| Title | Studies on the heat labile hyaluronidase inhibitor of monkey serum |
| Publication Type | thesis |
| School or College | College of Science |
| Department | Biological Sciences |
| Author | Wilcox, Wesley Calvin |
| Date | 1955-06 |
| Description | 1. The nature of the rise of non-specific hyaluronidase in poliomyelitis infected monkeys was established. 2. Animals infected with the virus of poliomyelitis by means of the gastro-intestinal route failed to exhibit a rate in non-specific inhibition. Those animals infected by either the intranasal or intra-cerebral routes exhibited a rise in serum inhibitors levels at or near the onset of paralysis. 3. A possible relationship between virus antibody levels and the level of physiological inhibitor is suggested. 4. The kinetics of the enzymic hydrolysis of hyaluroic acid was studied in some detail by means of an improved turbidimetric method. The reaction order, enzyme-substrate dissociation constant, and the energy of activation were obtained by means of these studies. 5. The kinetics of the inhibi |
| Type | Text |
| Publisher | University of Utah |
| Subject | Monkeys |
| Subject MESH | Hyaluronoglucosaminidase; Poliomyelitis |
| Dissertation Institution | University of Utah |
| Dissertation Name | MS |
| Language | eng |
| Relation is Version of | Digital reproduction of "Studies on the heat labile hyaluronidase inhibitor of monkey serum". Spencer S. Eccles Health Sciences Library. |
| Rights Management | © Wesley Calvin Wilcox. |
| Format | application/pdf |
| Format Medium | application/pdf |
| Format Extent | 7,142,536 bytes |
| Identifier | undthes,3781 |
| Source | University of Utah Spencer S. Eccles Health Sciences Library (print no longer available) |
| Master File Extent | 7,142,576 bytes |
| ARK | ark:/87278/s6vx0j97 |
| DOI | https://doi.org/doi:10.26053/0H-VBSZ-K800 |
| Setname | ir_etd |
| ID | 190902 |
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ItAllLI Jl!AL1fIOlW)ABB UUBItol GP KOIlUl! .- !he .pread1D.s tacto •• of b1010810&1 origin (pOlip A or Dlu;taa-aeJ'l'U!l1.) are belt.Yeti to olle "'11' aotlnt,. to the • ..,.. 1l1elm-onid ... whlch. the, conta.!A. It val thIlough 'tiM .tt •• , .t4. tl •• ue ,eztraota up_ the 0 ..... ot 1Dt ••• ,oup~o ••••• \hat the •• tao'er. " ... .rUI' 41aeover.4. !he eu1lUlUo ... tlv!'" dJ..plqw4 br 'the •• taotoI" i, ~1'.ete4 p.~lJ asalnt'bhn1 .. ens.a bld. Vh1ok. 1. aD. lllt_sra! .,.. .... , .t ... allea tl..... .. O1U,,. O'E ..... I\tb ........ lao ..... the .p .... bl11.,. Of 8.2.81 tta •• 1. ot aPe.' latere.' .. ...1I1 .... 1.1.g1.t &ad. baa le. Il0l'. OJ' 1 ••• 41rectl,. to the ocmoep' $I !D ••• I .... t1.'~1he4 tr-Y1r'Ql .. , iDle.tloa. l'u:fthAM' .... .,ohw ... ··.thN1.iJ ... when .... 0 ••• 1'7 ot the· •• But- ............. SA ... l •• ~i .". lat .. 'loa with. .,. po.-..cloa or htab17 epe.4tl0 .. ,1- 1P. ...i Da _u..,...,. '-1M...... Ito. .......' 11,.tll4l• • baY. _"".aft l•l._ ,. ....... . whteb. 1acU.o.ta Shat ~ bl... of ••u aber ot 1 .... lAp ·aboll' ilbetAaet19.tle ot ktd:areai. 4 •• :.. Ibla ..... '"7 ... '" dl.'fAa\daJaed. bOll ... t •• to .iJh.e ,...eaoeor a .,.oUl0 .. "1'0047 prod ... e4 sa .. .., .... '0 • 81 ... bplvold.4.... !bare 1. no eYl4.aee I. lati •• ,. 9hat thl, ....... ltl. or pJrplolos1oa1 tJthl'bltor 1. idle •• nl' 2 or apGo1.f1c 1nanuniaat1on. Relatl ve17 11 ttle information 1, lUI ,-e'tavallable regarding the chem18t~ ot tb1. aub.tance. It 18 fortunate, however. that non--apsc11'10 1nh1b!.tor oan e41117 be distinguished traa 8peo1t1c anti-hyaluronida.. b7 vlrtueot its J:UK. ofltab111ty at 56°0. Ant1-hyaluron1d .... . 11k. moat antibod7_1. o~~arat1v.1J heat stable. lnreOEtllt years the variation. of •• rWl ~8p.ol~lc lnb.1bltol'level. under a vu1ety of or.md1tlone haa been studied. 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CU) Sa 1941 that, llOIfUl ........... 1 ........ ,atae4 .. bbillt'_ ..... • , ...., ....& 1. ..., 1,8 , ..._ ....f *- .... la. ....t .taa G4 1 ..., .....\ 004 ..,• •' o• •f __ bUD ..1 • •'1 hJ1l1....u.... lfottltJ" ell) ..... .-a1f' .... 1.... thl..tt ••• , •• al. t ..... " ......... .... at--. .. h7a1\W011lo •• 14 A1I4P'O*'h1&te4., ~.t. .... ..' ...· '*111'1_ ... ,.Jaaltll,. 4_ , • .... tt •• , .... __ ,-, ... 18 ?~ l1ura VI- ».t.e ...,.. • .... ()S.48.49- Sa 1946 •• ."v ..... , .1dw ... ......... .,....'1. ,.-, the ... han_ t. Ile., lOU_ at .... ...... ta. theM.' .~.'b1."'11t'. -"W7. Ib&. o"...-va.'_ art .... .. beat ..... til .'P ... Uoa .t ........ uat"l-. .... __ be_' labile t ... ,loa t. pt ••• an,., ......... a- ., .. trio .. phf.lologlo&1 (ge) lDhlllltGJrt. On the 'ball_ of 10 .... a1Id -"r.~"" (31) ....l ucte. ...' i Dl. bdIllll'_ ........ 1a .. "1." i .... ...,... to .. port it'd. I ......... hal 0. ...........1 . ..' ....... of ....... ...,... b 'lUt. ~ 4 ................... ,,-laT .. S •• ... .... 1 ...... .u ... , ... ,., .t .... , .aoh .... w.u.a ........ eft .. , _OIl .. ..... It 1 ........ ( J tdaat "'.1', ... Ute -. •• , • ., .fall., __ " __ .' •• , .... 1; ... . "PI ...u .. ..1 J' .., .... -7 h ......' . Ie ...... hut4. tortua, .", ... Mae, (.;1) 01. " haYe .Y14 .... wt4_ rd. •••• , .., ,. •• lblll", .t lJaht'bl1J'oa « .......... ,... Stall ...... 1 .. 1 ... haW· ....... 1 ..... ...., ..... '1,- ." . ... k .... al. '~2) .... 1rJ Ba4l4Ua ... "",. (53)~ .,. Moea''''_ (S4l 1a1l .... :.: 'ba' 1l7a1v ......... ~ , •• tltN1w .. tala '0 ......... ,1'0'.01", ••• tlY1., Wbloll ....... ,. M •• "."S'ble tOJ! -.·11Iuaate .. a. ..... f ... bdIll>liftoa ot h7al.ai .... bf ........ Stl .... 14W. Bst ... "'11,,_ pobo17 14 .. Uta1 tIltb ..... ., •• ia.ula X· It •• ..,. UlI617 .... h ... r ....... ~ ....... _'hi_,. ...... n. .. ..)1 1ft It.., ....... · •• ,. .... , .. Ida..,. or _, 1 ... , w., d.l"iaw. ad .~ ... ...,... !be u'.P •• ~" .~_f4"_.'" -_ tM ol.'$!q lUHdl .. S .. itaI·· ....... 'et • .... ,u.. ... ... 1,. .. 19" .... (3S) ,..h1 ............. .,.. ....... 1« •• tUt •• ". of .... · .... ltl .... 1d......... Xa ·1948 ... , .... ' .......... 14eatlt., .. ~1tt'" "'ilho. .l ....' .. p .........l tla (SS).!bJ,1 AI ...' I. '7) MY. ........, 4ialpe4_na. lqal .......... ot ........ 1 •• 18\11 ... la u Yitro bJ" v1rtueot 1', 0.,u1t7 to counteract tile thPoml»pl. atie 61't80t of plateleta. thea. l1We'11gatorl belleve that the inteZ'act1on between h1alt:l%'t)n1daae and the olott1ng uehan1sm. repre.ents a 0..,8"1 ttv. 1ab1b1tion bew.en platel.et, aDd enaJ'ld. An. lDbibltlon ot the eUJ'lllatlc degradation ot hyal'U,'tt'oDic .. a14 dWl to tl... throrabopla.tiJ;1 hall allo been noted by Plala(S?). ,Hore r8e8nil17 Jto1;h (58) augg •• ta that hyalm-OD1da •• 1Dh1bltlon 1. intimatel,. a •• 001ated vl'bh proconve~t1n aotlv1'i1 de.plte evidence whioh ~e.'. that lnb1b1 tor .Ul4 proconvertin are not chem1callJ 14811t10&1. WI thboth contplement anc1 proconvertin prepare.tlona h1aluroD1d ... 1nh1'blttng aot1v1t,. 18 loet rap1dly \1pon rr ... 1ng. ~ actint,. can prompt17 be re.torad b7 add1tion of inactivated serum. Both Jillton et al. (,,9) and Both (sa) 1JU.gg •• t that this indioat •• thattull inh1bltion depend, V90n the pre •• nee of. heat stable eo-faotor whioh 1. pr ••• n' :1.1'1 lnaotiYa'bed •• 1'tR but il d •• tJto7e4 upon tree.ing. aeveral lav •• tts.tora ha.. atudled the vu1atlo.n ot hyalurGD1d.a .•• 1Dh1bltor lm4er a. varlet,. of cond1t101l1. Allons the •• are Gliok and Gellan (60) who have r.port.d.Vlldl •• on the 1nb.1bltol' level.. or human a:n4 moue,. blood atter tnteo\ion wlth the vil'U8 of poll_feltUs, Oral. ancl Gliok (61) who aVQdiedlDhibl'tor levele in a Tvlet,. of .kin 41 •••••• and fulton at ale ($9) who b.«f'e.w.4104 ,he 18Tel of hyalurord.dU. lDh1b1'ior 1. varl0Wl mll1 __ 0 ..... 12 The explanation of thesaf1nd1ngs 1. a" let obsoure. The ob.eX'vatlon b7 Wlnzle:r et ala (62). a.nd. Selb$rt et al. (63). that elevated levels of pla.!t"lUi mucoproteinaoceup 1n patients with a val.'*iet'1 of me tabollc and. 1Afeotlol.l8 dl ...... ottered a tempting solution to the or 1 g1nof the no~ specific 1nb1bltol' 1n view ot the oheenatioua' er Gllek and Gallen (60). Stlb •• Quentl1 work: b7 G11ok: et 'a1. (64) indicated, haveT8.P, that h:Ja1~on1da.. lnh1'blt1on 1. not .aaoo1a1ied wIth the.. pia ... mucoprote1na. 13 Preparation of Ens,.. Hyaluronidaae waa extra.oted fpOIIl .fresh bull teatea b7 ea.en:blall,. the .alD method aa that described by Kahn (27). Th.e resu1'1ns 11qu1doon.oentl'ates were stored at 4°0. for eonaldEJrable period. Qf time. 10 appl"ee1able 10 •• of actIvIty wu ob,erv$d over th ••• period'. F'p •• h enzJ'l1le p .. fJparat101\fl were m.ade for each ~.r1m.nt tl*om the liquid CGftOtAtrate. lot.allum hya1'tlronate waf prepared trom. hum..lll umbilical oord celleo'bed ln2 pes- eeat aqueous phenol. !Ph. pU1'lflcatl0l'l of h7a1urcnate depend. pr1marl17 upon the removal of yu-lou. protein. trOll the .."b111cal corda. Difterent •• thode of purifioation v~ in thema.xmer in whioh 'ihl. 1s aecompUlhed. The method d ... orilled by Tolka4ol't (41) proved hlghl1 .atiata.tel'J an4 va.. theretore I"Qu'lnell' u.sed .0 effect the prep ... tlen of the .'libllrate in tM8 1&1;). oratorJ'_ The p0w4ere4 Ilib.tr .. t. thtla obta1n4Jd. va. .tared at room. temperature. Fl'OII 'MI .took IUttl,81en'b 117&1:uronat. vaa ma4e UIJ tapH 6.0 butter 1io run 8aoh esper1aen'. Once 1n lolut1on, th..torage or 1".' •• 1$. 11 net preotical linee turb1dlt,. pro4uo1.ng prop.rvle. are loa- rap1411_ 1h1. neee.elta1, •• the preparation of .. 1're,.h IUbe'rate .olut1on tOI' each experiment thwt involving certain welgh1D.g enol's wbleh. in turn, are ttetlected In ,,. .&Age ot turb141tl •• obtained. c. Butter $01111:10118 A 0.5 M 8.08t10801".,o41_ aceta .. m1ztu:Peot pH 6.0 oont.lxdng 0.73 per ..... aodl_ Olll~ •• , and. a 0.$ K a.e,.te buttUt t:,£ pH 4. 2 .... Wled ~oughout the .xper1m.eu:be. Th.... butter. are k:Jlova '0 allow iiile developaeni; of max:1Jmal turb1d1t7 1n a gtvens.b.Ur.'. pr.,aratlQD UDd.~ .he proper oon41tlona. !he butter.oluttolUl .ere .terea. at 400. to prevent microbial gr~. Human plaama wu obtalned trom local blood bllU3.ka, acidified, then aged aocording to the method d •• cribed by 1'olkadort, et 81.(65). Monke,. sera was collected ••• ptloall., b7 oardlao puncture. from anea'bheUze4 expers..ental ar4mal.. After centritugation the •• rum. va. rea.ved and .tared in 8'11.pule. ai; -60°0. \111,11 needed. Se1"l.ml IUllapl •• ftP. cOl.lected. t:r-om. 20 no.ftUl. moDke71 aDd poole4. Th1. materlal wo then placed 1m aapul •• , ·,eal.ed, and stored at .6000. uau11 1188d. Sel'1JDl froa t.h1. pool va. 15 ueed t~o'ltghOut the entlre atmlt,. 1n ~4.r to· eliminate variations in lDhbltor 1 ••• 1a wbleh woUld InteJlfere with 1. Oolleculon or I.~. A.t 1,.8t two .81'uaaemplel were obtatnedtrom each aniRal previous to infeotion with poliomyelitis virus. Prom the •• samples, a mean norm.al resting level of1nb1blt1on v •• atce%*tained. Blo04 was drawn at two cia)" intervals following tnfection until ~hetl~at 81!UPtom.s of po 11 Oll1ye11 tis were noted. It luuJf()lUldnece.llar1,due to tll.8,crapld p:rogreB8 ot thAt 1nfection, to drav blood upon, the dar ot first IJla.Ptoma and each daythereatter until the height ot Pfl.l'a178is and the The 8er& thua coUected were •• &led In ampule. and stored at _60°0 unt1l the ,.1'1.8 of seruna samples tor the an1mal under obaervatlon ~aI oomplete. l'.b.eent1re group of .era were then rtm. in one Q.P$l'1ment With a ail'J.gla substrate &ad eu,.... ps-eptu'at1onto determine t.he eztentor the ri •• in 1n.b.1bltQr. 2. '.8t tlr nOD'peq¥,1g (asz.1()loS:oll) lnh1b~tqr. The technique used to alSa1the degree of h7aluponidaae lnhlbi tionwas ••• '88t1a111 that described b7 Bachtold and Gebl'l.ardt tor the a.aa,. of enz7lUG prepfUlation (42). For eachl.rum. sam.ple a eerie. of tubes was set up, each conta1n1ng O.s ml. ot aubst!'at. solution and 0.1 ml. or a 1.3 d1lution ot the aerum sample. Bothe the substrate and the 16 ae,.\ml ~r. prep~.dln ':PH. 6. o "'bUf£e:r-. 1'Q6aoh'bu~ "as &\idea 0.4 ml. at enaJ.m& oOllta1nirlg 2. 0 t~b1d1t1 roduc1.n~ ~t. (~.~.T1. ),. ~Ge tubes werEt tlum. incubated ·'£Qr vU"11a8 p • ...,.1od., oS: t~ 1n a Ill0c: water batl;... A au:1ta'ble sCl"-l.eaGt $QD.Q"ol tubes waa also i-un, each tube contain1ng O.5l.ill. of aUbatrate. 0.1 ml. of pH 6.0 'buf.ter and 0.4 ml.ot ena~ • .fol1Qw"';na the 1noubat1on interval tho reactiQn 'Waa atopp$4 by s..aa»~ the tub •• in ,a 60°0 water bath tor 10 .tJAU1ukl. ~ tuba. we1'e ohUled in au 10e bath then .3 al, ·of pH 4.2 buttez- aad 1.0 Ill. of .. ~41tled pl*o,"1u war. aMati. Atter a Jutl'lod or 30 ldml.fI' to allow sur'b1d1i;7 to 4evelop, 'ill. &b.ol'banc7 wu d.t.~d nih a C01MUUl Jua1ol'ap&o11r'opbo~ "'.1' Ie' at a wave. lens_a of $80 al. Ae haa ""·en IIl;l.own '0, o~ lnvaeU;ato1"a (42,66) 'bh4J ooaeenvatlon ot l!qalvoaate U1 .ach tue .an be d..'.~ tl'''' ihe ab •• rbaa.7 ~b •• ".4. !he· ooncent»atlon or bnlvoaa1Je pro.enti at ... 'ihe It.-loti of hJdrll>1.1-1a GIlA then 'be aub.ttt .. t.4 1n:'0 the 1'am111ar tir,. ..4er eqQ&'loA 8D4 ..... ' '5. ealoula_ the· ap •• Ule l' •••• loll .pate o ... 11utJ It .. .how bal., 00 .. 1ntttal oODCaatra'lon or $Ub.tl'ate 0t_ oODcentlta,tloaor ftbetraiie after the reactlen lats.pya! (t) Praa .he .ean k velue 8.ebtdae4 to!' each ............ (k,), the following :r.-ela:b10 •• hlp oan be .stablished with .... k yalue obtained tram the en;c1l'le oontl'Gl (k.) t 11 .. .,.bo1. "I, rev.".'. "be pep .ent lDb1bl~loa.t _ .. ,.. ,aetl Y1 t,.. ,. .at •• a .fa .. " .... tc&_I& .. III_ • .. itie aDtlhf'al_.u .... ret ••• ,. the 8 ... U1. _'1- bD41 totqa11.U"oft1d ... whte .. a.,. lie •• a«UJ' 4l.~aM4 hOIl tbeJh78101oS1o&1tD:blbl ••• f4' lqalvonl4UelB -,. at ... ..... • ..,1. bJ· ..... or 1" •• '&biU." at !),oo. •• thl • ......... \he pl2J.lo1oal." OJ' ~oUi • .tahllt1t;o1' 'a ... 1.,.1, 1aaetl •• te4 at U. eD4 .t 10 ai_.... ,., ... lUlalDs iahlbttor, .....' Ill• ••t ........4 .... eaaple ..,. \hu lite a,vS:bate .... l.17'lo .......... flI .,.eUl ... 1Jl-ll. J1l .. 0Dl4 ... . !htt ... .,. pro.eeI ... t.,. ••• ut. aat1b:ralvo:rll .... 1. ,he ........... a-l))e4I: •• pb.7.iolo8$.&11DbS.14ilOr with _, .... a.,'loa, 1m8 ...... ,-.18 Wi, be ... .,.. 'a !lea .... •• ,S, •• r •• 10 .iD ..... ,.1.,.. " ... 11.'1& or 1.)&11&- pH 6.0 lJatt.. !he heat ... 411ui;· ....... s. ~a ....... ... , •• ,.,.... ude'iorS ... ,. • .,1.Q1,.. 1. ltlilallBll1II at saaHIJtaa_1 a ... a,a"a-the op':laa1 ... ,.. ......... t1on 'Yul •• ct ..... Saa l.IpOa 'he natwe ot the .... iluau. U. in ..,. given exp •• llYaw, the .. ,.. ooao.a.ia'aUen tho ... S8 '1100 ~at. l1;J4rolJal • • t ... ~.trat. p •••• t ........ a. 4.gr •• dv1Qa ~ JIt ••• ~l .. 18.,_",&1 ... , 1t t.ltIp •• :-,bl. '" a •••• -.11 ...... the flDIO'Clt of rib.tn-a,ct·. P"e1b1Jii8'~.l.,..a.l'b.8· . MY •••• 81t:uatitJJl O(J0U78 ilhen.th..~~ COlic.Dt~at1on!. toolDl4U. ".-.ring in rUbe! th.r_eo.f~o17.i:. that 11, de81rable'in the ·propoa.d exp .... 1m.ent, a .ez-1esol tube. contaUing the pJlloperc·oneent .. 1ilQnot.11o.1B-.t.l. pr ..... d.. To e:ach tuba 1. added a 8110 •••• 1 •• 17 hlgb.e:r 4U~'1011 of . . ;t eDSJ!I6., !r.be J'eaetlon 1 •• UOVtKL1So pro ••• 4 overtbe· &11011'" t.ble 1ntervcl then ,,1Joppsd .. pl"ev1ou.l., 4e.orlbe4. .. . .-0..,.' ot 8uNts-au. J"_uaS~B1n each tube 1. d.~" .... t.OII 1*1. data the 4.~.ot 1QY1rol.,8ta lne<aoh , .. be··!. oCloulated. the·· anBJlU dUutloaoOl'pel;poDdlnato tho d."" raage ot h7d1'e1781. 18 thlle cmoa. dbectl,. or by 1D.ter.polatloa. In oM.,. '0 .S.t the p ••• l.. ~lI1dt. It._red top _ Idae" •• ~7 1'" •• ~ ........... .., to I'nt •• 'be p»ooe4-. .... t ... "he'tBbtbltloa IfIadle.. !he tutorl ....... 1"1. t01' ~ 1 •• c~&'le. ,~ ••• at in "*- ol'lg1na1 ... ~ 8,.8'- ..... pJt1aa:r11,. h. to tempera". "sriatlonl, 'b1:rd.ng aacl plp."1ng eno .. e. In 01'4.1' to 81lid.na'., •• tu .. poa.ibl., 'h8 eno!' 4_ •• plp.ttlng isbe ra'10 ot theyu-1QUI reactant. vu .,:tI ..... tPO!l that orlg1na11y \28... in ~1nb!bl tot' a1nld! ••• o !ha eoa.eentt-,,'lo1'l1 or au'abate a:nd eB&pla ware .adJuatea ao Ib&' for .ae!!. .. volume of 1 111. Va. \\te4. per tube. fb.1, lU4e po •• ln. tbe UI. .~·Y01""l'tc p1pe"'.. ... 'fa-lou Ill'b.' ra1itt coaaelleratSODa ne.· •••• ,. tOf! .aoh t •• t v.". ob'a1ae4 19 \'If .... t1dclUull .. ' ""11%;' Ul'.,.'ot"",'IU.tl ... v ... ,ip." •• llit.··'1:l6··" •••• S ....... ! ..... 16~ 411",t ... of ............. , .......... in • 0.2 al. Yo1 .... a\\Oll a lUll ... 01-. v .. loul4e ..... 1Jo .. peftll •• lb:l. au.. the ..... t .f •• .,. "II , .... $G.'" 1 ••••• 1"0&1. ..... , of ... ,.. of ...... tr.... .. tIat4 •• 1-. .'t ...... ,1. _ _ ,..'- ... tlda a.a •• ' ... .,., •••• '1.0 ...... Sa tlUt· -iaUal lBblbl"oa .11I4l ••• .. utl1i •• " •• of • biehl' .... ak .... ,., ...,. ...... va .... bath..te , ••• "'18 to •• , ............ tl ... ""10. -wl ..... te.aOo. .. .•. Bit maJ-lt;7 ot .. Ill. ..' ...............1 .. "' •• '-' ......... ~ fit ..... 0, '.,lSOC. li ... ,t ..... ........ ., t ••• ,..,·· .. ., ........ tv ....... 1 •• Ie ... ....... , ., low ' .......... , .............. 10.1'100\11. be •• 'aNed. to ·tile 4.81' ....... tale •••• '1 .. tlt.u s. .... a1 oould. '" p •• l ...... ..rtl.1 .. 'lJ ' •• atmt· •• _e IS-I ..... " ... .-I'las bell ..... , .. u" •• SaMnal. .. .... 1.". ttltttol •• 'l" hlp ............... , .... .,. '" .',a .... to 1aIve What .... _.aU .. ~. p ..... 4 ........ 1.0111 .. OJ" tiP.".N ... "e •• ts' .. !.be •• .,t.,' ............... -cat"*, witA a " ... 11 Ult ... HlPIl.''', • _shIP." ."nUs ............. .. or ... ( .. wei _"M., boll ..... 11 .,uu ... Sa .. Wldctla ... 11 pl •••• • , ", t ...... pla;.... ...~ __ .. M., $I .... qUacI.... ..·,.,tJh ., tdd.oh •• 7 ............ 1a .. bath. ' ......... >. ., ...' .., ·er' taw· " .. JIlIn ., ... , . .., ...... r .. ~ n-_ 4° 'to 31Q •• Otd.4 .... lIId.a'llilttd to withtn ~ ~ ~ .,! . " • • • In tlr4 •• to .altul ••• ' .... 'ton. •• b.l'J" 0» ... _ ... U. tS;ae ··1a'_,pfal·., ".,,"oa C.), .... 1a11Jla;L .... tr., • ........t l.n (c.~ .........., or ••. .,. .._ . ......a laa ., , ___ ..... 01 .tt.e. a' .. valo'· .. _" .. (0 J. s.th .. ,s.. la'ertal .aa4 th6 ·1a11Jld. .... · .... '.ooae.atr.1d"ea 'ue olio, .. lJ7 .... inT •• tlS .... ,· heDoe OM' .... .alloa1oul_ .. 'bM ... lID' ot a •• ' •• '. Jit-ablns ~11l.41.' .. eat 01 .. ~I c i' ••• O\'d 1a .... al. ·t.hla i:I 4on. b1 OOJIp"lag the .... "141'7 4e.elt».s, in ~ •••• ll". ~ .Iib. tha' Of a ,e,..I.. or pad... tub .... ,. eonoe.v.'l.... 1M ... laVi.Ubi, 'be ..... abt .. ___ , 8D4 llib ...... · ..... 8.1'.'10& 1 ••• 'abU"" 4t.ap-___ uo.u,,_ oa."7t tpoa .he re.n.lt;u' 0111'''', ............ Ule ..,.." .. 0£ eu.babate tr ... ..., elven ."'.orb .. , .,.81_. a.vine ....'b lllhAt4 the ...,., .t......,.b... rea&llWq at,,, • 1' ••• " •• ·taM"al of ,,) ... ar tb.e •• a1eul ••• vtl •• 1'7 ,,,. the to11olt.tas nlatl.lAlpt 2he .)l\d)~. v I'ftpH ... ta «10.1'7· ... 1 • ...,1" •••• , ill 1iI811. et ldUlpfOU h:JtIrol,...4 ,.. ld.au\e. !be yl1ue CO' .. Ch. ............. __ t .'1 ....... ,. lQ'ctrol,a" w1~ She r, ......... s.., ... a1(.,. ...... ht.flll_l··· .... ·,.·' .. 1' •• :·U'itjHHcl.\l.Hl we .. ' utill,U4 ...... ,.Iag .,._< .... ,'" ·tIii .... ~'f 7 .,' , ~ ..... 4 ... Nlnatl •• ot .. " .. 11 •• ' ...... 01: .. ~.1)".1. 01 qal ..... , •• Vlu.. .,-. • .. __ .t ... ., ... t-a'.of idle· ..... "oa ..... 1thtt ·.,..ttl ....... , va. .... _U.-. .. ,. •• '.l'II&.saa -.. tkl. ztate va tatl ..... " bl' ............ ~cm.' ot .... I't .. tlaa •• \aBee ..... ,........ yal .... t •. ... ........ U .. • t ~.l"'''' ...... te " .... "'alae4 .. . .. vi..,. ."-s..~ •. 4 __ ....... ti... 'he ....... .. ... ",.'" ........ 11 .. '\WJs..a hela ....... , ... ~ .. 08:6 ...... Ibe ..... r fit ~ .... U ........ 4 ........ 801el,. "" Ibt "... ti' 01 ... '_'a •• ...,........ flo •• ., .. .... .... ',.... ... __ 'lM ,Ca" laY_i.bl,. t"h ... o~.17 .,-... the .... 1- ru., ........ " .......... 1 ..... VI • .... .., •• taeat&l ,tadlfll ... ~ 1t pH"d ....... ..,. to el1o'll1aM a r..otiOlUl .~.. ro. ..,. ot Mle " ••• '1... ....u ... 6. at tall, .91. .••• _ ._Sl:*a _au __ xa6u&Ja:. b .,.... to oa1eulat. v..s. .. _n. ., . ...., • .. " 1 ...... ..,. -. ."allal" .. att •• t .......... ti. ~ .. tleaY.loe1',. .,. the 1a1'!a1 .. b"rate co.oe.w."", !.be ..... rd' auN. ..' ..........' 10. . Wbioh ......' SlUed. la ... 11. ...... ,.1. lWk4..,"'.-" of-" '-lttdid ... l • .. __ .t.'hr_ ....... th1 ..... a, ....... 1,. ....... ell ...... -.. o __ .e.." .. "1'. _ .... _ '" .. 411."= ot •• Wad ... lO.v.a ••• 11&"." III aII7 OM • .,..s..a' ,he ,he ... ,.. eoa.&e1l-'.'lOa, ...,.,....... ... tiM la-..ft1 of • ... u •• tfN'te he~.on.i.l1:", tlul oDlJ v&:rubl. belag ,he .. 1l .... ,. ooaoen1;rl;"on. .. ftloo11;Jot the· it.a •• loa t •• • _ ... 1altlal luN. ..' ...........s ..n vae ....a I.. .. br d!:rtd1ng 'bhe amQui; of aub .... te h74l'ol,..ed 1n 8aob .. .,. b7 th,e ,. ... 1;10& idae 1at ... tt1. .. ye1 ... 'hu o"Une4 .... pl •• te41n yarlou ..,.a _.au., 1Jbe o" .. :eoon4las $nt'lll 'nb.tl'a1seoo.acmil1-aU .... 4 •• eribe4 lID4.~ upe»s..a' iel. r •• u1t;8. 7. !at C.!'. st !B!! .... In order to gain aturthel- in.igbt into t1M naiNPf ot thie ~ol,..1. of hJ'a1uroaa .. b7 hTa.l\1JlltR.\1d .... , an a'b'emp' va •• ad. to detemlne the .Ifeot of temperat1U1eupon ,he ~ •• (ri1on.. Sub"r." and. .~ (toaaenbratloQl .81le adj ..... 80' that the reaction prooeeded. •• near .. po.albie te the tir.. o!'4ex- over All large a • ..,.~.tur. ..auge all po •• l"l •• . !he :r ••• 'lon ... a11ove4 ,. p.oo •• 4 1;0 'ihe .... 4_gr.. of oQllploticm (within upel'1taenlal llmitIJof e.."or) a .. aaeb. t..,.r.'1JN 16 •• 1. .. .,. .... 1.1>1 .. were ~.fQl'. ' ..... tv. UId t1me. au,... _dlllbawa". oon.eatrat1one'betngheld o out_t , the •• coll4itl ...... ~;. &ap0884 U .. de" '0 lIildmi •• the varl.:iloa tr_ 1ilIe ... tilt- YUiIe. ialat oe0UPfi4 (at; •• rt-.1A ' ... Jtainare 18.e1.) •• tibe d.p'.. 0'1 bydre1781s progr ••• ad. !bi. oocru:red "'O.U •• the r •• oid.t)a d1dne'b p.o •• " atl*lct17 1n aoeord ••• with tllIJ tlr.1J erdereCftl8'bl0. .t .... tempe2'.~e. but wu. -..Terthtl ••• , 010 ••• no. to jUa1Ju, ........ r tdd. ...... tS .. CP .. .u ...... sr •• t4. ~1J.l. 23 vaar:lgld17 ooa'roUtd). 'fh.nk- 'Value tot! the reaot1on at e.eh tempera'lnl:r*& level... ealcula'ed by mean&l oflhe tlr.t oJl'der rat. equation ... plotted agaiallt the OOl'l'.apoadiDg tcmIferat'lDt .. 8howa \1B4e. eapel'tm.ental reana1t.. !he l'ed.e4 ••• a7 'eelmlque waa "t1Us84 thP~, the •• a'tu.41.. 1n or46:&- to ob'bala ..... p.901,. da'a •. !he ettec' .t ,..,apat,.e up •• the 1DbJ.bltlon of the uaetlon b., n.~ moDkt1 ..... a. d.'.J."lI1J2,ed bJ •••• nt1a.U7 the ...... 'hod .a cte,erlbe4 above. !o a.ob. tube, nth the exoeption o~ 'the aab:,pelt, va. a4d.40.2.0 or a 11,3 dilution of ao~ .0Dk.~'.»". !be .eac\lon va. allowed to »1'00 •• ' '0 the .... desr.. otoeapleUon a' eacsh t .... p •• at'u.re •• vat , •• .,.10..,11 4 •• 0%'1 ... 4 u4 the 4.~.. or lDh1bltlon .al et.lovlas.a tor •• oh ' ...... , ... 1 ••• 1. 'el' cent 1Db1bltton va. ,lotted u the or4bak veraua the ocn • .,oadias 1;.., ......... . !he lDh1bl vl... of hralvoa1d*.e 'b1 'lUlhe.ised .. aka, •• rum( Ix) • ., 'be .'.1'1'*'.4 '" 1Ih... ot w" taotor., Iz = I ..... , .. I.s the heat labile, Bon-ape.ltl. lDh1'bltoJt (I ... ,) pl •• the heat .tol1e an'lbGd7. aatlh7al:tlFonl4... (I.s). An'1h7a1urontda'. (I .. ) ".. ob •• Jfyed 1D appz-o.zlJaat.17 60 per cent oZ 'bh...1mal. , •• ted.but 1a .... h low ley.1. that 1t val dlttloul\ to' 4e .. ot. In a4d1td.on, the!'. ha. been no ooa.l.,_t oo~.l.'loJl be.... .a. Pl'otP... of tha· polle.7ell111a tat •• tten aa4 'bheleP18 1 ... 1 ot cbt. aatlboQ. The rew 0..... in Wh10h a ~1.. 1A tb1. entlb047 .al obaeMed «rl~e 1) might .. 11 .. chua '0 tale .e.oMery invaa10D t.reQ;u.911tl,. ob •• J've4 1n '.J'IIlnal 1'81'111,.81 •• !!he GODvilla'lon ot ... thJ'a11U'onl .... to the 0"',.1' •• 4 'otal inhibit1 ... (IJf) 1U7 the:reto •• be a ... 14."ed autf!- 01.n'1, _all at to be dJ. ••• SarA.d ~ l..a~ 1".01.11 aI a funati"n .f the nea-"peeltte bblb1.o1- (1 ... -.. ), In •• d81' ,. q,uaat1t.1ie @IDsea in 8el*UJ.1. h7I\lurord.4a •• 1ah1bltorrl ... 1., 0_ w,' n. ........ b118h a noraall' •• 'b1Dg raD88 tor thi. oapaol'7_ fh ••• '.b11'''ot o~'1toha "alue 60 s:: 0 -r-I +> .r-I ..0 40 .,-j ,.... -a H ~t:.~. 20 ----.- .... -- ..... _--- -2 o complete paralysis physiological inhibitor • ------ antihYbluronidase - .. - . -- ... - -- ---.' 2 4 DAYS ,. ,. ,. , ,. 6 • 8 Figure 1. Comparitive levels of physiological inhibitor and antihyaluronidase in a polioElyelitis infected monkey. 26 depeDda Upoa tv. major tao'er., 'he dq"'o-d&, "ui.t1on tn DONal reltlag 1Dh1bl'OJ7 1ev_1. end· •• ooadl,..the •• , e:prera 1~ .... t 1n 87 8l\I.1"lcal t •• t of thll nature. The tollowlag e.er1aea\. 1INH ded.gaed to .... I"a1D. the ma..gn1tw!. Ul4 r=el.'l"e _ortu.o& .t iihe •• tac~ ••• fU1d heDee 'bo."abUIb. ·the uol'lUl. re,tUgl ..... l. within ear.full, d.fined lJ.al1 •• Blood .aa .. awn t ... iaxt •• ~ lIOake78 •• 817 ,eeoad dq tor 10 dar.,. thAt'ok1 Ublltlt.-,. 1.,.81 (Ix) va. then oalculat.4 t.".aoh ... 1ft (Iable IJ. l'h. ... value. "pH.en'. o.o!.D&U •• ot 4.,. ... ·4 .. ,. .... 1avlon an4 8111 acalJ1;lolal .""1'. Konkel bI,. 1 2 .3 1f1aber of a_all!; 1. 26.~ 37. lit. 34.1t$ IVd. Dey!a.io. t.£. tll! It,. , 0"11 l.~ 2,.6", 2.8:' It 1s app ... at ~rG1ll tbe.. cia". ·Ihown 111 !able 1 t)ha'b the yuiatlon 1n the 1' •• ting 1.".1 of 1nd.1v1dual lD11I81a 1, large. !b.e •• .ad", «e .. l •• loa ta •• lve" 1. alae.. Wic. 27 group. In ord.z. tode'.rmlne' ilhecontjlloutlGn 'ot'bhe lUlalrtloal error to 1m. obl.ril. .'aD48Hd •• S.atiloB, tOlD' • ...,1.. or •• ra .ere t •• t.d I'GpCU1I.411 to" h781UPoa1da.. lDb.1'bl to%'7 l.otlv1t7 'table II)., 1 2 3 4 li,t1aatl ••• t AnalJ1;loal IfttOP (a.pe.1;.d Ob • .."a'lo .. on Sbgl. a .... 8..,1.,) 37,3S,lS,)2j,)1 29,37,31,31,31 21,28,26,21,2.8 21,26,16,21.'0 3S,~ 26.41' 26.~ II 2.~ l.6~ 3.~ 3 .• ~ I 1 trpon oOlll)HlJ'l.on or the .. "' ..... ge.tandard deviations hem !'ablel I aDd II 1. t ls .. put_at tha1 the •• n1a-lbu;1l1oD ot the dq-to-4a7 1Ml'bltol'7 ... 11:'10118 '0 the oh •• n8 • • 'andard 4eria'loB I, '0 .Uptihat It 1aot que.'loDable .1gD1.t1eance. '1!b.. 1' ... i.'loc about the Mmal r.'tba 1 •• 81 au._, thererore, be due altIo •• 0 ... 1.-'17 'oa:nal.yt1cal errol'. It ,he .xp.~1aeD;'1l 4... oould b. ....U'lUld to tollow e10 •• 1., the aormal or ·& .... 1.' d1.~1bu'1.n. an7 1 • .,..1 ot qa1uron14 ... tnhl'bl'loa V ...... then or equal to tvo .tandard daviat1GB (2) abovethemDan. r •• ttng val't.1e oould be oona14eredtotal1Wlth1nth& S'p.l"·oeat(O.OS) 1.".1 of oontldenae (Plgure2). I1ne. ,hOWever, tlie exper1aeJJ:bal p.pulation 1arOll111\617 l'athtraull(4 ,.' 6,..,1 •• ) 1t .u deemAKi Ji.o ••• ..,.'~".,la •• tb1 •• flUe wlth -,. tandattd d8'f'latlttaa (t.cr). !ht'.alue plu the aol'lUl re,1;lag mean ... pr •• entls tlua 10ver 1 ... 1 or tl_tl ... "e. !!be "tit tor an e.xpel'i1Iental popUlaiion ot (, • ..,1.. (S d,gr... of freed-om.. B-1) 1. t011l'l4 to be 2.6 ." ttt.e 5 .G%tcent level ot eontldeno8J lWace. -," .taa4ud tievla'1oM 1. 8.1 pe1* oent (1; = 2.6 Jt 3.11'). Atl7 1aor .... of •• ~ 1Dh1b1tol' value. 1n excel. of'htl ._ of 'he .. an 1"6attng yal8 p1111 -t" ataD4urd deTiatl ..... at thflperore .... 1d.1" •• 1so hu110ate a.1gn1tloani a-l.. 1n ... 1ab1bl'iol'l 1 ••• 1 of ~ •• ra. ~I 1. 4..,11It1'8\$4 .~ PI.... 3. I' 1. 1Jlpol'1umt 1Jo _'a 'hat &1 the nab ... of • ..,1 •• approaoboe •. 20, the di.tJ'1'bution ot the aisn1tloauoe ra'10 ( ,,) appJ'oaoh.. ,he '.. ot Uhenol'lUl OUP't'e (61) .. An. ldcmtloal pattern in the ri.. ot •• 7tD. hyaluron1da.e lDh1b1kr(I ) va. noted 1n «ntmal. tnt.o'-4 v1. the 1ntranu-'; and tntraolufebr.l l'ou:b •• '(J'1~ 4). Thla.1a11a*lv7 1. tlot''tIP})r1aina .1aee in both I-Ou'." the vb1J.a Oil' ••• the brt.1n papldl,. aDd more or 1 ••• d.1l-eobll w1thollt a h~a1 phue. With e1'he1" me_hod "Jut p.~lo4 preo •• d.1ng » .. &1781. 1 • •••• ntlal.11 the _am. 2.5% ot area ! -z 2:] :i:Z ri -3 -2 -1 0 1 2 .3 Standard Deviations Figure 2. The normal distribution curve. 80 s:s ..0.. . +l ..... ,0 '0 ..... .Q s:: H 'tc..:..~ 60 complete paralysis . ~. 1st symptoms ot paralysis ~ ---_.- ...... --- ...... -_-_ ----e---...-. -- t---.~'- -2 inhibitor - - -- -""I theoretioal level of significance o 2 DAYS 6 Figure 3. The rise of physiological i~~ibitor to levels of significance. 8 fM o r-i 5 \D to> CD H tlD c: oM +=> I1l Q) p:j • t> is 3 1 -1 • • \\\\\\\\\\\\\ \\\ .~ • mean resting level -2 o 2 4 6 8 DAYS Figure 3a. The rise iu serUlli. iunibitor expressed as the num.ber of std. dev. in excess of the mean resting level. The dotted line represents "t" or 2.6 std. deviations. u complete paralysis ~ 1st symptoms of paralysis /:. ~1·, '/" 70 .. 60 .. t--....... ------_ -----. ,,) ._ - - - - - - - __ I _ _ _ " " 50 ," " -2 , - --I Intracerebrally intected - - - - - - in tranasally inte cted • • I o 2 4 6 DAYS 8 Figure 4. The rise ot serum inhibitor in an intranasally and an intracerebrally infected animal. rOI. ra'her abr\tptl,.. A .'SJ!d..tleant 1ncr.... in the eeJ'Ull 1nh1bl"'n level ju.' pl'f:" •• daoJ;' 1;1. conoom1ttant w1th the ollie' of paralya18 (Pigure. 1,3. and 4). Wh •• a "'.%111081 lJ.at 1. dJtda to the aboi .... b-oa tihe ~ polr:l1~ 01 ~'.~o.pc or the ..... ' lDhlblt;o7 ....,.. with'1lhe line l'epr ••• n'b_ .. theoretical 16 .... 1 ot alSDUloanoe, the t1ae neoe •• U7 ~or Ib.e '81'111l tori.. ;from re"1Da to at_tloat 1nh1b1torl ••• l. 18 1ndlea'.« (:rl~ •• 3 aa4 $). Ta._an yalue tOl' th1. pe:p1od in a l.n4:, o!tlO fIl'l.hlal. va. found to be 5.1 dq. 11'1 ooa .. rut 11. _an. 6.2 4a7' t~J' the· on ••• ot tlp., para1yblc .,.,'0118 IJ14 6 •. 9 de,.. lU\t1l.paral781. v.a oompl.' •• (Iabl. III). !he .. _tude o~ 1Jhe ria. In .. ~ h7a1woa1d.a •• 1nh1blt;ol'J' l.vel. o~ 1n41vl4ual ar4ma1. 1. "1 ... 1 •• 17 oon. t .. ,_ conver •• l,., the aoftUl l"e.tlag lDh1bl' •• 1 ••• 18 1n 1Dd1Y1dual .. animals vari, •• ,,14el:, ... 11111. ...... 4 1a fable IV. fhe mean value to" the •• gD1tude ot •• rum by.lvoaida •• 1nh1b1tor (It) 1t1 •• 1" 20.S per cen' po.,o.liAsa atllll4ar4 "evlatlonotS.3 pe ••• nt. It 1Jhe errol- due to the t •• t pre •• d._ ().1") ... IUblt",aote4 trom thl •• alue <".3" .. 3.1~)'he reau1ting deda'ioa 18 2.2p .... nt. tfhi. 1nd!oa'. athat the masnitude ot the •• l"\m1 1Dh1bl1Jor (lot) r1 •• ill IndIvidual an.tma18 18 .... 1Dall C)onatant. On tbe other h&tld, the :peltinc mean yal .. of 34.8 pei" ·e.at to •• e1'Ul1 1Dh1bltol', po ........ the extremalJ large .'Uidard deylatlon 70 60 50 • !svel ---------- 1 -2 o 2 4 6 s DJ\YS Figure 5. Method by which the time interval of rise of serum inhibitor was c&lculbted. The interval necessary tor the inhibitor levels to rise to signifioant heights was, as can be seen, 5.8 days. RiiDke7 I'UIlber •• 1 2 3 4 5 6 1 8 9 10 5,.34.,.' 6.2 ...,. S.O da,. 5.6 day. ,.3 day. 2.5 4&7' 6.3 dq. 5.24*,.. 4.0 day. s.a 4.,.. Sdq. 7 4&,._ S d.,.. 6 dQ' 6 da.,.' :) da78 7 day. S a.,. 6 d.,t 1 day. toiit Peral,.81 • S dq. 8 cla,.. 6 day. 6 d47. 1 d.,,8 6 """ 7 dq. 8 day. 1 da:r' 9 d&78 of 10.9 per gant. Corre.ilon for .WO~I of maa.veaeat tall t. reduce th1. Ya].ue al&nltscel'b17_ !b1. 1D410.' •• ,hat a l.I8Pked. vetation in the 1' •• t1l1.g1evel ot "0%'1311 1Dh1blior (It') e:dlua 8Ilonslndlv1dual 0111Ull •.• D. Pa.!lure to Ob.ene a 11,. in the Hral:urrmlcla •• IDh1'blt0l'1 Level of S.~a fhtt probab111'by ot an 1nt •• 'ed·a.rdaal e.m1bl __ pard.,,_!. os- eUnloel ..,., ..... 1 • .uoh 1 ••• in .he autre- 36 intelt1n&l.lyiD.tected i.D.llIal than'ln an' .m.m8.1'lnteoted b'1 either the lntitae61'eWUOrfntranajul'oute. 'Ord1nar117 9> per cent tol00p$rcsntotthe GlUulalllnnoculated bl" e1 tiler of tth8 latter twol'Qute'e.Xh1bl t lu:ae e'V'!denoe ot poliom,-ellt1e v1th1n $'bo 11 da7' after lntroduotlOll of the v1l'ua. Wb.en the rtPal 18 tntl'o4ueed Into ;a. ..... ell b7 .. ana ot a oathetftr oDl1 20 pel'a.n' t.,60 pel' oeai; ot ,he audmal. exbl.b1' olln1oal 'J2IP'''' or para1711. and the· lnoubat£Qll period veri.. trOl1l 1 '0 18d4,.._ iii!iiiiI _el£ 1 2 ) ,4- 6 7 8 9 10 III! •. A Oompar1aoa ot the H.8Dl1nl<l. ot the ft1.. 1n Serum Inb1bltol' I,e"el 1n Several Mimal. <iGsf1iii · .. ilzl· . 24~ 59~ 31 f, 24~ 32 " 48 ~ 32 % 32 " 31 " 29 ~ 1 J'll 4l;C 14 " 60 _ go" 46'; 69 ~ 60 " S8 ~ $2 ~ 43 " 1 I.t.il.e 1~ MIl 17 !t 15 " 2.3% 26 " 14" 21 % 28 ~ 26 " 21 " 14" a •• ting aean :: 34.8 " :10.9 ~ Hean relatlve ri.e: 20.S" '* $.3 ~ 31 Twen:b,. animal. w... l%ltG ••• tl by ,he ga'~lnt •• tlnal route. Eight antalla OP 40 per oent exh1blted paral,.a1, following lneubat1onperlode of 8 to 16 da7 •• lione of tbe 8 paralY'aed 'anlmalt e.xb.lbl "$4 ,. l"18e 1a •• ram hle.lur01Udaae 1nh1bitotT level .ither p~.o •• d1l\S Ott dllJ.-!as pu*al781. (Tab1e V). Total Inbtbl'ior7 Le.,.i. 1. Hollke" In.teoted with Pol1em,-e11 til vla the G •• ti'o*ln.t •• tUlal Ro11'_ AiiIiiaI i •• tlna It:,/ vaI".lm ••• a- %% '.es is. %neU'batlon Humber I.t 1ngrara1,..la(_) ot .. ,ub• •q uentrer1od 1 2 1 4 ;; 6 7 S , , . , 38% 26% 31% 28" 49'; I ,.".1 j' .' i " I I I~I '1!11Z!1. 'AI, 26,26, .23,28,23 24.23 10 4&,.. 3$,33,31,31,33 .32.33 10 48.1' 40,36,45,43.4.3 4$,44, 10 48.78 39,39.40,42,36.40 22,26,23.2,$ 28,)1,29,)5).33 31,31,28,)1,,28 $2,53,$0,46;49.49 40.39 2S 31,l~ 30,)1 SO . i 11 dq. 8 487' 10 447' 12 de,.. 11 d&7' 11' E. Th.e Hel.at1oralJh;lp 0'1 PoUQ111'Je11tl. Antlbod7 Leyol to theSe1'*tD1l llyaluro1i1due Irib1bl tor. i'he 1'18. 1n the h7a1m-on1d... 1nb.1bl'boP1 lev_l. ot monk.Jear.. 4u.»ias the lev.. ot ,oUOllfell '1. Val ma.rked.17 ,11111..- , in 'both 1ri,trtmiai1l>t:'ili(l "!nt ... ci:reb2:'allt tnt •• k .. utaala. Bov • .,ett" there 1. no oorre8pondlns 1'1.. ta 'the lnbibltot'r level. ot monk.,. l11teoted via t,he oral or g ...... inte.tinal route. It haa £requently been ob,erved 1!l tb1a laboratory .. veIl ae ell.where (68,69). that monk.,.. 1nfeoted 81ther tatracerebrall., or :1ntl?ar.t.a8.11y 1"ai1 to exh1b11; d6teota'bl. ot humoral ant1bod'1 to pollamye11t1. viru at the t1me of paralltl. or up to 1;wo :m.onUhaJ nbaequent to parallel.. On the other hand, oralll tnteoted anL"Ual8 rare17 t&11 to show deteotable v1rua aut1bodl level. at $he the or » or ahortly art.r, par8.1711e (70) • The t'a11ure to e:dl1b1t a vlrua antibody t.1te "alOo1a:fie4 with a ti •• in hJaluronldaae lnh1b1tory le.ele and, eon.- v.r •• l., high. vl:,WI antlbo47 \1\8.1* a.aoolated with the ~a11 .... to ob.erve a ri •• in lQ'aluromdaae 1nh1b1top level. .usa •• '. a reciprooal relatlolUlhip bet •• 6n enzyme 1ahibltloa·ud v1.rua antlbod.,.. Wheth ... or not thi. relationahip 1s a eo1nc1de!l.O& 1. not cleaP. !he analogy la, however, too t ... $1ng to d180u4 v1 thoui l.'Ientlon. P. Iab1b11s1on of Kra1u:rom4 .... 'bJ' the 11,drol,.tl0 's-04u.ot. of H7a1uro%llc Ad.d 1'.b.e r1 •• of •• rlmi hyaluron.1da •• 1Dh1b1to1'7 level, hu 'be.A oh •• rved,la avU'let7 of 1m-•• tto .. proo ••••• '71,61). 1be po •• tblli",- that. th1 •• 1 •• m1ghi; be due to the acc'\1llllP latlon in ,he 'bl004 .r the .... kdowu ppO«uo'. 01 gro\'tDd 39 .ubatance oJ,f,ln •• a.nee, the h1dz-olyt1c pl'04ucte of hyalw-omc 601d 1" worth lnvelt1gat1on. It 18 theOPlaed that under o~ta1n coud1tlotLI this eDZJmat1c naction nlq be.elt lUdt1ng du.e to the acoum.ulatlon or reaction bJproduct. whioh m.ar aard.t •• t an 1l\b1bitoX'7 effect upon the reaotion. ~ !'he etteeta ·exert,ed upon iqa1ur0.D1da8. b,. tlt., h'ydrolytlc Pl'04ue.til 0'£ h7.1uronlo 1.014 .. 1'. 4etel'1l1ned •• ahovn in J'lgt1Pe 6. It v •• noted that tollowlns o"ert11ght dial,._1. (atep 2). the ooneentratlon of }qalUl"Onio aoid in ~ d,1al11'ate, (:measured turbldhle":rloall,.) ~eae1ned oonatet (step 2&). To thi. d1al,..a'le e. &mall aJIOunt of' ~ extpemelr 411ut • • olutton of hyal:a.zlollldaae va. added (at.p 3). nu, va. enough to ... sure complet.bJteakdown of "he aub.~ .. te ..,,,t not enoll.gb, to Inserter. v1'h the aubaequent anal,.1. ot the dlalya.te tor ena,.. 1nh1b1tor-,. qualltl... Sinoe a tm&11 re.ldual IUIOUnt otaot1Y8 en..,.. 1lU 111 ".17 e.... ,~ ••• nt 1n the dlal".':b. a:tter h7dJtQ17118, l' proved. aeo ••• 81'7 to dev1,. a method. '0 •• t1ma~. the ~-V4.. of th1. :tt •• 14ue. B •• auG of thl. a ,8I'al161 eontJ-ol val F\IU In whteh em identioal eonoentraUon ot ~ •• pr..,ared. 1ft bUtrer pH 6.0 •• u .ubml".4 to 'he .... prooedur., ..... the original ens,..· aub,tl'at. preparation. It".. ..a'l.1J1J.e4 that at •• ,. h7dro1711. the aame am.ount ot active -cmmJl'l.8 would be pre.erst 1n the hJtb'Gl,..ate a. vas to'tllld to be pre •• nt 1n the eontrol prep81"ation. 40 R1dtto11jl. ot the .utl."'.'. 1Ia. tou.n4 '0 be o..,l.,t. at the end otl.O hova (.'" 3), at wbleh tllle 'he h74ro17- ... t. wu dtri.ded In~ two aUquo'b..Var1o"8rr •• t1on ot the.. allquo'. ".l'" t •• tMw1 th ..s. table •• ntrol. tor lqa1uronld. .. e 1Db1bt'bol'7 aotJ:,lt,.. !he. re.ult. of th ••• , •• t, aPe .hown in Table VI • .... )"JIe.Iab1blt.1'7.Le •• l. of VU'ioua ' ••• 'lou ot the 1174ro1.,1;10 PrOtt.",. of Bta1voa!.c Aotd &i.1I.: liiiB: . :: . ::: .: . ::::: Xataot ~rol.,.at. St.p Sb. (P1gVe6) Be.' Ina. O'lV •••. d a~l1 •• t. St.p Sa, (Pl~. 6) Dial:, •• t. of 'he Intact B74ro17.ate Step 4a,(P1gure 6) OOD.an'rated D1ttQ." ot the B7dro1r.a'. Step 4b, (J'1gtlPe 6) t .• I : : 1,1 The 1nh1'blto1'7 yalue' noted. aho.e are ellpr •••• d •• 1iM ptU"centage ef the theoJ'ei1oal17 ~ol"abl. nb'ilrate r .. 1I41n1ns tllSh,.dz-ol,. •• a. 1n the p.G ..... ot • glYen .. <nan' ot .ns,.... !he.. .&1U6, ... ealeut.t.d.. toUow •• X K ': ms. of .ult"tra"e« bJdrol,..... 1n the· oontrol. X k:: mg.. of nb ..... t. h.'fd,l'ol"'.4 in the t •• t. (I) ( 2) (3) Sat. soln. of hyaluronic acid in pH 6.0 buffer ---l-(a} ) check % transmittanoy I Overnight dialysis va. 10 vola. pH 6,,0 buf:fer,--2-(a} ) compare transmittancy temp. I4 uC with that obtained in step 1. Add enzyms (10 T.R.U. place in 37 bath. Check transmittancy at intervals until hydrolysis is complete lL2 hydrolysate • 1/2 hydrolysate • . i Dialyze hydrolysate Divide hydrolysate va. pH 6.0 buffer (5) into 2 aliquots 20 bra. Temp 4°C I I I Cheek dialysate Cone. diffusate to Heat to 60°C - 15 Cheek for enzyme for enzyme (4b) vol. of dialysated (5a) Min. and check for (5b) 1nb1bltory inhibitory check for enzyme enzyme inh1bitory activity activity inhibitory activity activity Figure 6. Flow sheet. This figure illustrates the steps in the procedure used to determine the errect of break-down produots upon enzyme activity substrate. ,-", ... - ECCLES HEALTH SCIENCES LIBRARY t:; T1me, tem;pera'bUre, .ub.'"..a". ooncent ... tloa. IUI4 eu,.. concen. tratlon &1'. ne •••• arll.1 the ... lnbotb the coat.al and te.t. It 1e obrtoua tb4t trI1 th 1he oone8atl-.t10. of hfdrolJi;lc pl'od\1Ova obta1J.'utd lr.t. the pl'eoeedins eaper1a.ent the lDh1bl '1 on oth1aluron1da.. b7 'the •• pr04uota in mln1malJ theretore, tme ril. in .8l'Ula qa1mtoD14... lmd.'bltGl'J 1 ••• 1. • .... t be attributed io the pp •• en" of I\\ch b,.-pl"oduot. 111 the blood ot tnt •• te4 aat.a1a. 43 V. EXPER.lMB1rfAL BBSU'L~S II, (Tal ItlUnCS OJ' '.fR'I llBIBlTIOJ' B1'A.1i'URO.IDAlE BY IOIJW, KOIUY SIBUK) ~ order of tihe re .. o .. ton Val toU'DAl to be 1D.tl1le1lOed by the nature ot 'hel1lb •. trate pepeatlon .. ed. The r ... ott,oa .a. ob •• "... t. bo ot a r ••• tlenaloJ1der with the be.t lulJstrate prep .... '10 •• "a11able 1a thl8 laDorator7. Ihct data, hewever" :tit, •• 010 •• 1., the ooD41'lona 1apo8ed. b7 ,he tir.t ord.~ .qua'loa in both the .81,.. .... b.1;I" ...... na,.....tlb.bat. plua 1Dh1'bltJor· .,..teq, 'kha1J" ... tel" juatltiabl. to 0810111&". 1Dh1bltlen on the bUt. ot the tile" value. o'bta1ned trOll the a'boye equ.tion (Plf&\\Pe 1, and ...eable vn) .. It pro •• d 1;0 be , ••• 1 .. 1e, with. 1 ••• hlSb17 refined nb.tlla'e preparatloDII, 'b. pUlh the reaotS.a lnt. tdle realm ot the .... Ord.er at detined below. Th1 •• al be due ttl the tact that it i. po..,lble, 1d.1Ih thi. t". of aub.tra'. pr.,aratlon, to get a h1e;h eonceatra:t;lon ot lubatrate without the fl0 •• ul.tlon whioh ••• '\Ira in th ••• .... .Gacentr.tien 1'~' ... n uatns more h1ghl,. J'.~1n.d au'bawate pxae,uatlOlUJ. :::..1a=' .======JJiiC===. ....'·..'2.! :..Jr,·-.' ..-,. ..·1 OiJIIIIII!01Ii.::"!':IiiI;·a;1,."1 ;lnllll:.=-. ',· · :~:: laB".. -Bnlrme B ••••• if'"!I"IIIli I I S lIlia. Sat .. to .1IIlA. 10 ldXl. 1S aJa. lS lda.. 20 lila. 20 Ida. r .k.. iO.1480 'O.lQ20 O.ot48 0.0120 O.G80S 0.0610 0.0682 O.tJSSS ,-" t 'J, ,- - ft" .... !he ett.ot ot the e • .,m"lfd1 ••• tl1lb .... ,. (1l7alu.OD1c e.eid) upon" ".1.001", or the, h1~Ul'Ga14 ... cat.alp.a re .. tlell 18 .llloWata' Pl~. 8. .... 'bu1. for the .. 'hulatloal. u. ........ n1J of ~ thi. ,,.,..t. · ••••• a. tu., t • ...u1.te4b7 R1o ... U. aa4 Heat •• (14), ~ .. r .... 'iaatioa ot ~ • ... "... .... 17 .... oomplu 41',le,.i·aU .. I.Ut&11' (.~) &ad. the Xlu!.latll •• looltr 'fl, 1. 1' ..... 17 aoeoapUehed 1fJ ~. 1 ••• 0_"1'." .,-., Of U ....... J* aal ItIrlc (72). 'P-:,: u. of tbS. ... tho4 ro'r 'be .4I1e1l1a,lt. ot 1.1 aJ1tt, V t •• .. _,.,leal h7a1woaid". 0 .. ,.1,..4 P • ..,Uoa la .... 18 ft. ..... 9. !.b.. 11opeot tht Un. 1. PI..,. <'I 1. E./f Ia4 ~ oP41aate In.. i ... •• " 1. 1/V. hoe ...... ,. ltI imd'l 'aM 1',.84117 obtataabl. SfW •• al ..... teftd.aatloBII .e.. Faa OIl dlf.terant 11781 .... D14a.eh7a1.o •••• id .,....... ... "wlt.w1th thetr 0.6 0.5 .p (.) "'- 0 (.) ~ H0 0.4 0.3 5 • ./ . " "'" 10 .... ..... / . enzyme and substrate enzyme, substrate and serum 15 TIME (min.) "",. "'" . 20 Figure 7. The enzymatio hydrolysis of hyaluronio aoid plotted as a first order reaotion. ~he lines were drawn by the method of least squares. The slope is equal to 2.)03/k in each case. N 0 r-I M ~ ,• , ,. ,"'- .. 2.0 ~ ,I 1.5 1.0 "" ~ 1.5 • -• 1.25 , 1.00 , ,. • I / I I l , I I I ,I J 0.75 - Log (S) x 10 1 . ,, • 0.50 0.25 Figure 8. Michaelis - :.enten log substrate - velocity curve for typical hyaluronidase catalyzed reaction. •• ...,anJing .'aD4aPd4ett •• toatU'e Iilowr1 U '.bl. VIII. ,.. lU'P " ... leo.. 4 ... _,,* .. '.4 in 'able YIll lD41..... iduL' tba .,., .. l,llat -••• 14117 wel1lUite4 top •• 11 ,"01 •• • iNdi... aow .. el', th. yll... ot.'a1ae4 hOll 'bh...experlUat • ... Id ..... '. to 1a41,e .. _ •. ·tbft· _a,pd.1n14e of __ phJ'Il.a1o ... • ,.,. 13\"011".4 "11 .,. hl&):U •• at ....... tJal,.ed. .•••• tt..n. tiil I :::' :::::.:. :Ii:::::::::::::: :::1: :1:::::: ::: I :: 1 2 3 4 5 '.lOs 10-2 3.04 s 10~2 2.21 •. 10-2 '.Sl a 40-'2 s·. to .. 10-2 6 1.42 .. 10.2 7 5.30 a lO·'2 " 'If. = ,.81"x'.2 'l*ul'I:.~"x ts=! Y a; 4.12 :t. 2.29 >.00 4.08 1.20 6.00 ' •• t~O 1.S6 1.81 I .•• *KI • th. tli .... laSlon conI'at. of the ... ,._ 1I\\b.'.atee..,18,J All t •• _. .... rur.r. 81 the 1n _U.... ott tplpl1ca"te. It vaa t.UD4n ....... 7 '-0 ada' Un -he l"eaotslona ., low t..,eJtat\1.Jtt) (40 'to 6Q~).. At bl~ ~a'ti'ar ••• heB •• neo .... u-117 lowe. ens,.. oODCen ... ,S,01UI, the naetlon p.oo •• 4.4 1a • 7 b 5 4 :3 2 1 • • o 1 1/(3) •• 2.22 x 10-2 1.)6 7 10 Figure 9. Calculation of max. velocity (V) and enzyme - substrate dissociation constant {Ks} by the method of Lineweaver and Burk. The line was dravm by the "least square" method. !ltmD.er anal.goul '0 .... ro'Grd.r J'eaotlOn. It val on17 when tM r.actlon' •• ~oel.tooU1d'bjr.lt:rlo'i.4 (lower t..,el'attrr.:.)'·th&ttliielSi,..' eOhCirtt.P .. tlon· '801114 'be raised. to the 1 ••• 1 Where' tbe""eact1.olll •• 1041t1 would rea· pond to oh.aas.' lJl. the etlba'irateoGaceau-aU ••• In 1881, Aln"hea1u ,o1nted out that ., reuona'ble e4Wli1on to'll the .ar1a'10n of rat. oou1umt' (k) w1th temperature mlgb' be !be quanti',. Ea 1. 1moWn .a 1;he .... SJ of .0':1 ... 'loa Dt the r •• o.10n. If Ba .an be ... .-..4 to be ' ... !'at~. 1n4epeD4ent the abo.. eq,uatlon eta 'be tnt.sPated to ,he tON Whepe A 1, ~ ,..,t_t ot lnitepatlon. Ddl I. the 01&'81081 Arrherdua .quatl' ••• It 1. e'brtou fIt_ th1.tha' • plo' ot ~. logarithm. ot the rate eona, .. t (k) asdaet the •• 01p.O.81 ot the abaolute ~.rattu-. aboU14 be .."81gb' Un. w1 ish • 8lope equal ,. so Pl'OD1 'hi. data the actlvatlo11 eileZ"Q' tor the react10h 1. readl17 oalculated. Fl~. 10 111u.atrate. the Al*rhen1u d1-sr_ tor the bJ'al'ttron1da.. ea'al,-.M .71.... !'he .1,op. or 'heIVd.ght 11ae 1s 3,Ozl03 aad trOlfl 1;b1s the .nerQ ot •• tt ... Uon (I.) 1, ealcul .. i .. lobe 1).7 Xl!ioalo~lel. I4eall,. in t ... ra'liUr..t11d1.. o~ th1. tJP. 1ihe only v81'labl. 111 • .,..,_ of ens,.., sube'r ••• , lDh1blto., and. time would be the t .. eZ'8:iur. 1t •• l1'. It v •• rea .... d* how.v~, that 1t the 1d.me lm.te.rval va. kept oo .. 'aat, 7egard. 1... ot the " .. lotll 'euaperat'u'.. .i; which tbe , •• ta were P1ll1, the end ot •• ch t •• t would t1n4 the reaotion In a d1tt.~8nt "ag.of 00191.1l1on. An enapdo "&01;1"11 lU.7 be a1nl41ed. at d1tt.-en,.tag.. bl veJ'1ng ~ t11l. lnuenal of reaction. Table IX 111410&'.' the variatlon in peroe.t 1Bh1bl'lon ;t01md at vu-101ll ,tage. of 8U1lJ.' .. ate ~ol,..l. ln .. t1P1oal h:Jal:u.romd.... catal,...d •••• '10a. ..... laPge .'aa4ard "ed.'ion ob.ene4 tdtbla ,he 5 db •• gJ'eup 1. 'to be ezpeo'e4. In tmla abort b:anal 1Jhe •• error. involYed in t;he 1;1.Sag of ,he te •• are ma8D1t1ect. the ~ overall l'budard d .... 1 .. tloa(5.06':) of the llib.1biv01-7 value. to\\Dd a' Vt~18U8.'age.ot 'be J'8.8tl.n 1. JIIath81' lears •• POI' th1. " .... on the r.aotloD. intend., at the ",arlo ... teap,epatur •• , were choa'en •• .a to allow the bJ'drolTI1. of the aub.trate to p~oo •• d to the ... degr.. ot o~1.t1oa in 8ve1'7 caae. C""\ 0 r-i ~ ~ M 0 H :~ 2.0 1.5 Slope Ea 1.0 ••• ).0 x 103 13,700 cal •• . ~ ••• ••• 3.1 Figure 10. Arrhenius diagram for hyaluronidase - hyaluronic acid system .. 52 the ,..,,1.b1 •• .in 1he ••• tud1e. vere, ~J'.t01"., teaperatture and '11M. !'.he l' •• ulu of 1Jhet..,.,..ature exper1manta aPe .Ihovn in Figul'e 11 Gd 12. 1 2 ~ i 7 6 9 10 11 12 tt 1S 16 PerCen. IDIb't"bl.t.;1 eo.r o.tlh .Jei 'tHal7. 4..J.'oa 1t7dl. 1.' ...iJ V. .1 0\Uf '. u 10 mln. 10 lI1n. 10 m1n. 10 min. 1$ min. IS ld.n. 1$ ala. IS Ida. 20 min. 20 Itdn. 20 lila. 20 111ft. I, 1$ 22 8 16 21 n 26 26 23 23 26 26 19 19 ~ -.an " lDb1'hl'lon ., ao.> _ O .... J?aU Std. Dey. I % 5.06 _ E. The Deter.minatlon or B1al~.D14a •• lrlh1b1tor 1n Konke¥SeFtlll A l1near l"elatloll.lh1p bIItw •• a b¥a1ta-on1d ... 1Dbtblted u IIl1d the ~t ot 1ah1b1tor vo1l1d ••• to 1nd1oat. that ,he bloOd pl ..... 1nhiblt:or tor.rua sUlohlO1let.rlo, non-dl •• oclable 15 "" o J / • I I I I I I I / •" '" ~ • c mean values , I 10 I 20 Temp °c I )0 " .-. 40 Figure 11. Effect of temperature upon inhibition. Per cent inhibition values calculated by "k" ratios. ./ ! / 20 1; 10 • o 10 20 Tempo OO )0 40 Figure 120 Same data as presented in Figure 11 oaloulated by the "least square" method. eompound1d.th h7t1Vto1tl.daIJe. 'U 'bh,1. piJe •• 4 '0 be 'ihe oa •• then a •• 'hod .lmlltt.P 1hi) thatct Xunl1f.(7) I might; be adapted to the detel'tl1natlon of h7tluremu •.•. 1ab1bl1;or ill anlaal 88l"" In 'order ,. dete1't4lne it ilhl.· 11r.lear pelatloD*' ship ex1e'e4, "arlo,. •• .P'ta dlluatloDl .... ma4. w1th pI! 6.0 butter 'bo obtllia vu-10'll$ 1nh1b1'boJ- oOllOenfn'·.,t.t,.one. ~e degr.. or h7al.\lrordd... l1lh1bl tJ..on Val .xp»...... III pel' cenu lnb.ibl'bloa. l'hS8 yalue ean be d.'.~d.ltbel' .tr .. tJhe a_gr •• Of IUb,t •• '. h,dRolf'18 (ngt\1"e 13) OJ' by mean. or the '1£tt .alue. oh\~ hOll 1Jhe tlra' order. pelatlo%1lh1p, th.e ._.en' 'beWeen the· y~ue. obt.~ ... b., the two utJhoda i.fUrl, 3004 .a .ho1m.1n Pigure 14~ When ouplo'. p •••• Xltag. 1ab1'bl ilon....... ct •• Hulag 1nh1b1tor oon ••• i,.at1oua. the r·tlatloub1p betw •• n the •• '1'&1\1.. apPl"oachea Ii Una.. ttm.et1on (Plgup. lS). Vntortunatel.,. _, fJbe •• rum ..... n.'t •• ts.. .. nee •• ,U7 ~. ,.04110. a eomple'e1711near r'elatlou.h1p, ~ dep •• of 1nb1'b1tloa e_ no lange be lIfUl .... d 111 th ppeelalGD. p. the TJpe of lnb11rJ1'iion Involved 1,n'he .,81m-onida •• SeJ'llll Sy.tem Inblb1tora ot ealJ!d.81'8aot1ona a:H hequentlr tU:f'1ded into 'biro t." •• , oompeUtlve·&114 lloa-oOllpetll1.e. 00.p.' ttlv. lDb.1bl1J1onaa7 be 4 •• o.1~ at the 0..,.'lt1 •• b ..... n the 1nh1b1tor and -.w 1N.b.-..t. toP .,e.1tl0 groupe on the .n~""Ji thu8, tmede."e ••• 1n GlW)l1I1o ao1Jl1'lt,. 1. dependent upon the relat1"'8 o:ol'lOeaR'a'iioa"ot both .u.l>.u-ate • ~ ~ t:-3 Q Ji-l • r-i 0 I> I I I I • I 0.8 • I 0.6 .. 0.4 po. I I I • I I I I I I • I I I I 0.2 '" / o U.05 0.1 •" / 0.15 . 0.20 Co - Ct (runt. sub. hydrolysed) 0.25 0.)0 Figure 13. Relctionsilip between amt. enzyme added and amt. Bub. hydrolysed. Used to calcula.te percent i~hibitlon (%I). 80 'd .(.1.,) .,...; 60 ,0 .,...; ~ Q H Q) ~ 40 ~ Q r:q ~ 20 o I • .2 I I I I $7 I I / • / / '" fill""" .", .6 ."..---- - - . .8 . Cone. Serum' ' 1.0 Figure 14. Demonstration of '% inhibition values obtained using Figure 13 (dotted line) and "k" values (solid line) to deterlliine enzyme inhibition. 80 't1 ..,Cp..D, 60 .,,Q.., ~ H ! 40 N s:J r:cl ~ 20 • ---------. / • _ . ." ". ". • ;I' " / , / / / • I I ...• • I M • I J I , , , / , / I • / I ,,' "" , . 0 002 0.4 0.6 0.8 1.0 Serum Volume (oots) Figure 150 The effect ot serum dilution and ooncentration upon the inhibition of hyaluronidaseo ..... 1/5 dl1 0 of serum 1/10 dilo of serum 1/15 dilo ot serum Ul4 1Dh1b".r. In 11-" cOlflP •• ltltYelDb1blt:1QD. tbeeODlb1aatloa .t bhlbl1Jo7 aDd ."..,.. 4 •• laot In;hrt ... with the.l;p.elt10 group. ot the eDlpewldth. ld. .. the lul\a"a' ...... hen.e, 1nb1bltlon ditpenda aole11 upoa·.the eon •• nt».tlon ot 'the 1Dbi'bl1ior. !.'he aethe4 ... 4 WU ba .... ,.011 the ... :bharla'loal t.~ 1.t1011 ot Ltaev ..... e,. ....... k (12),tOJ& .... 'lt1 .. 1Dhl'b1- 'lO1'l. O ... tltl". iDhlblUon 1, lD41 •• '.4 b7 1m !nor .... in idle 810pe ot the· ••• 1pPo.~.r the •• loo1ty vel'''' "he Peolpoou or the 1\1\\."a'" (1/.1 1/< s) plotaeoompcde4 b7 no .1plfloaau ohare. ·in inter •• p'. 8uoh 18 the 0". in 1lhe h7a1vom.4 ......... 8,.t-. .... ,. be •• en 1. Pi .... 16. !'he .• tteot; ot the · .... 1l1t1 •• inMbl'.1' 1. to·lno,.. ... · tbet elope of "he 80 .... 1 line b)" the qwm.tl1J·7 "d,l' Ita: <l1., •• 1*.loAo ... ,_1; "I' (,IS) (1) • eono ... _ •• 108 or tah:1b1ter ttl • 41 •• 001.t101'1 OOl1ll,mt of (HI) hom. 'bbl. relatloaablp 1 t lapo •.• lbl. tocalelllate· M. In. thl •••• e, how.ver. the: .... of '.l'mater lDhlb11s0Jl .. _eluded an a •• vaile •• &.1118'101\ ott X). 7 6 N 5 0 M H 4 I> ........... M 3 2 1 enzyme,substrate and serum ------- enzyme and substrate only ",.". ",.". , , , ./ • ",.". • ",. /'/ ",. . " / / / ..- , .. / • Figure 16. The competitive ~nhlbltion ot hyaluronidase by normal monkey serao 10 61 n. DISavSSIOI fhe.tUdl •• pre.eulea he~. ".re,e.1PQallD11to olarlt7 two elol.17,,81IltM .,our.D ••• , th.'.! •• 18 the •• 1'UIl level of h'faluron14a •• lah11tlto. «i-Us the 00 ....... or pol1..,..U'1. U4 •• 00_1". the ,h1aloal natuPeot the 1ah1- bitton of qalurom.4a •• 1>7 aeftUl ....... 'lb.. faIt ~atti1. 'blood of a ~:r of • __ 11aa .-peot •• "Usa aboui 1me lahlbl 'ion ot .h7a1u:PODld.a.. hal beea lmowA to,.. ..... tltu. !hi. oeow.no. 1. due '0 the , ... _.nee .t a ·~lo1081oal· Ozt rum-.,eoltl. 1Dh1blfior •• eenvuted te the .p."itto 8I1tlboclJ pr8tl\lo.« 111 reaponae to thean1l1gea1o atWaulua otaOll. ., •• !tic lqaluroa14.... A 1"1.. 1n Vhe ,eJ'1D l ••• lot th1a non-.,. •• 1tl0 1nb.1bJ.tUt hal been ob •• ..,.ed. in IIlOnlIe,.1 inJe.'.d .. tal tbe 71n10t' poU.,.Uti. bf vqot \he 1ntrana.a1 aDd bltra ....... l •• 11i... .... aD1mal. wh1eh tailed to e:d1t'bl' ol1n1.a1 poUom,..Utl .... well al all the ad •• l. 1.3 •• '1\14 b' ..... or the suvo-ut •• tlenu rou'., 4em.ona •• t .. DO .1 •• In,lq.lo1Ggloal latd"b1t __ 18 •• 11. Animal. la3 •• te4Yla the 1 ...... JlI.bral a;ad taiiranalal rout •• r ... lrahew a yl .... ,,'b1b04,. t1'e .. 1. 1 ••• than wo mon'hI following hlJec'loA. On the other haad, tho.. an1a1l. injected via the aaa.,.o-1n, •• Uonal route rOll'ttnel1 eSb1bl' a tltes-ahorU,. p,.. •••• Uag 01' ooneCtld'tant 1d.'h the tll'" 'JDlPUOl1t. of pard,.l,_ Whe po.albl11t 7 of a ",clprecal relatloJUll\lp exl.'las between y1rul .. tlbo47 aad ... ".. 1ab1- 'bl'or (mentloned p,. the exp •• :J.mental r.8lI1'.) should. aot be ignored.. It ,w.. relaUoaahlp oO\1l.d. be a........ ,. be oorrect 62 1 t would appear that the pz. ••• ac.ot hmaOl'al n.ru. ant! 'bod,. 11&7 be related to 'bhe .tallUXte of' pb11101og1oal lnb1b1tor 1 ••• 18 '0 rl,.. !be pre •• no.of a ~QUP OOllllU)f). to 'both the vlne par"lo1e and t~ ph78101oS1cal 1Db1bltor DlG160Ule 1I1ght then be &peoulated UPQa. !he rla. in the •• 1'UIl lah1b1top 1 ••• 1* ot 1al.oiled !hi. 1'1 •• el'1her oOGved with orpreoee4edlhol't17 the tll'.t ligna of pau-U,.! •• !h1 •• lD/ eonJunetlon vl'ih 'bhe·ob •• nat.1oD tha'i DG 1nh1'bltol' 1'1,. oco~ed til an1maliJ not exblblt1n8 oUa1cal eJ'llP''''' woUld appear to1nd1oate that "he rlae in 1nh1bltu31' i.not a d.£ ...... ohanlam but, on ill. ollher hand •• oon.equeno. ot cellular damage. The pr ••• noe 01 both lqalvon1due IUld h7al:urome a014 in aom.e t1aaue. (1.$). 'U88fJ8'a theeGMi.'ltte.,. of the ground aubat8Jl.08 lila,. beooatJ.to11ed 'OJ' a deUe.'''ll' belanoed. h1a1'dPon1o aold-b:Talvoa14ue 17"'-' What '-01. 1the ph,..1.- 10gloal lDh1bltor pla,.. in 8\lOh .. 1,.11' .. 1. DOt UB4 ••• tood •• ' ,.., the 1'01. of h7a1voald... in ylral late.tiona 1. 'boU ..... d b,. aom.e to b. 41tt.rea' thaRthat 1n 'baotenal lnteo'ilona. The 'Ppead1218 et.t.ot of the h7al'UZ'en14... can OialF ·d.d the rlPWI in Tl.v of 1ta 1nltJaaee11ul.ar habitat where.. the .am. ,ett .. " •• ,.... to real •• moe' 'bacterla mor. readily acoe.albl. to the 11oa' doteBee meohaDlllU. D\l:ran .. lteJBala (19). hal atatedthat 'the oritlcal coae.ntratlono~ yll"'U1 pep unit are. 18 au infeot1olUl UD.1' ot y~. 1'h1. 6) oplD1cn i.ba •• ' upon ihe ob.e .. a1J1onth8.t t •• 'loular extract always enliaDCedaad never 1upp,1f •••• d the 1 •• loDt ot vaec1n1., Shope tibroma. and. "11'118 III. Belaslv.l,. 11ttle inform •• ion it ... y.t .vallable regarding th. ,ohada'PI' of the h7al~oD14 ... 1ah1t4'or of .... al1an •• ra.. Th..ulO1U1 pvlt'lcaM.oD p7ooedv.. 8.1'. of .-.11 value •• oau •• of ~ apparent tnatabl11t7 ot 'hi • • ub.tan... 1'he taot th .. , tbi. lah1b1'o2' 1. heatlabll. hu, In taoti beea used. '0 dl •• hgu1.hth1. &e'1v1t,. hODl the antibodie • .famed aga1D1t a •• c1.tlc -hy'aluroD14ue which are lllUOh mOl'e heat .'a..,11e. '.!b.8 pe •• lbl1t1 that the Inh1bl'ol'J' p,..per'1.. of aerum ve.. .d,ue. to the pre •• ace or the breakdownpro4uo, t. of hlaluroa1c aold w •• lnY •• t1ga<i.d.~ the l'eeulta obtained. from. 'the.. eq •• hMn'. 1ndioa". thi. 11 ext ... l,unlikely_ 1'h.e aotion of .. r\UIloDh7alvon1da.e PeIU1ne an 1n:trlgu1ng IN.t poor17 underltoodphenoD&eaon. Altsho1lgh trequ.n' peterenoe 18 .. 4. 1k) ao~ap •• ltle OI'ttph,._101og1cal lahlbt ... " aotbtDS 1,' _ at .,., ot the· 1& ftYoao'l •• ot thl ...... , ..... ' IDh:1b1tlon of h7.l:u.oa1d. ••• 'bJ' a ... al h1.Ul ... tWa ha. been uiJJtlbed ~ salt fo...,1d.otl between .. rum alb'alJ1n and h7aluronlo aold augg •• 'lq tha, 1Db1hltl0."88 prlaarl17 due to an .tree' upon the· aub.tr.te rather thfw 011 the ena,.. (11). Ha.e(3S), on the othel" heAd. eonclude. (on the butl ot t1m. and ' ... :ra ..... )' ,hat the 1ab1bltor,. lUbetuG. in the blood 18 an euyme. !'he Haul t. or the kinetic atud1., rep.,.."ed helle indio.t. ,hat neither of the 64 above theori.. 1. eOI'~.ol. The insym.atlc h1drol.,.18 ot h7a.luronic aeid va. found to be e •• ent1&ll,. ru., order in natve when a hlghl,. refined aubetrate preparation vaa utill.ed wt.der opthaal OGD41'1on. •• It vaa found, hew • .,..., ,. b. pOII.1ble fop the reaotion to proceed ... a zero order reae".». tn the p....... ot a large exce'lof 8ublt1"'ate. 1. 1. !lOt unUkel,. that th1. lithe cu. in vivo. !'he .tteot of t ....r a'tnlP. upon the reaotlon 1n the p ••• en.e an4 aba.nee ot 1nb1bltiorvaa obaer •• 4. !he data thWi obtained proved adequate to%' the eal.oula'i1on o~ the· heat of act1 v.:ilon, an 1apor'an.' ph181oaloonatant.. A ~.t1n.d t'ut-bldi.etl'l. method. vu "evi •• d 1fh1oh proved preci .. enough to mea.1U'& the 81Ib.'1'&t. euy.m.e41 •• oo1a'i1on .onat .. t b,. either the ola •• loal .e1ihod. ot MichaeUa and Xent$:Q, (14). or the men retlMd •• thtxlof Una •• ave,. and Bu:t-k (12). Prout th18 ... _ ilnua or ezp_.laea'IJ 1t va. 11'01.11»1. to •• tabl:1ah the 1nb.1b1tion ot hyalm-om.d .• a. by a-rum u bell1g competit1ve in na~ •• The preel •• nel. ot 'he l"ea\dt. obtalaa<l in th ••• Jdne'lo ,'tudle., 8.,60.11117 those 1n Wh10h ..... was u.ed, would .ee to lnd1oat. that the:lahlb1torJ'.".b.tan •• 18 1.,. "ca.plex 1n na'bUre 'bhen prGrttJIWJ17ihoUSht. On the 'ba.i. of tl1e •• ob •• rvationa l' 1. telt 'hat more loph1,,'1.oat.ed me\$hod. I11ght be 8uoo ••• £u117applled towed the elucidation of the natul'e or the plt,.8101og1oel lDh1"1 toP of tthe "1'1IIa. 65 VII. StJMM.ARY 1. The natUPe ot ·tM ~1 •• of non-ap •• 1t1e h7al:uXtonida •• in pollGm1el1tla 1nfeoted mQl'lkeya W&8 eatabllehed. 2. A.1'J.SJIala lntected with the ylrua of poUomvel1.18 by meane of the gae:b!'o-w1at •• t1onal r011te tailed 'be exh1blt a :1'18. 1n noa-epeo!t!o lDh1bltloa. tho •• an.1mela int •• ted b7 e1ther ilhe latrma,uoJ:l intra. ceI'13bJ!'al rou'" eXbl'blkd a .1 •• in .e~ IDh1bltol' levele .'t or ntfl.r' the one.' o~ peJ'a178:ta. 3. A poaelble ttelat1onlh1pbetwe."!l ~ an:tlb047 le'Vela and the 1 ••• 1 otph1.1Gloglllal lnb1blt$1" 18 1I'Ugg ...... d. 4. The kinetic. ot the ensJllde hlUztol7il. of b:7a1lU'oDic acId va. studied 1n some detail by mean, or au 1mpro •• dtu.;rtbld1m ••• lo me~d. 1'.be reaet10D ot'del'. enuyma-substrate d18.ooiat1on constant.. an4 bh. enertn or actlv.tlon were obtained by me, ... of thea. 811141 ••• s. The k1netics of the 1Dh1bltlol1 of hyrUuron1da •• b7 nom.al :menker sevum wa". studied. fne type ot 1nh1bi1;1on 1mrolved in thte P8aot1ull va. to'Ulld to be cODlpetit1ve 1n na.t1.J.re. 66 fIll. RIlPBRIIOBS a. s. 8. 9. "'" "'''_':;.,' " .. , • "_",;>4 »v ..... J'D*1.j,. 1928 Baal,_ttoa 4. l' u,t"".' d.ll. Yb1J.a Y ••• tra~ ... ",,,, l ...... ~d' ...... "'aiDe 0Jt8..... OeIIpt. Iea.tl. 100. .1.. !1. 6. Bottaan, D.C., .... n... '., '1'. 19)0 Iae m..e.e.Ja aaont, .. .· .o.t 1 .. ..~... lnt• •U o. . 'b, knl.1e .. ob..... A,.L. I: . ~ •• ·B.a ... , .... Mt441 .... ,4f.2. 1940 fhe .. bl.logte.,.. ... .,,·Ql t •• _Sold .. ~ taoto7. Ilecllea. I •• a. 11+64. . 8011 .... B." ..... 01 .... » •. 193S A,f, ..... 1ft JUUcnaat t1 .. .,.. wh1eh.. ~ ••••• ' the p ...... 'btu,,. o~ ihe· ~~~ . fl. P.,b. ...... ,.,~. ~~l. ~aeJlUll ••. 1' .• , 19,3.)8 ...... on a ... 'u..' .... , ... tactor •• U,s._ ta. ta ..... 1a ... 11. .1p.ttlo ... . tep b •• t; .. J.l1b ........... ,J",lbp __ • ' .... &161. Sprtm.'. ».-.1950 the .... _ PlJ ..... ta tat ...... .... ». Y ...... I.i., iI, 1 '. Qlud.a.E., . ... Dl.1dd.., B.I. 19)9 .• l1ltleolJtl. ...,.. III 'be"I •• n~a.'.. • ...... , .. 977. ebB.a1 i. -4.. ..' .B).1• .4..0 . .4 .. D,.u.. ..tl.d1. .. ., .1 1I .•1•. ••.•.1• 9"'a"1n4.,.. Q ••U ,. a ..'. :.:... A.7.a .. ·•l .- ... . .. He, •• , IC., IU14 'ala.~. I. w. 1936 Qa 31;reepl'o'kiul p.l,. .... lUIJt14.. .f .y,..... ........ ... .t ua1d.1lol1 ool'd_ I • •• 1.m.a., 68,. 10. II.,..., It. ... 'ala."I... . 1,,60& the .. ~ .... r •• 111_ tlu1b. Aa. ,.Oph'hd •• l •• u.. 8S9. 11. 80"7. 3.L., a ....... a.a •• Ut4 •• ,.. •• It. 19_1 The relatlon.hlp betw"'Q,. •• 41Q8f.otor aDd. lqa1voJd.4U,6. •• _ ••••• 4 •• ll. 109. 12 01'0.,,17, I. 1'"- B7a1 .. ..s. .... p.oduo'ton '" !1eJIt)lJ1;l. avep, ••••• t ot __ orilla. I. 'ath. ·aact .... , •• 5!. 21. 13. OIudltt., . .l. c •• 1 ••• 1NlehU, a., .1111 ... ,0., ... ..... 1'. 0.'. 19.. .,.81 .... 1114 ••• p ..... " .. 07.'apbl.tto •• i. "1' I. _ ••• ,.Wk.. -. 124. 61 14- Dta.'bo., ft, J. 1931 .... a.t.l",. ., .... ot p ........ 1. Z. .. •• ReA., il, 811. lS. ~.,. z. a. 19114 _81m-onli... . lletlan' b7 pA81l1Ooo •• 1. I. 'ath. AD4. laet.. 213. 16. ",.r,K. J)u.bo •• a. ..... at.J'he. :LB. 19'7 'Ibe ~1,..1. O'E 'he polr.aeoha1'14* a.ida of .1-. ... a..l' of urablUe.:!. .orel, .and of ,"e,to ...... ot .the·aut.lnle •• JIle of ~ •• o... I. "01. Obes.. .l6!t 11. . , 11. Ev __ , E.G •. 1943 . '"teo'1v. propuU •• of a1pha aat1-'ozln ud ... 'tbJal .. oD14 ••• · .. , ... Uta 1. olo"l'ldlua ... l.h1i '''' A _'1......,. Patt&. aa4 B •••• ,11,4.21. 18.. lJtl:r __ B.~.' r. 19), the, u'Mat of 100111 dUpod'l •• o..f.. lat• •' l.. ~ ........1 .1. .••...•..I ...t .. u ..•• at.t. ... to IDle.UeD. I., ...,... ..... f 611. 19_ Dt.D-aa-IeJU1"'. 1911.2 11 ..... '."111.11111'7 and iJhe ep,. ... l .. t •• ' ... la tate.1i1oa. "ooa'pl_tlo11 to tm.e ho.-'p81'alS.t •• "obl...aaet.Rev •• i.. 191. lIoC. •• , I. 'I.. U4Dl1r..-Ie7Aa1.. r. 1953. Iaa.'t'Ya'iloa of .&ectal. .1.~ a ditru.al1)le. Often1; t .. om }qd.rol:p.a. b.7&1.- "aold. ael •. , ., '). uta, II. s., aa4 O,~, B.K. 19S0 Blooheml.U7 of t1uovomo 8.14. • .... mla PI' •••• I.!'. . DuP..-a.1M1"".u4 0.11.1'34 0. the .al ...... • t :~:-:.;~::=~.ii1~:-~!!1!.iaIJ7~ AlJ¥OH, P. L 1931 OhtId.oal na .... of \he •• lD81- 'P .... 1as tao'or trea ••• all.. '-•• 101e. hoe. Soo. ~. B101 ..... , 471. Lvie. H. B.19S0 .ec&l!Wl&", art.atlag·""." 1D. tuberou1o.1e. ADa. K. 1'. /Load.. Sol •• 5..&t 1041 • ... tll.,..i.tt.a, 1.!-9)8 8 .. 1e., fl. tI1ttu.~al t ...... uti •• pr6paP.'loaa from alJfll8l1u ' •• '1el. UI4 the&- bl.1o,loal .. a • .,.. B1oebal. I., lit lat6 • •• ~.a.B. 195) .~&O,..t," •• ol ,-,.tollla. h7a1uron14a... .100 .... Bloplrr. Aot,a:. 11- 524.- 21. llaAn, L •. 1'41 trb~4" ,l'ItU.801J'1,aohe ~ deft ...... h.4 ..... oobela •• , ..... , 5, 83. 68 11.7.1', It.» and Chatt •• , B., Bobb,.. G.K., and :D ... oa. H. H. 1941 B7alupon14ale of 'bac'erial and uSmal " orlg1D. I. _.1'. Xed., ll. )09. 29. lIe,.r, 1'..1946 ~'o!4 artbrltt.. Am. J ••• d., 1. 615. 30. Me,..!', It. 1948 111M,. '11 ..... 804118 h,.81: •• oa.' •. I. Biol. Cham., 993. 31. He,._., K., &Il1'h*. B. H•• &adD. .a on, X. G. 19)8 )Jatun of the. UO~lJ .• accJwo14" ot a)'M9'1al tlu14. Sel ... , Ai, 129. 32. He,..., 1'.., aM< Palaer. J. V.. 19)4 !he po171.00 .. 14. of the rltz.eoWi Itllmor.l. Biol. Ghem., l!l.Zi 629. 33. Kad1navettia. J •• ana. Qrd.bel1, T. B. 1940 the aotlon of t •• 1iloul,ar .~tr.ct. em 1he 'riaooei tl or v1treoUl humor pr~.fUtat1on.. Much... J.. lA, 62S. )4. Kealeen, D. , aad Kale,tl.V. 1941 fb.e h7a1._n14a'. ae1J1v1il'J of ,., __ 18111 .... ~.ot',ba ••• Jtlal culture filtrate. aDd other SS •• " that lne" •••• 'b,le.ue parae.hlli '1. B1.oo~ I., li, lS9. 3$. Baa., B. 1946 On She uehUd._ of layaaloa. 1. Antlva.ln I, and. ena,.. 1n plaam.a. J. 8101. Ohea., 121, 6:;.89-101,. 36. K"Ole .. , 1).19"7 . Method. of .a..,.. or hralurOD1da •• , fUl4 their oow.latloa with ald.n dJ.ttuelng •• tid',._ M.ohell. I., Jl, 169_ 31. Pu1ton.,' .3. It.,. Ha.r. .... S., Utd. l'itfblD101t,W. D• . 1'48 H,.aluronida •• iDhlblt;or. b1 bod,. .flu ... la 11.0:rma1 an4 di..... .t.'.a. Proe. 800. _... 8101. aDd Ked., 21. 2S8. 38. h •• , E. It.. aa4 Seaa'one, G. V. 1944 !he pol. ot the llU.Oold pol,..uohaJt14. 1n the viPU1en"e of group A hem.ol,-tlo abepklooool. J. -'.1'. Ked.,];L. 319. Leonard. S. L., , .. bp", I.., .... XUSFU, ll. 1946 A t1.IPbld11leWl0 .... 4 tor <i.temtllS_8 h7alvosd.d .... in serum. an4 tlaaue 8X~aetl. End&oJ-tnology. li. 261. DoJ'blan, A., .and Oilt. H.L.1948 The idnetl0' ot ehe ens,.. .. t!. hr4rol.,.la ot h7a1wen1o aold. J. 1101. Ohem •• !li. 361. 69 folkldort. S., and. Ca •• 141, I. W •• HeOread7, K •• aad KoCulleugh. o. 1'$0 In n.firo itft41 •• with h7al:u.ron1daaua. A:aa. 1'. :r • .load. B01 •• 1&. 1024. Bachtold. J. G., an4. Gebha1't1.t, L. P.· 19.$2 ~ determination ofh,.a.lvon1cl ..... 1J1Yltrude1'1.ed troa , •• react10n kin.illea. J. B101. Oh8l., A!lb 63S. HeOlean, D. 1943 3.......... ot 4ittulng taote1'a. 2. M.thod, ot a.,.,. of hta1u.:rcnld... and the'1I' eon .. 1.11101\ Vltm81dJtdl.tt..,1AC aotlYJ.t.,. Btoeh.em.. I •• ll .. 169. . .",.1', 1'.., .andOhatt... 1'40 .,.&1 ... 810 acid in the plewal tlu14 ..... 1*' ... with a a.u.saaat tumor In'l'olv1ng ·thAt ,1.". and. pe.!,..... J. Biol. Oha., m. 8). 45. H\aphre7, J. X..1943 . Atltlser4e propertt •• of h7aluron1o •• 1d,Bleehea. I., Jl. 460. Bauapbre7, J. 8.1946 !he 4Otloa ot h7al:a~oD1da •• prepU'.tloaa trom vuloUi .our... upon ... BUb, tUG... oUhe1" than h7811D'oDtoao1d. Bieehea.l. , MS.. 442. Du.Pan-Re7Jl8.l •• P. 1932 !btl.trect.14 antl-t •• t1oldu •• VlUIonche enh ...... l1:b value of 1Ie8tl01_ extract.. J. _ ••• Me4., 1it 10l. Baa., E. 1946 On. the .eoh&D1 .. of inva.1on. II "ol~ ... 1ft I. and ... ,.. 1a pathogeDic. bacteria and 1n •• D.ODUI. I. Biol. Oh_., l!J.., 89. Hu., B. 1946 On the •• chant_ af In ... ln. III Ant1valu II, • ens,-.ln pla .... , 1.]$101. Oil... .w,. 101.' . .. DortDum., A. 19S0 !f.he actloDof •• Ft1R onbJaluron14a ••• .Arln. I. 1'."ead •. lo1.,.52. 1096. , $1. Dol-tam, A., otit, K. l4. ,and Vhlta.)". R. 1948 The h7al'tJ%toD1d.... lnb1b1 to,. of h ..... blood. J. 11101. Ohem.., 174. 621. S2. Rechte., 0., 8D4 8ouU7. B. I. 1'41 5 ... 1 •• on spreading faetcn-.l. II·. 'J!he ettee'b or IJePUlrl upon h alUl'on14a •• aprea41Dg aotlv11.i1. I. Exp ••• He4 •• 19. ss. sa. 60. 61. 63. 70 Had.1ndIl,a.n..,. . Z. 195) 2he .,.lon ot 'YenOlA lUl4opp• ••o l7fJle 01\ the noa-ffP:.oltle 1nb.1laltor of hJal .. onld.... I. Gea. Ph1alo1., l6. 3"1. HarOllll. s. ara4 Pultoa,l. It.. 1'48 aela'tlon of complement to theJnl31abl1e h,.a11..1ronldale 1nh1bJ.tor ot •• rum. Ped. JlJ'OO., 1. ,401- P1ala. S. .)1 ...... ,D. B., an4 Roth, It. 19$2 2h.e Interaction of h1alvomda •• w11Jh tltroabopluUe c..,.neDt. of blood ooagula.lon. Iol..lAS.. 600. Pltl.a, S ..... 1' .... , D.it.'jal'l4 "'h, It. 1952 c ... lumptlOB of 'hPOlIboplastln in '01004 coagulation proo.... Ped. bOo., 11-2'10. Boith, It. L.. 1954 labilJ1tlon ot h7a1uroD1d ... a.1Ily1_,. b7 raoto~' ••• ocl.t.4 wlth blood ol •• ting. hoe Soo. kp_l'. Mol. M64. t 8~t 20942. Ptllvto..n , J. ~ •• Her.WI. s.. and IobS..,.., W. D. 19.$0 ot the .tr.,tooo •• al de~fJap814atlon teat .e a me~e ot tiutl"llOlab118 h,.a1uroa1da •• 1Dh1bltor 111 .e...... Ann. I. Y. .lead. 8.1-. .21. 1133. GllG."k.,'e mD. . anI4.G oIlDlabn1,l JlPt1. oa1 9o4f8 h yHaltllUe'oolD7'i1d0. . .e.n bs.y, ..a.e rum. 1ll po11omye11tl.. J. lAt. Die •• n. 200. GraIl'e.,' .H... .L . IaDI.d GbllhO1k1t) iDtl. on1 o94t8 h J'H81u'Uoo.ll'o7n'l1e4 ....n ba7., .. •• rum in addn' 41.,ea.... J. lnv •• t. nePlU.t •• l1. 2.39 ' W1~1.r, R. I •• DeTor.A.W., X.hi. J. W. aD4 a.,th. I. M. '1948 Stud1., on ,the mucoprotein. ot huun plaama. I. »e'.r'JI1D.at1on and 1.el •• 1oa. J. 011n. InT •• t.. ~, ~09. Seibert, P. B., ,seth'-', K. V. , A'~, A. I • • end Oatlpbell. II. W. 1941"arte.tlona 14 p.ret.ta and polysaconaria. content of •• ~a 1n" tJhe ,. obPOft1e , 41........ tulNpeulo,1a ,.·arcold •• 1_. andoero1noaa. J.OUn. Inv •• t •• !L 90. G11ck. 0 •• Good, R. B., Kell.,.. V. C .. ,W1mIle •• R.I., and Mehl. J. W. 1949 Lack of lden.tlt)' ot ' h1a1uron1da •• 1D.bi})ltor and c.r~ nut.op~ot.1A. 1a blood •• ru:m.Proc. Soo. _er. Bioi. Med •• ,ll. 412. 11 65. 'folk.dor!'. e., HeOpead,., M. B. , McOullough, D. R •• and Schwenk, E., 1949 !he ~bl4metz-10 a ••• 1 ot hyaltutonldaae. J .Lab.Olla. Ked... JJl., 14. 66. Dorfman, , Ott, H. L. 1948 A tl.'lrbldlletria method for- the as • .,. ot 1'l7eJ..11l'oalda... J. 8101. Ohem ... 112, 367. 67. Croxton, F. 1953 Elementary statistics with app11eatlone 1D. aedlclne.'"entlce Ba:U.. I'ew York, p. 226 68. Ho~gan.. I. N. 194.7 !h.. role or ant1'bod1 in expel'!m.ental po11o.7tJ11 til. Ill. D181U'1bUt1on o£antlbocl11n an.d out of tbe 0 ••• 5. in paral,.ed monke,.8. _.J lit alS-. ~. 396. 69. sabin. A. B ... f1nd. Cllta1q, P. K. 19)6 B'lmlo .. al antibod,. alld r •• 1.t81.lcu;~. of vaccinated and ooaval ••• ent moa.1te)"8 topoUOIl78l1tl.· vlrus. I. _or. Mod •• .2ltv.. 139. 70. Magnua,von, ll •••. and, H$ln1ok.J. L. 194.8 Antlbo<!:,. response ln monk.,... toll owing oral admin1atratlon of' po11om7t'J11 t1& virus. I. ~l... .22. S8). 71. AdDAr. L. 1946, from l'o.rtman, A. 19$0 .lotion ot lerulil on h7a.lupon1dase. Ann. li. Y. Aead. ot Scl.. ~ 1048. 72. L1neweaver. H.~ aaA. Burk. l). 1934 !.'he determination of el1&~ tU •• oeiat1on ooa.stan... J. As. ahem. Soo., .s.s., 6 S8. . 13. ltlm1ts, M. 194701',..'1111ne 801'bfUUl tirlPain 1nh1b1tor. II. Oeneral properUiea. 1. Gen. P117&101., 12. 291. 14. 15. Michae11 •• L., and Heater,M. L. 1913 Die ld.aeUk del' invert1nv1rkung. B1oohem. ZtecW., ~, 3.3. Heyer, K. 1947 The biological 81sn1t1eenee 01 h7811.U'on1e acid aa4 hJa1u:roDl4 .. e.!'h78101 • .Rev •• gz • .33$. |
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