprotein phoephetaee. Too empe. preparation were deeoribed by Hattenheiner (113) in 1953, one on Iron rat liver and the other me from he; etc-och. The rot liver preparation proved to be the toot active on B-glyoero- pmmu, 1...; «an on m1. or d-omm and ineotive on phoepho- pOTptoneg therefore, it one regarded as a phosphaocmoelteme. The he‘ltmohextreotnenotootiveonanyotthebondtype nbotratee bot eaueed phoephate liberation in. filole caeein, 4min, oeeein phoephopepteney hence it was regarded u a phoqhoprotein phoephetue. Inrthereere, addition at oatheptio enune extract did not enhenoe phoephoprotein phosphate» activity; consequently it had no relation to nordependeney upon the letter. In 1951; Mean and 3mm (76) chewed that on eneyle pre- pared tro- ox epleen would liberate at pH 6.0 incl-genie phoephate Ira casein, phoovitin, end vitellin. When attempting to cleeeity their easy-e, they found thet only a little inorganio phoephate one releaeed liter incubation Iith slyoerophoephote and none tree caeeio pinephopeptone. They believed thie reparation no not an ecid phoephoumoeoterooe. In anotlnr report the lone year (77) they med a preparation or rat epleen, tree or acid phoephoeono‘ «tome. it p3 5.8 equal manta of inorganic phoephate tron Ihole eaeeie, dueeeein. and 6 oeaeein eere hydrolysed. It nae alee active wee pheevitin, but not woe glycerophoephate. Since it eplit pheec phone free -caeein, it on elixinated tron the liet at phoepho~ We. flopheephataee canoe, metronpetatoand the other (meal! ape-on, were new by M, Roche, and m in 1951s (81). The potato reparation at a pl of 6.0 eplit inorganic phoephate tr. m1. caeein, d-eaeein, phoevitin, o'valhnlin, and a caeein phoephe- peptdde. It had no effect on eeeeinophoephopeptone or diphonyl phoe- phate. So they concluded that the enyee eae a with phoe- phuonoeeteraee, not nagneeiee ion activated. the eoeyae tron calf epleen had ite optin- p! at 5.5. It catalysed the hydrolyeie of mm phoephate trol m1- caeoin, 0! -oaeein, pheevitin, oval- buin, and eaeein-phoephopeptone. It had no effect on oeeein phoe- phopeptide or [3-31me It 414 hoeever mm phenol tree dipheeyl phoephate. i‘he aethere decided that the cal: epleen eneyee eae an acid phenhodieeteflee. . The Meet of nogghataeeo on all! Fractions -- Gertrude Perlnann has rather exteneively inveetigated the effect or plmephataee eneyeee on the separated fractions: of caeein. Ber reeulte have helped to explain why in certain inetancee phoephataeee heretotcre mentioned have not releaeod inorganic phcephete fro- ehole caeein. 12 In 1952 form (61) reported that d-ouein eae about he per» cent dephtphoryhtod in the on range or 5.6—6.6 by proetate photo pheteee. m “acaeein when the dophoephorylated decreaeed in eelnhility and ehoeed eeverel new empomnte electrophoretdoally. rreetate phoephataee had no effect on Bacaeein, but up» 2:: hour eapeeure to vhole caeein 12 percent inorganic phoqahoree nae tread. When 0(~oaeein and b ceaeein were W eo that fl-oae'ein eaneeded 30 percent, eneyne inhibition developed and inoreaeed proportionately. it tb 8min- on rheepheree notaboliee in 1952 Perl-amt (62) revealed that intee'tinel phoephataoe nodal]: regarded ae an alkaline phoephataee, actually releaeed phoephdrue tron the phoephoanide 11.an R—ltglo-R at either Pl 5.3 or 9. 0 but only flight]; at pH 1. o. Thie behavior ms round with each mIbetratee ae fl fl-chloropheml) ' mono-photo, «M. and d-cmtn. With 4 ocean digoete at ’3 true 5.) to 10.0 (option-e at 6.1 and 8.1;) 10.0 percent of the phoephorae eae readily liberated. I with reopeot to ,5-oeeein'rerllann (59) dencnetreted that metate pheephataee (an acid, medu- ion activeted phoephaonoeetereee o: opts- pl 5.0 to 6 ohm net liberate any phoephor'oe. leither on W pm“ found dun fl «celeb we treated at p! 8 .l 111th “rifled phoephodiaeteraee tron rattle enake venom (Gro talg W). louver Ihen 5min one pretreated with the one}: veno- phoephodi- eeteraee at pl 8.2 followed by proetate phoephataee at pH 5.6, from Sh.O-72..o percent otithe phoephea'm in fl-caeein nae rotmd converted to tug-nae pheephete. m- led Pot-Luann to conclude that 64min contained phoephorue linked principally in the dieetor form. Pox-1mm (60) in 1951; pointed out that 0‘ -caeein contains a certain number of phosphate groups with ionizable hydrcxyle which oontribute to the not charge and thus to protein oleotrephoretio nelbility. Such groupa are readily attacked by the suitable phoe- phataaee. Those condition- are not fulfilled with 5 oeaaain. Dephoephorylation of thin protein fraction results in the formation 0! trichloroaoetio acid solnble nitrogen (N. P. I.) products. In another publication in 1951: Perlmnn (63) chewed that ho percent of 4 «coin phoephcrue m mom by prostate phosphatase at. p! 5.8 to 6.0, but that none was given by purified phoephodi- eeterace from rattle enaka ”non at pH 8.5. however when 0( «main ‘0 first pretreated with phosphodieateraae at pH 8.5, then adjuated to pH 6.0, and incubated with prostate plne intestinal phosphatase 78.0 percent of the total appeared ae inorganic phoaphate. Inteetinal pmphtuu canted reaction on the phcephoanide linkage. rm. reenlt nae con-idemd aa hydrolyaia or the phoephodieeter We followed by liberation of phoaphorua from the reaulting phoephmonoeater groups. Iaaet pyrophoephataee alone with Hg“ at pH 1.2 on not free my inorgaflo phoepha-ne from d-caeain. But combination of yeaet pyro- pheephataee together Iith proatate phoaphataee at pH 6.0 releaacd 59.0 percent 01‘ the 1311th in O( min. $111. nae regarded an action upon phoephoanhydride bonda followed by phoephcnonoeator cleavage. Perl-a- reaeoned tron these reeulta that h0.0 percent at the bonds in d-caeein are plwephcncnoeater, 20.0 percent are pyro- phoaphate ester, and 130.0 percent are phosphcamide eeter. flue Source or Preteolztic Epsom - Taka-Diaetaee ie prepared from a culture of the nold mun: emae according to the patented prooeea ct Talc-nine (79). The mold culture grow: on wheat bran ie extracted with water. Talm-Diaetaee is precipitated {ran the enter eolntion by addition of alcohol to 70 percent. The dried product hae been reported to contain at leaet twenty-three enzyme ayetena (80). the iaolation of preteolytic enzyme from the product ha been reviewed by Liner (35) . Some acid pheephataeee have been reported to be in Talon-Dianne. Guava (82) ehoved 13-1932 that tannDiaetaec contained a phoephomono- eeteraee of optim pl! 0! 3.24.0 on monophonyl phosphate. The em year he denonetrated (83) Pinephedieateraee activity on diphenyl phoephnte at pH 5.1; to 5.6. Iceberg and cemrkere (53). (53), have demon-touted that fab-Dianne also contains a pyrophcephataae active on trieodim pyropheephate at pH 3.24:.0. 15 III. EXPERDIEKTAL i. Edam Ueed Thermostat .. The oonetant temperature bath in; equipped with a memir bottle to maintain automatically at eon-tent lml of water. the thermoreguhtor (richer-Serra” Electronic Belay) mtrolled the temperature at 295° 3 0.10 C. (unaware - Lll pipettee and glassware were the Kimble 01a” brand. _ $3233 - The reaction period: were timed 11th a ‘Heylan atop- itch. 25 Meter .. i Becknan model B 2, glaee electrode, line operated, p3 rotor we need in asking pH neaetn-cmente. - m «— Lll dialyaes' were carried out in viehing tubing on a rotating external dialyzer cenetructed in thin laboratory. e tre hotcnet - 1) A house model a spectrophotometer nae need for analyaing for total and inorganic phosphorca. The inetnnent m calibrated for percent tramittance or optical denlity unite. 2) A Bedlam model DU spectrophotometer was need for analyeie ct acid-eoluble preteelytic producta abaorbing at 280 em. The inure- lent me calibrated for percent transmittance or optical density mite. Beniglioro geldahl mute .. The 50 I1. digeetion flaeke and a rotating digcetien rack (manufactured by American Inetrmnent Ce.), were need for digeeting total phosphor-no or nitrogen eanplee. The dietillatioe apparatne wee the nodiIied type need in thie labmtery. conductigtz 31-1933 .- l nodel 33-13 mommy bridge (mfactnred by Indutrial Inetnncnt an.) no need to:- reeietance and conductivity eeaeurenente. Conducting Cell - l tn ll. capacity conductivity cell (Parkman-er cerp.) vae need for aeaeuring reeietance. The bulb eeetien contained platinu- eupe which acre emoted through aide one to cape. The caductivity bridge eae «mooted to these cape. Il‘be cell content 1. 0.11893. ' I. Hateriale and Solutione “the hem Source - l'aII-Diaetaee 1e (nominated by Parke, Davie e do.) a yellow, once-phone, m-lvgreuopic powder analyeed for 1.51 percent nih'egon, 0.21 percent phoephorue, and 0.10 percent noieture. Ital-aroma; ulnbleieuterMMudaoleu,wDMmeolu~ tion. ‘ _ ‘l'he Caeein at .. Caeein eae made according to tle di- rectione of Du (12). The air dried product contained h.8 percent eedeture ae determined by overnight drying at 105%. n We for 15.5 percent nitrogen and 0.83 percent pheephcne (both expreeeed on a noieture tree beeie). meetrcphmtic analyeie ducaetrated that l? the preparation wee the em ae that of. minor (85) both in amber of. coapenente and their nobility. Oeeein Stock Solution - Six grime of the above air dried casein val weighed into a 100 nl. volumetric (leek. Seventy-five ml. of “tar we introduced in uall portion until a emocth paete formed. To tide 20.0 al. of 0.2 l. eodiua hydroxide nae gradually added, Iith choking, until a clear eolution of pH 7.0 eae obtained. The liquid .8 cede to vole-e, filtered, heated on a boiling water bath for fifteen ninutee, and a oryetal of thynol an added ae a preeervatiu. the eolntion nae alwaye etcred in the cold room n 5.0%. take-Dianne Stock Solution - The pro-determined mount of dry ponderneeeighedcnenanelaticalbalance, anddieeolvedinredie- tilled water. The final concentration eae lexpreeeed in terae of milli- greee per ailliliter of diceet. Ireeh eolutione were eleaye prepared 31m prior to tea. Fake-9cm PEEL“! Reagente «- than we prepared, with nine:- nodiiioaticne, ae deecribed by Kabat and finer (31). LO log), rgchloreacegc laid.“ One hundred eixty-tln-ee (nae (Eeetnan) triohlcroaoetie acid eae eeighed out on analytical balance and treneterred quantitatively to a 1.0 liter volueetric {leek and diluted to the nark. 5,0 won-1 cadm- made .- 0» hundred grate (usher) codin- hydroxide fie weighed cnt on an analytical balance, tunererred quanti- tatively to a 500 ll. telnetric flack, and diluted to the ark. 18 0,5 Normal Sodium mud. - Treaty grene (Filter) eodiun hydrodde was weighed out on an analytical balance and traneterred quantitatively to a 1.0 liter volumtric flack, and diluted to the Dark. 10 I0 2292‘ 02:11:31 Chloride - Ten grene of (Hallinch‘cdt di- hydrate) duelin- chloride was dieeclved in an manila chloride butter. (prepared ae beloe), diluted to 100 1:1,, and eaturated with (Fieher) calcite: hydroxide. Ihie reagent nae prepared at but once a week, etored in a pyrex bottle, and filtered Just before nee. '. eh .. i one to five dilution eith dietilled water of the above 10.0 percent calcime chloride eolutim nae made. 0,95 l Sulfuric Acid - This me made by diluting 5.0 ml. of (Baker) 5.0 1 meme acid to 500 .1. Ammonium Chloride Hg» 23 2, .. The buffer eae prepared by dieeolving 26.7 p. (Fieher) .cniun chloride in 60.6 .1. of (Du Pont) concentrated unedite- hydroxide and diluting to 1.0 liter with dietilled inter. Bran Mel Blue - ‘the indicator for centralisation in inorganic phoephate analyeie nae prepared by dieeclving 0.01: p. brcn thynol blue in 100.0 :1. o: 95 percent. ethanol. ' 0. mate). Hethode W -- 1n lemma“ win-o o: 6.0 por- cent oaeein wee pipetted into one am of a bifurcated test tube, and 19 into the other arm nae placed an equal volume of chemo eolution. When those were nixed the resulting digest concentration of enzyme wee expreaeed in tonne of ng./n1. of digeet. The reaction vceeel we placed in the thermostat 20 ninutee before mixing. Digeetien eae begun by tilting the two-branched tube back and forth ten time. no. time or initial contact uae taken as zero digestion time and noted by etarting the etOp watch. Suitable aliquots were inactivated at e‘pecific intervale by pipetting thw into two volmee of 1.0 I tri- chloroacetic acid. Dining a period of one-half hour theee emplee were ehaken periodically and then filtered through Miatnu‘i #2 filter paper. The filtrates were analyzed for total acid eoluble phcephorua, in- organic phoephorue , and acid-eoluble proteolytic produote ae deeoribed below. 1. The Influence of ham Concentration - A eoriee of digeetio. were portomd with the initial comantraticn of «coin ail-eye 3.0 percent. Alienate oi' i‘aka-Diaetau producing 2.0, h.0, and 8.0 ngnl. of digest for each experileut nae tabn for 'etudy. e) hulleil fal' aid-Soluble homes Dredgi- - The liberation of acid-”labia eplit producte, other than phosphorus, W determined on the above trichloroacetie acid filtratee. the aero tine ample no eat for 100 percent teen-inion or core optical deneity in a nodal a loom Spectrophotmeter and the euoceeding tine eanploe eclpand at 280 an against the blank. The resulting changee in optical deneity arereportedinrablenandahomintigm-e 2. b) Anabel; for Inorganic Phosphate - l'he inorganic phoaphcm we determined by a procedure similar to that described by Roz-berg (56). Five nillilitere (occasionally 3.0 ml.) of the protein-free filtrate nae pipetted into a 15 .0 Ill. conical centrifuge tube. The aliquot “a neutralized by dropeiee addition of initially S .0 H and finally 0.5 l Iodium hydroxide to the green color of brass thynol blue indicator. (he milliliter of 10.0 percent calcium chloride in 0.5 M ammonium chloride buffer at pH 9.0 and calcim hydroxide saturated was added ee precipitating reagent. After 30 minutes the precipitate was centrifmd and washed with 5 .0 ml. of a one to five dilution of the precipitating reagent. Finally the precipitate was responded in h.0 ml. of 0.05 I culturic acid and the inorganic phosphate determined by the method of Fiche and Subbarow (15). The milligrams of inorganic phosphorua '8! obtained Iron a ataxidard phosphorus solution treated in the cams manner. The results are recorded in Table II and sheen in Figure l. o) gaggi- tor Total Acid Soluble Phogphate - The procedure described here, with minor modifications, we described in Hawk, Oeer, and Sunnereon (26). Five milliliters (occasionally 3.0 ml.) of the above protein-free filtrate nae pipetted into a 50.0 ml. micro-Holden digestion flack. A glass head (to prevent bimping) and 2.5 ml. or 5.0 I eulturic acid were put into the flask and heated on the aicro- Kjeldahl digestion rack until charting and fmnee appeared. The eamplee were cooled 9O eeconde, treated Iith a drop of 30.0 percent hydrogen peroxide, and heated again until the color disappeared. The peroxide 21 treatment was repeated until the color was gone. Three milli‘tere of dietilled water was added to the cooled flasks and they were boiled ' momentarily. The flaeke iwere cooled, rinsed: into a 25.0 ml. volu~ metric flask end the total acid soluble phosphate determined by the Iieke end Subbaron method (11;). A blank end a phosphorus standard were run in the em manner. The resulte are shown in Table II and Figure 2 . 2. Influence of mutate Concentreeion «- A eerie- ot incuba- tion. were conducted elm-o mbetrete ooncentretione or 1.5, 3.0, 13.5. one 6.0 percent euein were prepared from nook eolution. The digeet mutation of 11W us My! 13.0 ng-l. The acidcloluble mum. predate, hereunto pheephete, end total eeid «m1. pine-photo were deter-bed ee deeorlbed previouely. Reeulte ere given in 2.131. III and Figure 3. 3. Igluence OfJH - A series or digestions was carried out after the pH of the casein stock lolution had been adjusted to 6.6, 7.0, and 8.0. The digest concentration was alufiye 3.0 percent for casein end I: n3./ml. for Take-Diastaee. The mid-soluble proteolqtic producte, inorganic phosphate , and total acid soluble phoephate were determined In described than. The reenlte are reported in Teble IV and Figure h. h. Phoephodieetereee Pretreatment - Ten milligram of lyophflyeed nae-*1". the rattle eneke (ex-amu- mam-mm) m diuolved in m receipt of e oeuplinentery enple tron theRoss Allen Reptile menu, Silver Spring, rum, to gratefully «knowledged. 22 10.0 .1. of 0.01 n. upoeitn chloride eolntion. Three 1111th of thin m fled to 25.0 ll. of 6.0 percent emin n pl 7.35. um incubotion for :1: ham, the pa had dropped to 6.95. Three Innate-re were then mud end treated with 6.0 ml. or 1.0 l iriehleroeeetio acid for tote]. and inorganic phosphate onelyeie upon the tiltrote. At the one tine o 20.0 ml. eolution eonteining 200 lg. of Toke-Dunne wee added to the mining 25.0 ll. of digelt. Ire- then on the prove» of phoeplme liberetion end preteen-ii nu moored ee preview described. the reunite ere lieted in em. V end ehon in Figure 5. S. gondnetivitz Chenge - ‘l'he mime change during hydrolyeie In worded et 15 minute intemle on e 3.0 percent end it Ic./I1. hie-Dionne digeei et pl! 1.3. {he epeoifle conductance nee colon- hue end in recorded in Table I and Figure I. 23 TABLE I CHANGE II coumcnvm DURHG CASE]! PROTEOLISIS A. _h A“- A M. —M Digoetion Tine . Reeietencee Specific min. ohne. Conductancefi _ nhcg.n, 0 388 1.26 15 386 1.27 3° 379 1.28 BS 378 ' 1.29 60 378 1.29 75 37h 1.31 90 V 372 1.32 105 369 1.33 120 367 1.33 135 367 1.33 150 _ 367 1.33 i. .O..-.Q-QO..§-QCQQ...--QO-QC 165 361: 1.3!: 180 359 1.36 195 359 1.36 210 358 . 1.37 225 358 1.37 aho 35h 1.38 'Iennd (nu dividing the cell content 0.14893 by volnee in calm 2. ii. - ~»Pcin$ Ibere epeleeoence‘begen. 2h .aztanazeififim-ooe 3 m3 8.” am e 53... an 3 3“ w t. 8a 34 3 am up»... on R on“ 8 a «a; 2 3 mono a... R as me.“ 8 2. .. Rm...” .. .. MN: n .. wane 8m... ma m... emu 84 .3 a. 36 3 an «26 «a 3 8a 3.“. mm 3 and ma mu . 800 e an 8 .. a new». a .. lam .. .. .. flu. 8a... a in 5.0 e e 8 Smd ... 2 ~86 m a §.o n m on , 3.4.6 m S .78 a o .12 a n ma g g o o o o o o o. o o o nose.» 0 e." .. e .538 use." ... e on: a: i again..." .a 83.03 IH‘ J august 95. Bags «.232 8.5 53530 as a g a NH 3 25 ll- .533 3.9093."er onen- andom I e R 3 an.” R a 8; 2 a a.” am a an an 3 34 S Hm 3.." mm am 2.4 3 a. 0.3 3 on 02.0 mm . E «a; mu 3 an.” 3 am 8a I. 8 n3... 8 3 unmam .. I .. man s .. wane 3.“ 9 cm o? 3 2. mad 3 mm 3.0 .2 an «3 .2 5 2” l. 3 an... a , a a: S m... 34 nu «n 8 m m on... m 5 £3 a 5 334°... .. a no: .. .. :3. s. 8 n a . 8a. 9. m 3 8a.? m a 33. n 3 on N o 38. a 3 #8.. a o Ede a n me 20 fig o o o o o o o o o o O o C {q C {q COCO 0 9“ O O n. o C {q 0 ‘ O Q0011 0 4 C {q mmmfllfi flung as: flammfllfi fig 3.5 $.th WW“. 3:” mwmwll “Rum .5- - a 25% m .539; 333 .639... 3:3 m Leeann e333 .25. 53 once» 0 o 53.5 amazon} .: adeeeo «13.8m— o 550 nausea} H none-ems L}, @333 95. fig mg 8.5 $030 NEE a as NIH H a.» 26 «ado 8 on SA fl .8 54 em 8 3a 810 .3 a... 34 2 am and am on ca 5.0 i 3 «a; 2 3 S .n 3 2 8H 30.0 3 z u we a: .. .. Mu... .. .. w: n .. uan... .. .. mu... .. .. mm... on on? a ma 0.8.9 5 am Re o 8 mm 8” 25.0 3 2 H26 ma mu 93.0 ma 3 8 3a.? 5 n 02.9 a S a... a ma 8 ‘03.? a g 80.0. m a «86 m an on 3.0- ~ 0 .918 a 0 So on a «a mg kg . o o o o o o o o o o onoo 0 q 0 {Q oqo o {q I {q 1.11 0900 0 {Q o . ‘q a: E :5 mfil ER: .2: E .5. .688“ mpaéfl -839» “53053... 588m 32.33% .83. ad a DJ. mm 0.0 mm} noun-cg mama—88E ea Banana «3888.. 8.5 .1 B has in has 27 TABLE V THE mm 0" CASE]! PRETREA'DIMT WITH PIESPEDIESTI'RASE 3mm: 3! TAKI-DIASTASE DIGESTIOH A Fl Fw r x w” F“; r A. t: Digestion Tm, {3%1101'“ Libented Prateolyaio min. 0 or c 0.9. 37¢? . 7/7551? *_ +— 1“ 360' 70 18 0.19? z.- I o o o o 15 10 11 0.12:3 3o 15 12 0.303 60 17 16 O .62 8 9o 35 22 0.936 120 1m 23 1.170 150 57 36 1.1m 180 61; no 1.63 210 72 as 1.80 21:0 89 60 1.99 .4 A.— a ___ M. ' pH deem-ed tron initial 7.35 to mm 6.95 a. _ _ Point more updanoonoo began. T0130 4 pg per mlo Org. ’ A #8 per 280 80L 4O 80 “—4—.- § /:”:9 /’/O C) C) 2 o . (D i C '4 . .. 0’. . O l L l L l i E 5 ’,,,(} C) 2 , . e 5 / , o 2 ./ { /0/ Cr "1/ . / . r‘. , .,,/ // /‘ ’1 “A- O ‘ ’ / /0 /(~ I,» C .~ / r ,. ’ (; l/rr’ _//"" , '4 _;"J” {a /C 1 1 L L 1 L 1 " 2 3 4 Digestion Time, Hrs. Figure 2. PHOSPHORUS LIBERATED. A total ac. sol. P; B inorg. P; g opt. dens. Curves~l,2,& 3 represent 2:4,& 8 mg. Take-Diastase per ml. of digest. Toto 80 P; A 218 per 40 ml. In-. org. ; 40 A pg per ml. 20 1.6 A 001). At 0.8 280 1191 O -004 ~ i i (l -/ "I O , b‘ /" I .A/ ’7’ ~ | ’w” / ‘ J) l -"’ i r F !- A\ / D 6’ (N . ,fl» -<‘i/,//:~ 6. CL////’ ¢ ;<:T:::Z:~’Jnfl4 ; I- . _.\ A” A) ' , ,//- ‘ l (‘ g) .//,<é£:73<fl_ ' / | “€54” \ ' W I - 7 ‘ 1 1 g l i I L U Figure ,,r, Cké? i” % i I L l 1 J L __ L , L ,f5/1 / N,,/ / P .9 ”/Cj' 2 L. 5. L... -.L l 2 Digestion Time, Hrs. w— EFFECT OF SUBSTRATE (CASEIN) CONCENTRATION. 5 tot. so. sol. P; B inorg. P; C opt. dens. Curves 1,2,5, & 4 represent 1%,3,4%,& €'% w/v casein digests. TOto 80 Ana per ml. In- OPS. 4P8 per ml. 1.6 0.D. At 0.8 280 31 2' M it s g ; l 1 jLL I0 Figure 4. ‘ :5 m/ Arr—r -—--Q —————_o——-:-—C~—-':mbo mHmMA¢Z< mDmOmmmomm Qm¢QZ¢Bm .0 mhdwfim maponamonm Mo mfiwawopoas OmH OOH 0m 00 O¢ ON 0 a a fi 1 q — O \n l \MV\\nN\ 1 s.o NV\\ x\\\ a 03% mxxx \ .1 m 0 O \\\.\ i N.H J w.H nw 999 49 fiatsueo IBOIQdO 100 90 80 70 20 10 3h % of total casein phosphorus r \\\t> j \ to C) _Digestion Time, Hrs. Figure 7. FORMS OF PHOSPHORUS DURING PROTEOLYSIS. Curves: l, protein P; g, tot. ac. sol. P; g, inorg. P; and 3, ac. 8010 CPS. Po 35 Figure 8. ELECTROPHORETIC PATTERNS (TRACED) 0F:§ CAS- EIN; g PHOSPHODIESTERASE TREATED CASEIN. ASCENDING DESCENDING A in 1 % cone. in 0.1 M phosphate pH 7.0, 0.05 M NaCl, 6500 sec., pot. grad. 7.15 v./cm. g in 1% % conc., 0.1 M phosphate, pH 7.2, 0.05 M NaCl, 5610 sec., pot. grad. 9.14 v./cm. 36 IV . Um SSION The tailoring dieouenon '11]- be concerned with the releaee or the various tome o: phoephme during proteolyeie of main by rah-Duets”. The hetero influencing the phase or investigation that em be eoneidered are: A) Hethode or phosphate may-u, I) Gen-n1 obeemtione during phosphate release end preteolyeie, c) Influence of enzyme concentration, D) Effect of metrete concen- tretien, B) Optiml pl, 1') Cuein proteolme in renuee to phoephete liberation, end 0) Tut of reaction in “69 one with phoephodieetereee. MW flue Hethod for Inorganic Phosphate Leela! - The method of Hebe end Subbem (in) for inorganic phoephate determination we not directly lppliceble. When the triehloroeoetio eoid eoluble phoephom of e digest mqmt we treated with the lolybdete meant e white precipitate teamed ditch contained neat of the pinephom. Other workere (63) have euggeIted thet dth ouch elnplee the. mall molecule:- veight «genie phoephopeptidee new be removed by preliminary edeerption upon Done: 50 (1: percent ewe-linked). Such we tried with Dove: SO (12 percent emu-linked) eveflhble, but it did not elter the ebove cheer-red inter- ference. Triehleroeoetie eeid concentrations in the tiltrete up to 16 percent were tried and no improment we noted. luleroue modifi- oetlonl 1n treetIent of the £11th were tried end then included! one hour tine elepee before tiltretion, addition or S .0 l sulfuric coil! to lower the pl before filtration, addition or eodiun hydroxide to dieeolve the nolybdennn precipitete in the tiltrete , five unite heeting of digest aliquot with trichloroeeetio eoid on boiling tater both before rum-Ina (59). cooling in ice beth lS Iinutee before filtering (13). nee er nods.- tungstete with trichlmeoetic eoid (11), end mum of acetone with trichloroecetio eoid before tiltretien (3°). All of theee were unneeeeeml in preventing or redieeolving the muted precipitete eeneed by edditien of nolybdenu reagent to the triohleroeeetie eoid filtrate. In 1950 lerberg (56) indicated hum experienced einihr dun- cultiee. He introduced the pretreehent of triohloroeoetie ecid filtretee with e reegent eeneieting of oelciun chloride dissolved in main curiae butter “tented with eeleiun dee et p! 9.0. The reegent eecoupliehed e successful eeperetion of inorganic phoe- plnte fro- erpnie phoephnte u en insoluble epetite . To eeoertein the reliabilityo! thieprooedureverione Menominetioneof inorganic pheephete with euein end elee e etenderd phoephete eeriee elene tore teeted. theee reenlte 0W within experimental error with thoee e! e direct Pinko-m enelyeie upon Itenderd phoephate eelntione. fhereenlteeroehemin’igm'e 6. Whenitweetonnd thet the inorganic pheephete eontent or e digest aliquot eneeded the meltheetandu'doumemeepondinglynellerdigeeteuple wee taken. 38 Hethod goa- total Acid Soluble Mints .. The lethod adopted tor total acid eoluble pheephate endycie is deecribed essentially by hwk, Deer, and Sexual-eon (26). It wee foam! that no color eeuld develop win the quantity of trichloroecetic acid filtrate wee in- ereaaed and treated Iith proportional anomte or magenta union the final voltme wee aleo emeepondingly increaaed. for acne detenina- tiona the liberated phcephea'ua wee eo low that it we desirable to take eanplea large enough to contain a non accurately neaeurable quantity or phoephorua. It wae found quite eatietactory to do ao provided the cane mate of reagente and final volue for color de- velopnent were aaintained . I . General Obeervationa Protecfln a. he noted previonely (35) and in these experilente, when 3.0 percent cuein was treated eith Take-Dhaka (2-8 “451.) at pl 7.0, opaleeeenee cemenccd after one to two hours. thirty ninetee thereafter the digeet took on the appearance at ailk. Pretee- lytie activity neamenante at thie prooeee by alcoholic potaeeiu hydroude titration , rate of turbidity (nation, viacoeity change (35), and electrophoretio change! (37) have offered the hypotheeie that a tee etage reaction ie involved. In addition to the aforementioned memento , with con-- dnetdvity changee during preteolnie eupport tide hypetheeie. the reeulte are a... in table 1 and plotted in Figure 1. The increaee 39 in conductivity may be regarded as the result 0: the increase in charged species (18) upon hydrolysis of bonds and groups in proteins. figure 1 show a linear conductivity increase until the opalescence time is approached when tor a period no change occurs until after nilldness has appeared. It nay be noted (acre details later) that simultaneous inorganic phosphate liberation round the cone pattern. It suggests that inorganic phosphate might well be determined by the mob simpler resistance neasuromcnts. Another standard method of proteolytic activity measurement is the determination or trichloroacetic acid soluble hydrolysis products by optical density a: percent translittanoe change at 280 nu in the Bosnian ultraviolet Spectrophotcneter (55). The progress of protec- lysievae deterdnedinthisnanneronallsalples involvingthe neasuronent of total and inorganic phosphorus liberated during digestion. Hence all curves involVing phosphorus liberation are aoccupanied by the corresponding plot of optical density increase at 280 nu. These proteolysis plots do not show two stage reaction as suggested in the previous emerinents. Instead they correspond more closely to the nonoprotein nitrogen ncasua‘enents made earlier (as) . However there isamthornnrhbleandyetmlainedphenomona. Thisisthat during initial stages at casein pa-oteelysis with lower ensyse concen- trations there is a negative absorption of aromatic residues (such as tyrosine or tryptophan). Christensen (7) hee observed the sane tdth trypsin, chmtl'nlil, and planin and 2mm it to be only applicable to casein substrate. LO Phosphorus Liberation 2951.3 Proteclzeis - The following ob- servations cm from an inspection of Tables I, II,- III, IV, V, and Figures 1, 2, 3, h, and 5. In practical]: all experiments the total acid soluble phosphorus released on produced at essentially a constant rate during proteolysis by Talm—Diaetase . Hence the protein phosphorus (precipitated by tri- chloroacetic acid) retained was linearly related by negative slope to the digestion tine. . is previously mentioned the inorganic phosphate liberated in nest cases showed the two stage reaction being involved in casein proteolysis. The increase was linear until incipient opalescence and after onset of nilldness assumed a higher rate of liberation. The organic phosphate liberated during proteolytio degradation of casein can be seen from Iigure 5 to be initially liberated at a con- stant rate until onset of cloudiness when the value dropped to a lininus before reaming an increase again. This was in accord with the observations or wington and Kay (72) who stated that organic phosphate approached a mun slofl; and then diminished. CM . son of Lrgteolzsig wand Phosphorus Liberated -- The formation of acid soluble phosphate and hydrolytic products of casein in most cases occurred at a constant rate. Binngton and Kay (72) found total phosphate and amineonitrogen liberation to be linear functions during tryptic digestions. The inorganic phosphate liberation rate has al- ready been compared Iith conductivity changes. Curves in figures 2, 3 and h support the two step reaction hypothesis («and by other! (35). c . Influence of magma Concentration One of the criteria. for atflyds by enzymes is that generally ' indtiel nativity 1.. proportional to we. concentration in the . presence or adequate substmte (21) . The protoolytio measurements of optical density chenge et 280 am a shown in Table 11 end Figure 2 sstisty the above condition. There is of course the notable negative absorption st lover snsyne omen-t tretions thst units turthsr emlenntion . The total laid soluble phosphate released u shown in Tobie II end time 2 sinilerly supported the oriteris of rate of relssse being smntimy linearily proportional to the mount of Islet-Dionne and. However the setivity concerned with release of inorgenio phoe- phnto did not indicate the single lineu- dependency I. the shove. it auteur onsyne eonoentrstion tested the two stoge release mm mom on it boom less apparent on the lover mutations. the met of cloudiness and eventual nilkinesl followed the identical dependency described them. Then oboemtions seemed to suggest that phosphste mention us sppsrsntly s. result of the proteolytio dogrsdetion of cmin by virtue of its purulel easy” concentretion dependency. D. greet o; Substrete Concentntion In lost my» astound «notions the velocity immsss, but not proportiomtely, iron substrate concentration is node greater (2?). when s11 owls nelsonles hsve booms converted to myse- substrate complexes, nexinm velocity is etteinod end becomes indeo pendent of substrete concentration. Oocesionelly e! dininntion in velocity my be caused by further increase in lubotnto concentretlon. The results or this verieble on proteolytic «may end phos- phete liberation ere reported in Teble III and Figure 3. The dete shot. that when the retio of substrate to enzyno concentration Ill constent the total end inorganic phosphete as an no the optical density chengee bed the some rote curves. This hey be cmtmod es evidence for the solo ens:- boing operatito in phosphete libsntion end protcolysil. . Int the opponent observation of increesed substrate concentration invoking en inhibitory effect with respect, to mom liberetien end proteolysis is inconclusive. Sinner exporimte by other workers ere not known uni respect to phosphate release but the proteolytic dete is centrediotory (27). fhis “poet of the investigetion bears further exteuiee experimentation. E. gm 2! In connection eith the effect of hydrogen ion eotivity upon eneyne cetelyeed nations one should consider its influence on stability of the 01.51“, on eneyne ionisetion, and also dissociation o: the euhstrete (17). the effect ct pH on protoolysis end phoephons liberetion ere mmumh nomplouooiongm 1:. Am enact drove through the optioel density values st pH 6.6 since there no meson to suepect operetiond error in the instrument at the time M the analyeis vac made. Rooster the reliable values clearly Inb- stantiated previone optimal pH findinge (17) . An interesting addition- al obeervation is that the higher the pH of digestion the more pro- longed was the period of negative absorption at 280 In. The phosphate neasurenenta seen to vary in the same manner as the proteolytic. Hence if the influence of twdrogen ions upon enzyme ie as stated above the reenlte support the belief that the em ensyns is operative in both phosphate liberation and proteolnis. Further-ore should one think that the previously deecribed phos- pheprotein phosphatases were co-aotive in this digestion, then A - certainly p! optima Ionld be In. 5.8-6.0. since human» activity in this range is actually hydrogen ion memo (17) this possibility scene remote when considered together with the findings reported in un- study. All cther phosphatase: annoy of mm- WhawbemnpwhdtoMotheiroptinUpHatflenlom values than 6.0. l'. Casein Proteclzeis in Relation to PM‘ hate ghee-anon Uptothispointetidenceregardingthenannerefmlease of total acid soluble and inorganic phosphate together sith proteolytio data has been emined. It can. that the liberation of inorganic phoephate together with formation of acid ionicing groups neasnred either condnctcustrioany or titriaetrioally supports the hypothesis of a too step reaction. The first step come to precede nilkineos fox-nation whereas the second develops uith onset of this digeot appearance. By electrophorctic analysis it ha been found that the fi efrection of the substrate in totally consumed during the rm: «hp and collagen; Iilkineee the o(-peak disappom. During mom proteolysis the formation of acid soluble organic phosphate is most predoninant in comparison to the inorganic fraction of the total amount liberated. Following nilkineoe this relationship is reversed. Hence the phosphate release so close]: allied with formation of new ionic species or new acid groups can only logically suggest that groups or linkagee involving phosphorus are associated with the protoolytio procees. During step one where the organic form of phos- phate release ie predominant and where principally 5-casein is in- ' valved, it suggesto, as does also Porlmann’s work (59), that the phosphodic eter and come peptide bond cleavage in principally involved. Then folloadng onset of cloudiness proteolytic degradation may principally be 430de with phosphmnonooater and peptide bond hydrolqli: of the d-caeoin fraction. Such mld account for the greater rate of inorganic phosphate production, conductivity change and other proteelyeie data. 0. Test of Reactions i_n_ Stop % With l’hoephodieeteraee If an enayns specific for the nature of the reactions of step one were available, panel: a phoephodiesterase with none or very little proteinase activity, the preceding speculation could be tested. Snake venom fro. the Diamond back rattler (Crotalus filamentous) has been found to fulfill these specifications and was incubated at pH 1.1: for 15 six hours with mole casein. The results in Table V and Figure 5 show what was predicted in the foregoing discussion. The pH of the digest decreased, organic phosphate release was predominant, pro- duction of new ionic species nae slight, and optical density change demonstrated very little peptide bond hydrolysis. in electrophorotio pattern showed alteration in the 5-peak only but unchanged c( ofraction. Then by addition of Take-Diastase the onset of nilkiness was ad- vanced one hour as well as the increased production of inorganic phos- phate, charged ionic species, and hydrogen ion liberating groups. he Y.SJHMARI l) A method for separating inorganic tron organic phosphate in tri- ohloroacotic acid filtrates of casein hydrolysates was developed. 2) The rate of liberation of inorganic and total acid soluble phosphate during proteclysis was studied. 3) Inorganic phosphorus followed a tan ltep reaction rate. ch. first stage of slow liberation occurred before the onset of cloudiness and the second of fast liberation developed after nilkiness appeared. 1;) com acid soluble phosphorus showed a constant rate of rotation throughout the period of proteolysis. 5) Organic phosphorus was produced rapidly until tin onset of cloudi- ness when the value dropped to a nininun before it resumed a slower increase. 6) the protein phosphorus decreased it a content rate throughout the reaction. 7) 9peoific conductivity or measure of charged species developed as a two step reaction. the first step was before cloudiness and the second after nilkiness. 8) Proteolysis products and phosphorus liberation shoved identical dependency upon casein concentration . 9) the liberation of acid-soluble proteolytio prodnote and all types of phosphorus compounds from casein were similarly proportional h? to TahnDinstase comentration. 10) Hanna activity for casein proteolysis and phosphorus libera- tion both occurred at pi! 6.6. ll) Optical density increase at 280 am or measure of tyrosine and tryptophan residues liberated by casein proteolysis was essen- tially constant at sole initial periods. 12) The release of charged ionic species is related to forsation of inorganic phosphorus products because both followed the two step type of reaction. . 13) Optical density and total acid soluble phosphcrus increases sees interdependent since they shoved siniler linear increase through— out the reaction. 1h) The optima pl of around 6.6—1.0 for maxim proteolysis and phosphorus release elininated the possible participation of low pl active and stable phosphcprotain phosphatase or other phospha- tases. ' 15) The negative absorption values sometimes had during initial proteolysis were affected by either concentration of enzyse , substrate, or hydrogen ions. _ 16) Total acid soluble phosphorus liberated during casein proteelysis eupared closely with total acid soluble nitrogen fomtion . previously reported (35) . 17) m evidence to date snggests that the initial reaction preceding onset of nilldness was the disappearance of 6 -casein by catalysed 118 hydrolysis of its phosphcdiceter bonds and peptide bond cleavage. Also silultanecnsly at reduced rate there was some hydrolysis to liberate inorganic phosphate ions . 18) cloudiness iteeit is believed to be due to the relatively en- gmgod deflection. 19) The reaction renewing nilkiness in the digest nay be regarded as further hydrolysis nee operative upon the phosphorus and nitrogen bonds of 0‘ casein. 20) A specific phosphcdiesterase preparation ct snake venen acccup- lished the sale results as postulated to be in step one described in timber 16 (above). 21) .‘ phosphodiesterass pretrsstlsnt e: casein desenetrsted thst only step tea as described in when 17 and 18 (above) resulted teen Tab-Dust“. m added. 1:9 3131mm 1. Am, 0., De Verdier, C. B. and Glusot, J. Crystalline Phosphcserine tron Casein Hydrolyeate, Acts Chen. Scand., 2, 32110351). 2. Anlrod, 3. Citrus Fruit Phosphatase, J. Biol. Chan" 191, 57 (19h7). 3. 30m, 2. E.‘ Theory and Application of Colloidal Behavior, Chapter 33, Hearst-Hill lock Cc., flee Iork, 1921:. h. Chsrbuliea, E. and Jeannerat, J. Beoherchss Sur la Cassine. , 3dr ls Practicnnsnent de la Cassius st dela Paraeaseine an Chlorure d'a-oniu, lelv. Chin. iota,._23, ’52 (1939). S. Chsrbnlies, E. and Meyer, 1’. Rachel-ones Cor 1a Cassius, leie. Chin. iets, 19, 600 (1933). 6. Chorbolisa E. and Schneider, 1!. L. Rechsrohss Sur la Cassius La Caseine n'sst pas on Corps Bc-cgsne. Etude de son Iractionnenent par le Chlorare d'uoniun, hale. Chin. iota, 12, 597 (1932). 1. Christensen, 1. n. the Action or Proteolytic Myles on Casein Proteins, Arch. Biochen. and licphys., a, 128 (1951;). 8. Cohen H. R. Dr: flinging of Proteins Arch. Biochen. 2 355 data). ' ’ ' " 9. Duodaran, H. and lanachandran, I. V. hepatic Proteolysis. “in? tags of Casein Phosphopeptone, Bicchsn. J., 35: 122 19 . 10. De Verdier, C. 5. Isolation of Phosphothrenonine tron , Bovine Casein, nature, m, Bet (1952). 11. De Verdisr, O. I. The Isolation of Phoephotln‘snnins tron Bovine Casein, iota Chen. Scand., 1, 196 (1953). 12. Dan, I. 8. Preparation of Casein. Carter, I. 1%., Editor. liochenical Preparations, John Wiley a Sons, Inc., vol. I, lav Iork, 191:9. Pp. 22-21;. . 13. reinstein B. l. and Vol]: 1!. E. Phosphoprotein Phosphatase in nee-aim tissues, J. fine. one... .111. 339 (19M). 1h. Piske, C. n. and Scbbarov, I. The Colorinetric Deter-ination e: Phosphoms, J. eiei. one... go, 315 (1925). 15. Fields, C I. and Caliber-ow, I. Pheaphocreatine, J. Biol. Chen. g, 629 (1929). 16. Pacts, II. N. and lind, C. A. A Phosphcprotein Phosphatase in the Chick teem. Arch. Bieohen. end siephye., to, as). (1953). 11. Proton J. 3. and Sis-ends, 3. General linemen-y, John Wiley 1 one. 1nd,, see Iork, 1953. r. 261. 18. (Rick, D. Methods of Biochsnical Analysis, Interscienee Pub- lishers, Inc., as- red-k, Vol. II. 1955. r. 255. 19. Coo-don, W. 0., Ss-stt, W. P. and lender, H. Aline Acid Conpositicn of Y ~Casein, J. An. Chen. see., 15, 1678 (1953). 20. Gordon, W. 8., Senett, V. P., Cable 3. 8. and Kerrie, ll. Aline Acid Cuposition of ol-and {5-Casein, J. An. Chu. Soc., 11, 3293 (191.9 . 21. Gortner, R. A. Outlines of Biochemistry, ed. 3. John Wiley & Sons, Inc., lee Kerk, 1910. P. 1011. 22. oi-eh, J. Uber die Praktionisrnng des Caseins, z. Physiol. Chen" 33g, 32 (1931:). 23. Golland, J. H. and Jackson, E. )1 Bone and Who Phoephodi- esterases, Biochen. J . , 13, S90 (1938). 21;. W, 0. and lsdin, 8. C. Textbook of Physiological - Chemistry,- ed. 7. Handel J. A. Translator. John Wiley 8 sons, 1110., lot Pork, 191‘. P. 652. 25. Harris D. L. Phosphoprotsin Phosphatase J. Biol. Chen. 1.6.2. 5131 (i916). ' ~ .’. 26. Hawk, P. I., Gear, 3. 1.. and ”room-W. I. Practical W logical C , ed. 13 fhe Blanltiston Cupany, Ins... nee zerk, 1951:. Pp. 629.535 27. teen-eons, r. eieebenistry, Join Huey a Sens, Im.. In York, 1955. P. 363. 28. lanrorita, P. Chenistry and Biology of Proteins, Academic Press Inc., Publishers, lee Ierk, 1950. Pp. 202-201.. 29. nipp, n. J., Groves, n. 1., costar, J. n. and nausekin r. 1.. Separation e: Y—Casein, J. AI. Chen. See., 1;. 1.928 (i950). 30. Jacobson, C. P. The Activation of Chynotrypsindgen, Conpt. rent. Lab. Carl-e. 32, 335 (19M). 31. Rabat, E. A. and layer, 1!. 1!. Experimental Mochenistry, Charles 0. Tho-as Publisher, Springfield, 19118. Pp. 292-297. 32. may, H. D. Bone Phosphorus Coupomds of 11111:. the Presence in hill: g Organic Acid Soluble Coupounds, Biochen. J . , .12, 33 192 . 33. Kay, 3. D. Kidney Phosphatase, Iiochen. J., 32, 791 (1926). 311. Laidler, I. J. Introduction to the Chemistry of key-es, Monroe-Hill look Comany, 1110., In Park, 1951:. Pp. 69-72. 35. Lieser, R. C. The Proteolytic Activity of Take-Diastase tron Lsmfi%lus O as on Casein. Unpublished I. A. thesis lliohigan Sta e ofiegs, 1952, 63 nab. leaves. 36. Lorene, P. A. and Hill D. W. On a Dipeptide Phosphoric Acid Isolated Fro- Casein, . Biol. one... 101, 111 (1933). 37. Lillevik, B. A. and Liam, R. C. Casein Proteolysis and Electrophoretio Changes. Abstracts of Papers, 128th. National Meeting Iberian Chemical Society, limoapolis, ninneeete. dept-oer 11 to 16 (1955). 38. Lillevik, a. A. and Mnderstrfi-Lsng, I. qublished Data. 39. Linderstxfi-Land, I. and Kodana, 8. On the Solubility of Casein in Hydrochloric Acid, Coup. rend., Lab. Carlsbsr‘, .1_6,, lo. 1 (1921). 1:0. Lindmtzin-Mg, K. On the Fractionation oi’ Casein, Conpt. rend., Lab. Carlsberg, 11, Io. 9 (1929). 11. tips-en, 1. Beer die linden; dor Phosphomurs in Phosphor- retain-n. Bushes. 2.. 291. 3 (1933). - . 1.2. Lomdes, J., Res laoara, 1. J., nit-er, 1.3. A. Analysis Preteine, Caseo-phosphopeptone, Biochen. J ., )5, 315 (191d). In}. Hattenheiesr, I. Die Dsphospherylisrnn; Von Casein end Phosphopepten dnrch Phosphoprotein-Phosphatase , z . Physiol. one... gag, 216 (1953). of 52 Mi. lattenheinor, 3., liter-anti, I. and Zahler, P. has Lab. and Seine t'irknng an! das Casein der Kilch. Uber die Phosphatase- ‘eirkeng des Labes., we. Chin. Acts, 15, 1970 (1953). 15. Hcfleeltin 2. L. Chap.~l6 hill: Proteins. Isurath, H. and Bailey, in Editors. The Proteins, Aeadeeis Press, Inc., Publishers, lee Pork, 1e1. 11. Part A, 1951.. Pp. 389-1131;. 116. housekin, 1'. L. and Polis, D. D. Hill: Proteins. Advances in Protein Chenistry, Acadenic Press, Inc., Publishers, lee Pork, Vol. V, 1950. Pp. zoo-225. In. Hollander, O. Chuistry or Emu Milk, Honatschr. linderb heilk., 21, 111 (1919). c. 1., y}, 9122 (191.9). 118. Hollander, 0. Chemical and lutritive Differences in the Casein Iron Breast Milk and iron Cow Hills, Upsala Lalo-ree- ‘0’... Path” 2’ 10? (19h1)’ co to; 91' 37” (19h9)e 50. Hollander, C. mektro horotische Untersuohung Von Casein, Biochen. 2., log, 21:0 £1939). 51. Hollander, O. neotrophoretic and keynatic Fractionation of Casein tron Hanan llilk, Batu-e, 12;, 60h (191(5). 52. Iceberg, C. and Pischer, I. A. Ensynatischo tong Allergen- ischer WW, impinge, g, 191 1931). 53. Iceberg, 0., Craner, A. and Handl. I. Per-tics! of Pyrophes- phats, Ensnclogia, _1_),, 157 (1950-51). Sh. Nicolet, I. H. and Shinn L. A. Abstracts of Papers at 110th. neeting e: inerieen Chemical Society, Chicago, Illinois, 1916, r. 20 a. 55. lorthrcp, J. L, knits, 14., and Herriot, 3. ll, Crystalline May-es, ed. 2, Coluieia University Press, lee Icrk, 19168. PP. 303-309. 56. Norberg, B. Phoephcprotein Phosphatase in the Rat, iota Chen. Soand., 1,, 1206 (1950). 57. Osborne, ‘1‘. I. and Waldemar), A. J. Sane new Constituents of 11111:, J. siei. one... a, 21.3 (1918). S9. Psrlnann, C. E. Phosphodiester Linkages in Proteins, Biochen. st aiopm. Acts, .11. 1.52 (1951:). 53 60. Perhann, G. E. floctrophcretic Studies on Enzymatically Modified Ovalbunin and Casein, Discussions of the Faraday society, lhlb, 61 (1953-51.). 61. Perlnsnn, C. E. may-atio Dsphosphorylaticn of Casein, J. An. Chen. 80s., 13, 3191 (1952). 62. Per-leans, C. E. Enaynatic Dsphosphorylation of Phosphcproteins and the Nature of Phosphorus Linkages. Mommy, W. D. and Glass, 8. Editors. Phosphorus Metabolism. The Johns Hopldns Press, Baltimore, Volume II, 1952. Pp. 167-185. 63. Perlnann, C. E. Phosphorus Linkages in 0(«Caeein, lature, 111,, 273 (1951:). 611. Peterson, R. 7., mirrington, B. J., and Hdieeldn, 1'. L. Separation of Primary Products Formed tron «Casein by Action of bypsin, Abstracts of Papers, 126th. lational Meeting of use; enerieen Chemical Society, New Iork, September 12-21 (19514) P C. _ 6S. Pli-er, R. I. A. an! Dayliss, H. l. The Separation of Phos- phorus tron Caseinogen by the Action of amylase and Alkali, J. Physiol., 3;, 1439 (1905). 66. Pcsternsk, S. The Phosphorus luclous of Caseinogen, 310011.. J., 31, 289 (1921). 67. Posternak, 1‘. and Pollaoaek, I. De la Protection Centre l'hydrolyse hynatiqm wrese par les Groupes Phosphor- les. Etude de la Degradation Mystique d'nn Peptide st d'un Polyoss Phosphorylo, Helv. Chen. Acts, 33, 921 (19M). 68. Binington, C. Action of Alkali on Caseinem, Biochen. J. 3;, 201. (1921). 69. Rimington, C. The Phosphorus oi' Caseinogen. Isolation of a Phosphorus-Containing Peptcne tron Tryptio Digests ct Caseinepn, $2.151.” file 1179 (1937). 70. Rinington, C. The Phosphorus of Caseinogea. Constitution or Phosphopeptons, Ibid., 31, 1187 (1921). 71. Rinington, C. late on the Anise Acids Present in Phospho- rous». £212.. 22. 320 (191.1). 72. Rimington, C. and , H. D. The Phosphorus or Caseinogen, lbid., 32, m (1925 e ' 5b. 73. Roche, J. Phosphatases. Stunner, J. B. and hyrback, I. Editors. The Enema Acadenio Press Inc., low Pork, Vol. I, Part A, 1950. Pp. 371-516. 7h. Scheidt, C. Zur Cewinnung der Dipeptidphosphersaure aus Casein, z. rmioi. Che... 22 , 86 (1933). 15. Schmidt, 0. end minnows», J. Intestinal Phosphatase, J. Biol. one... _1_h_2. 369 (191.3). 76. Sundararajan, 2. A. and Canes, 2. S. Phosphoprotsin Phosphatase, Diochon. J., 16, 125 (19511). . 77. Cundararajan, '1‘. A. and Carla, P. 8. Phosp tein phospha- tass, Biochen. st Iiophys. Acts, 11, 588 (19 ). 78. Suterneistsr, E. and Brenna, P. L. Casein and Its Industrial Applications, ed. 2, An. Chen. Coo. nonemph, lo. 30, Reinhold Publishing Corp., Ion Iork, 1939. 79. Pumice, J. anemic Cupositien, 8. 3. Patent lo. 1,1160-736; c. 1.. 11, 28914 (1923). cc. Isobar, E. The Chemistry and Technolog of Ensynes, John Wiley & Sons, Inc., New Iork, 19119. P. 1101. 81. 'i'hoai, l., Roche, J. and Pin, P. Recherohes Cur lss Phospho- proteines latnre des Masons Ester Phosphorique de la Cassius , aenetis de la societe de Chinie Biologique, pg, 1.83 (195k). 82. Users 8. Phesphsesterasss of Iran J. bitches. (Jepu) 1 “1935); C. in L6, 2991, (1933). ' s is 83. (Jeans, 5. The eta-ass and the Phosphodiesterase 11:19., 15, 19 (1932 3 c. 1., 3.9.. 2991. (1933). 8).. Warner, R. Chapt. 17, Egg Proteins. leurath, I. and Bailey, 3., Editors. The Proteins, Acadsnie Press Inc., Publishers, Poet York, 1e1. 11. Part A, 1951.. Pp. hag-has 85. Warner R. C. sticn of (x. and «music J. An. Chen. see. 9.5.. 1123 (198:). p ' 86. Van Slyke L. 1.. and Bosserth A. 1.9. Preparation of Casein, J. Biol. (its... 2, 203 (1913 . O ii ‘1’ “VFWSTRY W3 Date Due Demco-293 IlilHILIIlhjlfllfllflill[Lllfllljlllllllllflllllllllillli 68 9130