A Thaarg of the Stnrcture and Process of Formation

of antu.?odiss*

  Dwlq this past far yaare I ham been makinir: an effort to mderstz.nd md

Interpret rerolo@al *enomsna in terns of raolecmlar rtn~0tu.m and molecular inter-

act ions.  The field of immunology is so axtensfva and the eqeriglanta2. obeomt~ona
are eo oomplax (and acoasionally contradictory) that no one has fonnd it ~oasfb3.o to

indwm a theory of the structure of antibadiee from the obsarrvatioml mtarinl.

-Aa an alternative method of attaak we may yonomd and attmy& to answ?r the

followin& qmst%ons:  What ijr the ariqlest structure which can be mqypstad, on tke

bassfar of the extensl-e infamatian now available about intrmolectiar `and. inter-
muleau~r forc02, for a nolmmLe with the! -pro:)erties observed for antibodies, and

1   so0, fo      zy of thi6 inforztion, L. Paull~, Bl%e Xatuxa of the
Chemlaal Don        1e Stmcttxre of Noleeules and Crystals,* Cornell University
Press, Ithaca, IJew York, Second Edltic.., @&.

what la the &plast reasonable p~ocssa of fornation of such a mlecula?

Pmcead& in this way, I hape dsaeIqed a detailed theory of the atmcture and

pmcass of formation of antlbodfee and the nature of serologic& reactions which

is nore deffnite and more widely a-+icable than kWW1i8r theorirs, and which is

con~tlblrs w5th oux present knowledm of the strnctura and pxuyerties of uf~@a,

moleculeer as well 8~ with most of the direct mnpirlcal inform&ion abotnt anti-

bodies.  This theory is detmribed and discusrsed below.


2

   when an antigen ia fn$xWd into an ani~~al f3ostle of its; aMlectxlar am
caytumd and held in the rs@~n of anttbody produotion2.  Ax antibody to th%e

Bordst ,

foraaa of qqmmiabl6 rrtrerugth betman the antibody ~~~lwul+ aRd tho anti&en

surfwe configuration oanp3tmQatsry to that of the antigen. The rnle of ptarrinuray
(the use of ths mfninm effort to achieve the xwmlt) -eat@ that them are only

two such reg%onog that Ire that the antibudy realeaulsrr SF* at the mott blnerlent.

The pzwpossit thbary la tsww% in this rwwmbls armqtlon.  It wMd, of uourae,
be posadbls to expand the theory in mwh a way aa to proride a rauchkirln for the

formation of antibow latblsauler .tith ralsaue h%@er than two2 but thir woxilh


antibodies of other olmaes 8~61 obvicms.

The affect of (811 antigen fn dstomining tha rtruatlrre, af MI antibody

that in the normal @o?mlia, ar amgmetsd by Breinl

amuse, howtsver, that this ir not 110, but that all


4

antibodies with consfderkt4u tq;ecfficity for an apparently ualimitsd nurdwr of
dfffemnt antigt2ne8, withcut the necessity of invoking 81~0 a variation in the

T It hws been pointed ox& b A. Rothtm md I. Landsteirmr, Suieace x, 65
(1939)+ th8t th e posaibilitr of dlffment; wsys of foldinp: the uunc~ palgrt;rqptf&s
abin to obtaia diffarsat antibodiew lo worth conaideri~.

1
           *ss3 % I tio 0 t   hii38 .--xl& us a.t30m tlz;nt Cha

~;lobuliri molemale cunsfsts of 8 rrlngle poQp3ptfde ain, contaLfnizg severs1

handma amirm-safrd realdues, and thst the ard#r of m&to-acirl x.6 i&ma is Budh

twt far ths ountar sf the oM.n one of the msersibl6 amf@m~tianr ilar xwh

more e&b%% t&-m w other+ w&rem the two end -rtrr of the chafn me of sash

a aaprtmrs that theme oxlet for thea nasty cuafiguratitmer with aearly the emte eaerm.
(TUB Faint irr d%rm.aeed fa dstafl 3s Section XV.) pour steps in ~IW poetuJ.atad

p344@4 ai ftb=tw 0f 8 ~4-2 ~i0hiiin ~i44d4 434 fnu8t3ui4a 4n th bit

da0 0f ~1~0 1.  At atage I the polypeptida ahz&n hm hem qmtheaited, the

amiao-ncid awiQuoa luwiw baea mm&alLed into the proyor ordez~, pembly tith

the aid af ;xQymytldasee and protein ttm&&aa, SacT the two ends af the ah&x,

h ma C, each WXW&XL~~ gezharp~~ tw kmzred r@AduBs, hme bow lfbsnt& with

tha -z&able extended cmnfi@uatlon,  (The horixont8l lin4 in 48431 dr4wi~

eqarater the rugion, h~low the line, in which the pGypeptlde -ia it not atizs

to ahaage its aonf~tioa from the region, above the line, where this ir possible.)

%ch of thaw &sin snds then coilrs q into the moat stable or one of the pgoat

artable af the accerrnible coniigamtinnr (straQ;lt, 11) tend is, tied into thirr

coafigxration by the fonarrtion af Q-drogen bmds ,znd other mak bondo between

parts of the ohain.  %`he central pati 1 of the ohain is then liberated (atap,e XXI>


aad aammes' it8 atable folded wnfigkmstion (rtage IV) to give the cosrpleted

There em rrlro indiaatsd 3.n lQu.re 1 rix stager in ths proasr# of form&ion
                 .

of lm aatiboag molo~r,  In rtage I there sre shown an antigen molemle held at

a platm of globulla pxwdtrcrtion and a @kabulin swlecmle with itr two end8 A aad 0

liberarted with the extreded w~igu.ration.  At rtap If each of the ends har

srmnned a stable ooiled wnfiguratian.  These stable aoafigur&ions AD and Cl are

nat, howawr, idoatiml with those A and C asemed in the absenas of the aatigm.

The ataar and mupl which fonts the surfam of the antimw wlll attrast certain
.oomplemntory part a of the globulfn chain (a ne~tivelpkarged group, far sxzusple,
sttnrating a petitlmly-oharged group) and repel other ye,rts; as a result of these

intemationcl the configwatianr At and C* of the chain endr which are stable in

the pretmm of the euatigen a2td uhioh are aaaordi@y aemmed in the preseaao of

the antigen will be euch that there is attmmtion between the ooiled globulin

chain enda and the antigen, duis to their ooinplementarit~ Sm dww.re. Y2m BOPI

figuration atsum by the ohain end nmq be any one of a large zmber, depending:

upon whiah pax% of the aurfaco of the anhigm happenr to exert itr ifluence on

ths chain sad tmd hew lwge a regiea of the starf%a happsnq to bs aorered bfr it.

  Vhen the aontml pm% B ef the globulin ChclSn is liberated from the plaae of
Its ttynthealr (atage IIf), one of two procosser mafr ocuur.  If the forcea of

attraation batwean tha antigen aud the portions A1 and Ct are extmmely atmong,

the7 till rmaia bonded to the aatigan for SD indefinite time, 9nd nothiw further

of interert will happan.  ff the foroer are somewhat maker, however, on4 will

ih time break &wky-=-dir6ooiQtte frrna the axtti8en (eta@ IV).  Then the portion B

of the chain wiL1 fold up to aahiew itr noma stable configuration (stage V),

asking a aompleted antibody swlwule.  fn t ims thir will di5sociato f mm the

aritigoa and float away (rtw vX>.  It is rorsrible thart an auxili.wy mechanic


6

  The middle part of the antib&y molemU thus gro8wwl umlb be WCS that

af a normal glo'ltnrlin mohmite, and tha tmr bnda w&id have aonff@ratiaar n~ora cw

lm3r camplQmQntary to part" of the BurfaCit of the antigen. 9h06tJ two aatirs Qmll

am affwtiro in tiffsrant Birthions, w that, after the euatibbdr is compXstely

Samad, oal~r ane of them at a time tmn &tnasp a partlaal~s antigen aobmula,.

  The mtig!m molecals, after fts itrssertfaa by the newly-foxwd antibody

mt3lecule. may tmm as the pattern for another, and. uontlm6 to rem6 until it6

tmrfacs ia sarsmd by rary etrongly held ant2bodtse ot portiona of arntlbodfar or

until tht~ conaentmtion of antibodies bsqmer 60 Cjred that emn with weak forear
aptmating; the antigen is carmbfnad tith antibodier most of the tim (as illurtmted
in l%um 11, or until the ant ie;sa ~~~leuul~ lr dscet zvyed or ermpes f mm the m&on

of &.olmZin fomation.

of the tturfacft of an antigen mlilbaale.  Great Paristy in this consplamntary

conflgnmtSon WI&% be expected to rmi%t from the acoidmtal appmxi;rrstfon to

one or another mzfrma m&on, an& further varisty fmn variaticrn in poaftion a2

the antit?;en salmuls mlativa, to ?&a point of lfberatlan of tha &hulJs chain

end md from acrrld6mtal eol;lfng and lhk%ng of the ah&% ~lrd before It QOBMHI m&w

the inilnaaO6 of the i%digUa.  %UQ of the mtibadzy m~]Leatxrau w&d be UXliYd6at.

one of the ehain end6 having, bwauw of its too gr6at &i&awe fmtt the antigen,

fold4 fnto a normal pJ&mXin eonfigtmstion.


  'Bhl3"4t~rb~iCSti0~6 aT@ WXifi&i v 6%J?tWiJtl~tti XV3Sdt.tlt.  It i6 W&3, bOWl3

that an i.ms a4arw~ to on6 suntigan wlI1, a6 a ti6, rmct with a mlatsb

hetsmlogma antlgm, ouad that after ektmustion with the latter thare ramfar

a frwtfon which till at113 react with the original antlfleza, S.4wdstafner iund
van dtbr sa&a61r" , neixg as mitfgenr amprotein car3yixg marfour ha>tmnr mmtaini3qg

10

K. Lan&stsiner and J. van der S&mm,

. a, 325 (1936).

the $&se sctim gmwp, have &WWB tbt tha 24ntirsrt.m fOP on6 antig6n contab%

varfouft fmctl6n6 diffe 7 l.`in& in tb sb%~@h of *hair atti~atibn fbr the hapten6.
;Sg tbe cpmtitativ8 study of prcbuipitin mactionr H~i~slberger and Km&&lrz

  M. Rsfdelbergw and F. E. &m&U, J. &QJ. Med. j&, 559 (1935); &, 467,
697 ?o??? o

mached tha uwtm co~cltaaion, and thawad in addition that event after `prolm#I

i-~p;sltion the antiaarw6 cstutied (anti-egg albumin) aontainecl lnuah luw-mds

antibody, inctqmble br Itself 41 fomtin$ a precipitate with the antigen, but tith

tht, pruprty of bdag card& Qua in the precripitata flamed vith a mom mactiw

f ractid2.

____,_.
73.  Th Biva     of                          ,-ha theury

8truatUral Christ it is O&~MF that thi6 pi&IX0 of the prtmfpitate imat be tm?XWt.


a

T~Q gmst ~eaificsit~ of aurhlbody+mtig6n, fnt6matimr mqulraa th2Lt di dsfinite

band be formed b&men an antibody m3bi?uls and an aatigm aolsc~le.  If anti-

badlea of mti@M6 v6re animblmt, this would lead to CObT$116%66 of one &nti@n

ntalecmle mad one or nare mtibody molebats,a (or of arm antibody molsdrs md ona

OT wre a&i@m mleculer), and m kmw f rem tPxperiaurm with protsina that them

aggregates would in general IpmmiB in mlution,  If both antibody and anti-

am mltivalenCt howemrr the cmplex til3, gzcav to = aggragats of indeftite

8i66, aich is the pr6c;ipitate.

This pXYOC666 i6 obwrved disaotly in tha %@.UtiRation of cell+. 0x1 th6

addition of su1 &&glutinin to a osll nzapm~ion the cell6 arta reen to clung together.

ft la obvious that thn aggJutiti& aplecul6s which am hoMing tha cells tomther
am biwLIent" -eat& bar two aotiw ends, with tonfigtm&ion complarwntary 40 that
23  Z%llowi~ H6idelber&w and Kendall, I u&e th6 terminology of chemical wlenrire
theory in dirc!nsrifk& the ageaific nmtua~ attrmtton of antigen WI& antibody.
The antibodpanttgen %alentse Imd# am not, of course, to b6 uwfu66d with
ordinary comalent &umioal bando: they am due inatslrd to the integmtad weak
forces distmwed in hs. Iv,

of a partion of the &m6 of the cell6; the ag#Jutinin mol66ular hold th6 oellr

topcethsr at their re&iom of a(lntabt, ~6 shtavn in Blgur6 2.   &s-j.

ft 66dm6 gmb~~ble that sell antilmifea ham thi@ 6truatum=-t&i+ they 6x6

bivalsnt, with their two aatiw ~&&WAS oppositely directed. H~bdslbar@s and

  Pmfeswr Eeid.el?mr@w baa fnibnted n&e that in their tp&ntitatioe treatsmt
of d&a ML the prscipitin reaation he and Dr. &uui.aIl have found no incanpati-
bllity vith this metricrtiont In their paper% they Bircutmed the fienmrral CXWJ
of mltiv&mos of antibo0y am well AS of mitfgeY%.

(ignoring the sxmgMm%l cam of the attackxmnt of two or nom anti@tns OT

h,qtana to the mame wd r&on of ;urr mtibody) hzss not pretiously been made.


9

aa in the cuprwua o;rida arranhFaraQat (copyer and oxygen &vms beI= rqlaaed by


10


11

have the valsnca Jj = 12, If a31 regions of the euLtigen aurfave were active m;d
if tha antibody mleemlee wem vphvrlva3.t t!m aseunption of elongated antibv&
mvleculaa would pm&t the mlence to be vowwhat larger.  The ValuB 12 of g
oorwsyond~s to the W&XB 6 fVF the ratlo ,R.  lor l8lpptr antigem ZaPger oaluer of

,B wotAld be axpmtedt ad for tm8llar one8   ller Y8luQs,  hen for ant Lgem dth

molscular weight as mall at 111300 the pradictad ~TB&RW mlns of 2 is 4 tfar
syheriaal mtiWUar) or larger.  In f<mt * zs sirtpla calLulntian btbsed on the

p%.cking af sy-4Ieree l5 1-8s to t&s nmxlt I even in Tebla I 16 . 1t1ssecm

          abwut spherfcal Antifym &lsml;ss

2fumbsr of antf-   lw&mm ratio of   Ninlmm malecul8r
bvdy sloleculer   antlgiwa raditu tv weight of antfgva
abvut cntigan   ant ibvc?$ radina   (zmtibo?q 160,CXXI)



   12           1. WO        160,ooo
    8         0.732        63,000
   6         .4f4         11,000
   4          .a          1,m

cigitats
6        6
4         10
3        44

  2       178

thc;t wur theory provides a simplr eq-&matfan of the fact that for antigens of
moleeuls w&&t sqwal to or leoe than that of the antibody the prcrdpftste cantalnr

conalderrably more antibofly thm aat Qen.  The mlaee efrsn 1~ Table 1 are not
to be considtsred PSI hrming rfgorouo qw.ntftatlw sl~if9atwma. %%e ~lculated
EX&JIWR mwlmuhr mtfa waltI be li~rger for elcmgatad @Albody mleatiler than

for +lwb8l `a.r&ibwdg mo1%uula5, and lmger for non-spheric& than fvr epherfoal


12

antigen saol%anl%%, and, mreover, ln may aepa the antlbodlss at&t ba

CoqlaPrsntak'y iA thu asin 0fd.y t5 UertaiA 6~ft400 x'tB&OnS of the antl@%n, th@
number of thaas dstamlnlng ths rsl%zaw of tie antigen. That thirs 1s 80 I.8

idicatsd by the        17
            obmwvatlaa  that after lone %mmnlzatlon af a rabbit with egg

l7  M. H%lbl'b%rgsr and p. & Ifandtkll, J. z&L Id%&. j& 697 (1935).

albunrin eel~138p wae obtained &lying a pre~lpltsta wfth 8. eonsl%ebrably large

mlectzlar ratio than that for earlier blsedlnga. 18

  The %laausrl~n of tha n&are af this phenomenon of &mg% in th% 8erw1 on
continued l~l~ation amst await the detailed tnsgLtrent of lntracellulax
mlt lbody4nti@!Jn lat errace isate.  The phsnemenrnn nay imolve the mm&l~ of the
nom effective emrfaae r&ane of the ia&mted antigen molecules by semm anti-
bodies prodwed by earlier &moulatfone, laarfng only the 1s~ tsffectfve raglone
arallable far taraplats actian.

Obsarrved mlueo of 2 for prwlpltates form& la the aqultience son% (with

aawunte of antlgaa solution an% eamm so choeen that neither oxoess antfbo%y

wei@ s betweea about &or>0 Qnd 7001W0 lie tmtwesn &boat 2.5 for the smaller

antiF;sna and 1% for the l.ar@m 5~~458 19 .  It is man t-hat the walnsa of: are
T9  It IS our sestr~crtian of the val%nue of the antikrdy$kthe Rlgdmsa v&m
two whleh 1%&s to oar axplanatfon of the antlbdly-antl~n ratla$, `Fha g%taral
observation of mkluer of g aem5lderabl~ greatar than 1 18 not aoatmktsd for lay
a fmmmrk th%ary in whfch anti'body and anti- rnelsaalsa am both sultlvabnt,
Ud488 8onw SW%i3ialFJr ~08tUbkf@ i6 inv&ad to &%&I the effeativa Vabxm~ of
antl~n cansldmably greatar than that af aatlbodg,

soaawhat fsas thaa th6 corr%8po~%ina; w~lamr from Table 1, which indi5ates that

n5t all of the %WfaC% r%e;ians of th% antigsns am effsotiY%. '!h% data gi~@n
la Table 2 ark thorre af Hsl%.elberger an% his craU.aboratore$ the ~1~68 reported
by other iavestfgstora are ritmllsrr in rm&tra&e.


Table 2

for PracipitatQ8 froa B&&bit kti8W@

  IA 8 precipitate ftmB8d fram a Ml~tlon C@At&itiA#-$ AA mm611 of antibdly Aot

an of the aastibo%y WA&JXl5*8 will be 8&ttWW?d.  bt the limft cf antibody exam6

the grecsipitclts till be a network af linear ag&regaterr 4th a rtrwsture mch a8
t&t rUpm8fHtted in ?&gim 4.  &we &a& aAtig6A Btd.BtiS (tith am aooarioti
axaagticm) f8 eurrrwnded w E antitmby iIl5lWU&38, OAly tw5 of which band it t5

nsi&borfn@ ant i@m IB516CtilQI.  'he prrddsd 8triIk($s forned in thie way ar8 tiod

tug&her by an ouuartonal cra~.t-link to form the preicri~itate. l%e antibody-

antifln muleaular Yatio $8 80%~~ to be eloa8 to ; - 1, aiich i8 OAO let&s than twicQ

th8 value y2 for the nrlena+-tatarst;sd preuigitate. ai prdicted relertisn

betuwn thssr ZVAtiO8

      &t ibOdy MCQo88 - ~qubalema XcmIb - l
$8 man from the %&t& in Table 2 to be verified ayprox~t81~ by expsrfmunt for

the ant%gem other than thyme;lobulia.

The bframpmay &awn by i+?qcro~l~bUlin is, fndwd., ta be expected for aa

sntl@m tith raalecular weight @mt:4r than that of the antibocty. The reqnizvmanta

of getmtryare such that an amaqymemt in which sachaxtt2gen irbondsd


14

Haicra for large antigen moleaules tha ~~Gecular rastio oikn exceed 6 fa the vale~5e-
saturated preoipitatsr only if two or more antibody aaolsctllsrr are shared between
the %WX@ pidr of a.attfym B?d@ti%%, Wh%M8 in th% ~tibo~--~xaO$e re&On the
entire swf%c~e of the large antigen ssq be crav%rs% by antibody moleonles.

varies between 1 m.td the tinismg value k/Z.  XC i e, obserwd that in general no
precipitate fame in the ragian af great antigen %m388s, and Heidelbsrpat aad hfr
csallztbaratosrr hsrve in faot aroigned mlues 1 an% I/:! to the molerzular ratiae for

the coxqler-ae in eolution.

  pchaX%fks pre&$tatiOn i8 iahibitud b i%ZhtigQn u4081, it UsaeLlly OC@kWI %Ttm
with @`az%t aiatibody ~~888, althou& dohbf8 WQ&83C86 with n&mulaa ratio z
and th8 8tWturS OhOWn iR ?i@U'8 7 tW8 %q@Ct%d tC 0%%8t.  It %%@m% prCbmb&B
that ths biffarmwm ia bekvi5r of ripItem in %he excm~b aatiasf, re&w aad 6~~8s~
antibodg retion fa to be attrlbutad t5 the fast that the m?i48C~h~ ratio for
yreci?itate ;wn8 eolttble cxNEp,lex %iffers by a f~mtor if8 great i?k8 tw5 for the fmmer
cease, an% by 5nlyIJ!@ - 1) for the latter,
#b&i ?l b e
d            T
                            8 a8 the 'f                Molecule.-
Thsra are two ~pgs in which a~ antibody moleaulrs with two opposed arative, reg:ions
w?lementaFg to the ant&&en @ght be prodwed. One is the wsy %ercribe% in

&GtiOA If.  !I?he other would invalrs the EIMmfactwe of the antibow molecule
in its final confi.guwtlon h&&en two antfgen #ioleSUfee, one of which would eem
S8 the, pa+,tcpm for OA% &Atibd.y an8 aA& thta other far the ee5ond. &Zt attempt t5
de.cide botwwn thase alte~&tivee eaezw to Hera been IWL%~ beforej thesre, exist8


evidence, howmm, soA6 of which ia mentioned below, to indicolte that the first

method of antibody productioA, 1Avolvlng only one antikym niol8~ule, oocurs

prdt%oMi?laAt ly.  It is for this reauo~ th\.st I hwe d%velo?%d the rsther complicatad

theory described above, with the two end :~orfiana of the antibody fanning first,
me (or both) thQa saparatlA& frost the antfgtm, and the 5entml ;art of the anti-

bo&y then r8sn'&4g Its &ape mui hold&g- the active ends la :~osition for

attaohmztt to two tJ&i&OA mokculer.

  Thf6 thtt0~ X'O~Ui~ti th%t the fOZ'!tBtioA of antibody b% 8 rtWCtiQA of th8
first order with r~:spect to the aAti@A, w!ww&e the other nlternatiPe would

require it to be of the second arder.  There 4%istt very little o+fRence ati to

whether on immmiaatioA wfth i~~~btll. aAotmta of antigctn the antibody production is
~rwpartional to the anoant of antigen iajected or to ita BQS;"Q),  S5me tbupport for
the one-antigen-m5lemle theory is provided by the exp%tieAta dealiqg with the

hjeotlon of a XbiX+tW@ of aAti&eAs.  Xf two antigea m0hsl81) w%re requ$red for

antibody fomw.tion, it wmiL% be @%pe~tebd that antibodiear At-Et, At-C*, EI*-Ca,~~*
complementary to two different Antigms A md 23, A Eurd C, 3 and C,-ae a# well a8

those A'-A', B'-38, C'-et,... CoE@BB%At9~ to a 8iA&% iSAtf@3A wOti% be ionned.
The CSV~ihh~8 eviclence speaka strcmgly qainst thirs, !&ua Deep, Taylor, Rnd

hdafr 20 hstve reported that th% s%mm produaed by immmlzation with a alxtww

H, 1. Deal, Q. L. Taylor, and M. P, Adair, k. *giens 3, 69 (1935).

of egg albunin and s%ntm aDu~ia oantaina distinot antibodies h5PablogOus to the

two aatigtm6, +%A& that proaipitation dth 5nu aatigsn lsanss the #mouAt of the

hetsrologous antibody ansltersd,  ah @WA SKWO ri@!WUS d~Af.!It~tiOA t#%I

Yumished by Reidelbergm and &data, who, from the @%rum of a cow whioh had

  N, R%idelbarg%r and E. A. G&at, J. Xxp. we?. 6& U51 (19$5).

been iA,jecteh with types I, 11, rarrd 1x1 pA%umococci, isolated in mooeselon, with
the oorraspondiae; tpeaif tc -pol$laactiri%a, the three antiosrbahgarates, 8acpfr in
a~ appawntly -pure state rsnd tith 110 a~pr%*oiabLe 5rore-mm5tivltg as to


16

pnmmocaacur tpa .  gn another striking experiment Rektaen an4 Boor 22 fcmld

`22  L, P-aktoen wnd A. K. Boar, J. XRfect, Dia. s, fjB8 (1931).

that the smwm obt?+iasd oa irtjectiw a rabbit w%th ar. mixture of 35 antIgear
reacted with 34 of the aatlgens, zsnd that absorption with any me had in the main
little effsat on eubeeqymt mctfon with another. &we oa the tw-Lantigen-
molecule theory the wwmnta of antibodies A&k', 33*-B', o . . cqmble of cmsixyl;
precf?ltation with B sfnale ant&en mmld be ~:~11 co-ed with the total
amount of antibody (of the order af I&, for2 mtigene-about J$ in thfs care),
thersa qualitative observatianr pravida significmt e~5donce in favor sf the

alt sraztlve theory.
B
`3                      ,-There has lmn m&ensiva, d.lrsaussion of th6

qwtstion of what laake$ a nzbstawloe CUL antigen, but x~o gen@rnLly aoce:-Aad
concltislana hsve `bean renuhed. Our throw -pmnit$ the farmlntfm of the folkw-
lng maenmble ariteria for eratig~~%c ,zctitityt

   1. The antigen molemle mot contain attim .qmups, cs%ble of mfflciently
etrorf intar-.r)tion with ths &~bulin chsin to irrirttance it8 mniigwatian.
  2. The ceafipratloa cd' the anttgen aalemile must be well-defined over
snrfamt regions larget snowh to glw rfee to ,m integrated antiboP+antim
force mfffaPent to hold the m&m&m together.
  3. The zntigan molecule must be larce enou& to have two OP more such
surf~,ce m&ons, and ia ceree th%t the antfgenie activity de:mnds upon a pwtfmlar

aacaharida~s, invertebrate &m0&@+8k~~), and txme lipide and carbohydrate-lipid


17

prvttAns in the body.  Man-aatfgenic mabstmam km been rqmrted to beaome

aatZgmi(3 Vmen adsorbs& on pwtiolea (Farsmwn antigen on kavlin 24 )g  in this

caw the ymrtiale with adsorbed haptem is to be &mridemU the antigen *m~~eoule~

of our theory,  I pmwllct that raletiwly simple mvletiee ovntaining two vr

more -tens till ha fotmd to be antf,genic: e~xpmkaeats to test thla preifietion

are now ttmlar my.

ctut mae pmro a@# 26 that the well-UefinmI prvpvrti-ts of n&im protsiaa rmplm

a A. 3. Nireky snd L. Pauline, Prvc.
Ghiness 3. PhyriaL 3, 32l. <X9323,  xk&, AC@. SC%. && 439 (1936) : H. f9iz,

that the&r lacrleanles have daGfinite conflgcmtiona, Ws pulpqtibar uhaln or chaincl

between ymrts of the chains.  The phenoareav~ of duastuxxtivn $.mml~~~ the lvea vi

mnfQtuvitloa through the prttq or evxplete ~~alltrg of the t.zhains.  Of the

forces iamlmd ia the retention of the nativtt configurativn thvm ~!.eecribed WJ

hydra&en bonds abre probably the must fxpvrtannt.  our knowledge of the pvp9rties

of the, hyF.rvgvn bond kts incream to such an extent dnrlag the pet five yerarr


aar to Justify wme epecul~tiva :,.a to the n?&ture of the stable cvnfigumtianr

af protein mvleaulee.

Rydrogea bonds C&XL be famed by the pqtide ctrrbonyl aad i&no groupr of

a po1ypeptid.e chain, and alrt! by the carboxy, amino, hydrvzy, and vthsr oxygen-

and nitrogen-avntaiaixq group8 irn the side chains of the anrim acid rearlduea.

Xa a rtable oanfigmxtian am 18ou3;tt rtroq iqdrogen bonds as goreibls will be

present.  Cne, aonfiguxvMon ia which all of the paptide carbony nod imino

grouprp are forasine: strong hydrogen bonds ir that ehvwn in Bfgure &. Bern

extended ohaine are bonded together to form a compact lqer, with the aide &sine

extending sltenrate3.y a'ko~e aad below the Tlane of the layer .(protided that the

levo cvnfi~ation ie the onlg one represoated by the tiaa-acid residuee).

This configtm&ion hae been as&mad ta B-keratin and other fibroas rrvteinr by
Astbury27 on the barir~ of x-ray data.  Althwctjh %he atnmtuxe hmd not been
27 W. T. Astbusy, Tnsns. Ba.raw Sot. a, 193 (1933). sad other ?crs.

verified in de@il by the analysis turf the x-ray data, the agreement in the

-- ---

  It is to be noted that the 4IIMO-c~ smpmce alternates in altc?rn&s

lines in m.ch sL layer, so that e layer of finite rite could be cwmtmcted by

muming a ain,~la plpptide ahafn b&c snd forth.  A pJobul.ar protain could then

be made br building sem%l etch layers piam~llel tv one mother and in conbaet,


1ilC8 a otack of -p2Iw&ea, the l.&pra bsinp; held. tog8ther by Bider-chain inter

actions ao well aa by the ~o2ypytids ch~fn ituelf. 4 protein molatmle tith,
for 81+~mpl6, m&?$l;$ the 6h1%pe &ad $i26 Of a c??&Q k) i on e&q might c~ntaia

four layers, each with about ei&t strfarpra of about twelve msfdmta each.

  do f8W mr0 MQ x ZUItiC@Jd, v StmiIIg Bt318cn3bcP nxCrd#316, t-hat 3% ;~X'O&&CI Or

hydrolgrprcliae res/due In the chain would interfere with th8 structure #ham in

Biqars Is la mah a way am to cmxse the chala to tend to turn thmqh l$O*; heam

if these rssidusa WIMVJ suitably distribu%ad alorq the chain dtiag lte eyathmiar

the chain would tead to ammum the coafiguratian discuemd &me.

  Layer strtmturem other than %his oae mai@t aleo be ammmed, in which the

ohaina ar8 nob extkmw,  dom8 fibrow ?rotsias, euoh a8 c&mratfn, are lmowa to

have stmaturee of this ,ymmil ty?e, but th8 nature of the foldin& uf the ohrias

ha8 not y@t been d&8XBin@d.

  We ham portulated the elristenca af an eactramsl$ large rnnabar cf aocrbrrsibls

caafi~tlona With mmly the eanzlb ana~~ for the end y&s of the @obaUn

yo3gplcrptids ohaln,  A layer mtmwture, with variety in the type of folding &a the

l&pr, would not, it mms to PM, giw snsu& conflgumtianal psaibilitlss to

8X@kiXl the @-&La ObISrrSd YtVr#?&%litJt Of tkw Wi%ibCtdy ?X'8CUZSM tn h~~UBti%'#

itaslf to the aatima, aad I thiak that &a# canf'i~tions ianst be fnvokd.

But rimpls consider&ions aho~ that it would be dfff$cuZt for the ahain ta asmane

a mkew configumtioa ia whilch most of the pqtl&a carbony and imiao boadlu tti

prt fa fomixg lydro,gpa bandy, P.E they do in the layer struature~; and la

aoasscp8a~e the skew aoafiguratiaas would be mu& lam stable th&n the layer

confi~tlona.  The way out of thic difficultr is protided ?q~ th@ ~OFtV.kLt8 that
t& * narta of th8 globulin pLyr,
         -c-       1 sptlde chalas contaia 2 rery zarg Q yecf'J3gioa
(pmhape one-third or oaHmlf) af prollna   hydroqpolias regMues and o thar
                                                                           --
rarsidues diich pwemt the awmmptioa of a stable ,layer coaflgmation.
                                          ---
  Ua IBY, indeed, Antiol-te that g.loMkr groteias mr be divided into two

Blrsin claem~, ampriming reayecttfvely those in which there la a layer rrtmctum


20

and those in which tha satAble oonff@m%tlan of the chains ia, m0r8 oomplex; the

lttter IS-~ show the hi& praline md hydrtmypraliae content yoetulated for the

~loImlin chain enar.

About one-third of the residues in &.&in me praline md &ydmxyaroline.

natwe B, 421 (194~~).

contains LIB atm~ antiplm &roup8, a d8fidt8 eonf'i$q3ratioa ia a requirits for

aatigenlc actlritg.  Theee coneidemtions thue yrovlde +a poe~ibSe axplmtion of

the tmlMmown fast th& Eel&in is nut &'ftctivc! BE ED anti&m 32.

amprotsine, have pwM.e8 resulta of mt elgnifiemce to the theory of anti-

bO+ i'dXT2QtW8.  l&my of the arppmmtr based on these results are ;maientad in

cbesrly %y the strong croae-reactions between sirailm chloro and methyl hq@ene.


21

The dzta on epe~ificity of antibodiem witfx mqmet to haptenr indioatcs etroagly

that the hqtan group fits into a pxket in. the antibody, aad that the fit ir a

clam oae,  Xt ehould nab, be coauluded that all sntiboPI=antigen bands arw of

just this type; for exswsple, the fitting of an aatibtdy gxo~xp into a pocket ia

the antigen ;aray &lse be cr%tea of %~~~~%aaoe.  .&&enriva vork will be needed to

determine the dstailad nature of the Plntibody structures ooq&maatavy to

prtlcnlar hqtear aad aatigearr.

     m.
        v  J'n.rth 0

       3
             0 sib163                 Pre etia   .

*
A   lb hodr              V 6      t b d.ielr .-The folloting mB thode may

be proposed to determine the valence of eatfbohies. First, let a mrum be

produced Iqr iajectioa of 8&a amprotein of the following typr  its hapten ir to

be eufficiQlntly atraag (that is, to interact suffi6ismtly ohraagly with the
hoarologoua Ptntibody) that oae tiptea grmxp fomr a sertiisfactom aatibody-aatigea

bond, aad the amber of heptea grotqm ?er SX#le&8 is to be anall emu& 110 that

in the main only one group wilZ be parent in the 251'88 seroizq as er ~s'&tarn

for aa antibody end,  The same haptea ia then attached to another protein, au&d

this azoprotsla ir precfpltsted S&W thi3 sernn.  fi if ba samned that fha

prscipitate is mltmse-ratntrrd, the nrrtia of haptim gret;rpr to aatibody rrolentlsr

in the precipitate gimm thg ,ammge wAmce of the antibady.

  lhte of essentially this mrt, obtaiaed w%th arsanilf~ acid as the hapten and

the rabbit ata the expa;Sfaental a~iml, have been publi&md by IIerurawitx an&
hi.8 collabomtorrf4 In a pdqxw reporting many int0Fertlag exprim3ater.  The

34

3'. lbrowitt, B. Krms, and 3'. Bkwx, 2. fzhyslol. Chem &* a3 (x93&

equiva&mt welet of antibody yea ~rs~~ilio mid residue was found to vary frcr-aa
23,OUO for aatimns with very may attached haptear to fil,UUO for those with

only a few.  !I!he 8xpecltad mlns for 8&xratloa of all haptens by binslent aati-

body aolstmlar ia one-tilf the antibody walemlar weight, that is, aboat 79,000.


22

!Phe low ceiq3erlsental wlmr aru probably due to failure of 6osie ha~tms to

mz~bina with ~tibodiaa.  Ia ~r4ianlar. if twc haptens are attmhud to the

aaae tyrostns ox hfstidias rasidue +tsric in8uractiona of antfbcdier mar perrrit

only one of the hqtena to b6 effrctiw f5*

35  From tha data discussed abow, which fa 0%~ c-&ion Indicate that two
haptam ermps aomblne with a blvalent antibody molacule, 8amwlta drew tha
different conclu&on that: oae amenis-uontainlng gmq in the antigen comblnatr
with one aa4ibofQ solaaale.  He reaahsd th&s mmlf by aseneri~ 100,000 (rathur
thm X57,000) for the aobcrular wui@at of the antibody aad by asswsiag fbet the
WtiW @Qlrp il3 #Q aB41@ilk iWZUtiSt8 cf tW0 hspttBllS att&Ch4d to a tJTOUiX!l+3 OX'
histid%ne m&dab,  It %a, of ucmme, likely 4hat this aamm ie antigem with
hi,& arsenia conten4, btx4 ft mmt probable that the haptens are nainly atteehed
to eeymte wrlduee in the tmtl~ens mn$iining only a few haptbns per ~lolemle.

tw6 haptsn RrmJp8.  Pertinent data haw been obtained cm tkts point by

Hamtite and hte collaboratorr, who fotxnd that affuetlw sntllua-ptaa precfpftln

mrum flsrg Froducad by an arapzotein, ~&adm from rxaamlic acfd and harua gltOm.Un,
aontainfne 0.24$ arsenic (4,S haptenr per swraga mcllroals af molam3ar
weQht I57,000), and a tmcs by one containing O.l3$ arudu (2.6 haptens psrr

~.dPhe criteria giwm
b   Th Pas ible A i

ab~w f6r antigsnfa adtirity wmlb bs satiefis% by a subs*ce of rulatiwly

bs qns~fed aho 40 show the precripitfn zmwtion with lto OM semm or with

A mbstance rith oLLy twi hapten grmpllr ym inolecule might be expaatsd not

to give a precipitate with ?A* holsologmsr cnClbaQ, but rather to form loag


23

remain In solution, and wonld amfar on the solution the proper4y of pronounard

birefringenoe of flaw.  If, howewr, thaw were In one end of soma of the antibody

laolesular omplemeatgy regions for two hapten groups, makIng these ~oleculer

sffectimlg trivalent, the Irtrltyya would be tied togathet and a preclpitats w&d

be famed.  It is probably significant In this connection that Lmdstelner an%

wn der &&es 9 haw obeemd bo4h the preuipitln reaction and aaaphyla~tia

3b

  X. Le.n%rteIaer amd J. v8~). der So&mer, Proc. Sot. Z&L 3101, Me%. a,
747 (1932); J. %P. bd. s. 399 (1932) o

ahock (in gu.Inerp pIc:r mnrritieed with th5 oorrespondInC: arzoprotefnrr) vith azol?ycls,
euch SB resorci~ldfarra-E-m~~ll~ mid, (oa),c,Ea,(~C,rr,~(~=~5~~)*,

forned by croupline; two anllPc acid nolecul.ecl with r+-?sorcisol. Lasdatelnsr

hiaaeelf hcer axpltnlnsd them obatlw.tions as resulting from the low-aolubIlIt~

of the dyes, but the ezplaau4ion advanosd abow mmn mara) probable.

__.
0.    e mmta wit                                    .-We would

predict that if there were nted aa an antigen cs aolscule to wikI& cweml ?uqttea

e;raups A and asversrl bifferen4 hapten group 38 were rattwhed, thu serum obtained

would contain three kinda of bivslent antibodies, AU', B'-B', an% As-B' (at well

as tha univaleat aat%bodlsra A'- and 33'.),  Ckr plctum of the process of formation

of antibodies pmita little or no correlation between the two chain ends of the

glob-nlin in the selection of etsfwe rep;ionr of the antigen molecule to oerw as

templates, sxcspt that the two m&Ions mue4 no4 overlap. we accor%k&iy prodlot
that for $ aad a+ croup A and B rs BpectIwlJr Ferr ant Qen mole&la the mmbers of

bivalent antibody soleculea of dlff swat ldnds In t&e eemm would br


24

& & equal to or slightly: gm?ter t&a twIo5 the ~metrla 18851 of the mtoants
             --                    --s            --1111

of AI-A' and B'-f)*
c-s The only data pensitting a qumtitatlw tsrt of this
                    .

rsl&tiota wUah have COIW to rag attention we those obt&md by lkrotits aurd

collabomtoss (loa.oIt.) by use of aroprotelns aada fmm arsanilla aaid.
                --

A serm~ produo by injecti= mbbits with a~ a;ritIg%Fan m&l) from arsanillc acid

aad home globulin was found to omtaln antibodies to the arsanllle haptan
(prealpitatimy: m eamtsla sta%s from arsanilic acid md rabbit globulin),

others to horse ~1obU.n Itself, an% still others to the homologous antigen,

them last greameatbly being oe@emeBtary la structure both to the tipten and

to the actfw groups of horse gMnilln.  The qvnntltatfw resulta obtainsd are

the fo11owI~ I  10 ~1. of Immms ssrm mw 17-U m,g. of precipitate with 4he

zaarIma1 precipitatiayy momt of asoprobrshn from arsanIlIa aci& and rabbit

globulin, and g mg. of pmafpit;ats with horw globrtllin, there ammts b5&n$

Indqasndaslt of th5 order of 4hs two prseI.pitatIoaa. Af45r exhms4Io~ with thees

two antigena ths MWWJ p=aoler 17 mg* of precipitate vf4h the homologous a&I-.

If we assum that the aondftlons of em& preclpItatIon w5re mmh that only

suIta?)ls blvalent antibody moleculas mxrs Iaaorparatsd ia ths pmaipftats, ths
equation abovs would reqzirs the 4hIrd pmeipftrats to wsigh aboa 24 w.: the

sJlrp6rIaMab aeeordIx&y pmwibm SOEM eupporti far the theory. Tha quastIt;atln

bfsorebymoy sray possibly be rma to the inaazyorotlon of FAOEM affscMvsly

Palmlant antibody molscules In the first buo preclpltatas.

The qualit&Ive sz@erIslcsxrtal results vrhidrh haw bma rtqortsd are in par4

compatible an% In prt incompatible w&h the thea=.  ae most intersstiq 61 ths

sxperlmnt s are those of Lmdstefmw and mm %er [i&et+ $7 , vho prepared aact-

37  g. tidsteinar aad d, VZM %er &h55r,              . &, 709 (lg8).

. .

proteins aontainlng two dIffarm4 kinds of haptttaa rd. studied the antiba%ies

prodmed by then.  fB ~ll!#t C?W$'OBly UnB Of thU htQt8B6 Wi%S ibffN3ti-W in Mltibdy

fomtatlon.  With the muprotein m9ds from ~Iacs-~a~~l~~~Inobb~oy~~


25