146
H 1

              Report of Dr. boebcl.                  

   On tho nature of pneumococcus antibol'y and its union mith hapton.
The older concepts regarding the non-protein nurture of antibodies, and the
hypothesis of Buchnor that antigen itself formed a part of the antibody COG
plox rtlust nofr be regarded meroly as evolutionary steps in an attempt to
undorstand tho true nature of these biologically active substances.  During
tho past decade these oldor theories have gradually given way to accumulated
chemical evidence rlhich at the present time clearly points toward the fact

that antlbodies are in reality modlfiod sorum globulins.
   The Ideas of the honch School of Immunologists sponsored by Bordet
accounted for the Interaction of antigen anL antibody by the phenomenon of

adsorbtion.  This concept, however, failoc? to o*ain the specificity of

immunological reactions.  More recently the specificity of antibody pro-
duction has been explained independently by LIudd In this country and by
Dreinl and Horowitz in Gerrzw. In their opinion, the normal course of
serum globulin synthesis la altered in the animal bo;`:r when antigen reaches
the site at which the ayrithesia takes place, Under the influence of the

antigen, the.serum globulin ie altered In a way characteristic for the

foreign stimulue.  When the modified globulin eventually encounters the
forelgh antigen,either in the circulation, or in vitro, interaction of the
                                                   --

two is possible.

   From the following interesting analogy - the results of certain

experiments carried out in  our laboratories - it seems not unlikely that
the synthesizing function of a cell may be specifically oriented by a given

stimulus.  When the %" form of Pncumococcus is grown in the presence of a
`1s~:)ecific activator" dorived fron a cell-free filtrate of the "S" organism,
the former is induced to resynthesize the capsular polysaccharido.  If the


147

H 2

nature of the activator ir: changed, homcvor, the "R1' organism can be made

to synthesize a


from that which


Thus it is scan

conditions may,

products which,

polysaccharida quite different in its type-specificity

it B+roducod when stimlatod by the original activator.

that bacterial cells, under tho same general environmental

by pecans of specific stimuli, bo directed to synthosizo

though chemically rclatod, are biologically specific.

    In regard to the non-protein naturo of antibodios there are even

today certain imnologists who maintain that solutions of antibodies may

50 socured which glvo no chemical tests for proteins.  It must be pointed

out, however, that the sensitivity of immunological reactions far exc'aedo

the limit of sensitivity of chemical teats for proteins. Until weighable

quantities of non-protoin antlbodios can be secured, the evidence for

this point .of view remains inconclusive.

    An analogous situation has oxistod in t,.e attempts to dofine the

chemical nature of enzymes.  It `has bocn the method of tho European

investigators to purify enzymes by adsorbtion on colloids. Solutlone

thus been secured which become lncroaslngly lower in nitrogen content

without loss of enzymatic actlvi ty.  This group of investigators has

failed, C however, to secure by'. thcee methods protein-free enzymes,. `nor

h&m4

have

they obtained `eubetancesi. th& Chemlaal' tiompoaitlon and' biological potency` +

of which boar any'conatant ralatlonshlp to ono another,  Honever, a new

approach to. the problon` of `enzyme chemistry has mde possible the prepara-

tion of crystalline proteins bearing the biologically active conuonont of

crude enzyme nixturoe.  Thoso crystalline proteins shov both constant

physical properties alld biological activity on subscouont crystallization.

The SUCCCSE of Northrop a:ld his associates in isolating from

mixtures of proteins a crystalline derivative of unique biological and


H 3

chenical properties has wrvod as a stimulus to us in attempting to ieolato

an iri~~~~s protein frou antipneuuococcus som.  It is conceivable that this

protoin night have certain properties which ~louli' differentiate it fron the

acconpanying inert sfmm pro toins.  The nature of scm globulins, however,

is a problcn of groat conplexlty.  Fortunately tile investigations of the

Carleborg laboratories have afforded an Insight into the chenical nature of

globulin corlplexos as they occur in norm1 horse serun.

Frorl extensiw studies on the protclns of nornnl horse serum,

Sorensen cane to the conclusion that the globulins occur not as nixturos

but as labile conpounds of 8u- and pseudo globulin. Ho found that these

conpounds are easily diesociable and that by mans of fractional precipita-

tion and dialysis the constituents can bo partially separated as protein

fractiojls which becorm, nith each subsequent fractionation, increasingly

richer either in t-m- or pseudo ,+,lobulin; as the case nay be.  Al though
globulins have nevor ken obtained in a state of purity,  it has been

possible to dissociate the covlexes of SONI globulins to such an extent

that fractions have been obtained of which either eu- or pseudo,globulin

is the predbdnat constituent.     : , .     .     .: 4 :. ' ..' ~.' _-    : ' ;.

  W4 have rlado'uso of.this `princiylo of the,dieeociatlok of the hvra

globulins in the eepisratlon' of the antibodies in Type I Pneunoc~cc~s anti-

seiun fron the acconpanying' inert globulins ni th which the inuune bodies

are associated.  The Ffator insoluble globulin fraction' of antlpnknococcue

scruff, described by Felton and  comorrly used in the therapeutic troatnent

of lobar pnewonia, has boen separated into an inert euglobulin fraction,

and a pseudo globulin fraction.containing practically all of the anti-

bodies prcsont in the original nixturo of the two ECIYKI proteins.

When finally dissociated and separatod from inert euglobuliq, the


14.9

H4

nntor soluble immune globulin is considerably more potent in antibody

content than tho parent subatanco.  Half a centimeter of a solution of the

purified antibody containing as little as 0,005 mg. of protein is effective

in protecting mice against 500,000 minimal lethal dosee of virulent

pneumococci of tho homologous type.

   In adaition to increased biological potency, tha immuna globulin has

certain chemical properties which distinguleh it from normal serum globulin.

Determinations of tho basic amino acid distribution of normal and immune

globulin reveal no etriking difference In the chemical unite which con-

etituto the txo proteins.  Honevor , tho Immune globulin hae a very alkaline

isoelectric point (pH 7.6) nhoreaa that of normal serum globulin lies in

the more acid range (pH 5.2).  Thus the most distinguishing property of the

immune protein, its alkaline isoelectric point, is one which can be

accounted for only by a relative lncreaso in the number of basic amino

group8 in the protein molecula.

    Efforts have been me& to crystalllzo tho immune globulin, but

thus far without convincing results,  Solubili ty measuremonte have

indicated that the antibody in Its preaont state of purity is not a chemictil

entity, despito many,ropeatod fractional pracipitatlona,  It may be for
this reason that attempts to crystalllze~the antfbody have thus far been

unsuCCe88~.                                                                .
   In view of its unusual basic propertiesit 8oom8 not unlikely that

the amino groups of tho antibody protein may play some role in the union

of tho antibody molecule with its type-specific haptcne - the capsular

polyaaccharidc of Typo I PncumococcuB,  In collaboration Rith Dr. chow,

it was subaequontly found that when tho amino groups of tho antibody wore

acetylnted nith ketone #LB, under conditions r:hich climinntod any


150
H5

possibility of protein donuturation, the resultant acetylatod antibody

protein failed not only to precipitate the type-specific capsular poly-

snccharide, but to agglutinate suspensions of Typo I pneumococci, and to
protect mice ngalnat infection nith homologous vi&lent Type I pnoumoco~~i

3s uell.

   The basic amino groups of the antibody protein have also boen

coverod by the grouping = CH,, with a resulting loss in serological activity.

`;Phen the grouping = CH, is replaced by two hydrogen atoms, honover, the

Lunino groups are restored and the imrm.nological proportioa of the antibody

protein are fully regained.  Tha chonlcal reactions rrhich are involved are

as follows:

  (1) - NH, + HCHO  > - N + CH, + He0 (serologically inert)
  (2) - N = CH, + H,O + - NH, + HCHO  (serologically active).

Ronction 1 goos to completion if an aqueoue solution of pneumococcua anti-
body is treated rri th formaldehyde at O*C. and at pH 8.5.  The excess

formaldehyde is removed by dialysis, and tho solution of the resulting
"formalizedw protein is ad,juEted to pH 7.5. The protein derivative Is

perfectly stable under these conditions and shone no serological aotivity

what soever  ff the aqueous solution of $& formalized antibody is      . ...'

adJusted to pH 5.0, however, reaction 2 takes place rapidly, When the

reaction mixture is dialysed and the pH s@,n AdJusted to.7.5, the lmmuno-
logical properties of the protein are fully restored. From these oxperi-
mcnts it Appears that the preeenco of basic Amino croups icr of utmost

importance in dotermining tho biological activity of the antibody protein.

    Studies carried out in this lAbOratOry on tho chemical nature of

the typo-specific polysaccharides derived from encapsulated microorganisms

have revealed the interesting fact that all the specific carbohydrates


thus far investigated contain uranic acids as constituents of the poly-

saccharidc molecule.  The invariable occurrence in those bnctcrial product::

of &lucuronic acid or its isomers su,q.csts that tho highly polar carboxyl
group of tho urklc acid and its stcroochcmicnl relationship to other groups

in the moloculc determines tho biological specificity of these substances

and actually ontors into chemical combination with tho basic amino group

of the homologous antibody phcn theso two substances are brought together

either in vivo or in vitro. Thus, if the carboxyl group of tho Type I
       --     --

specific carbohydrate could be covered by an aster grouping, the derivntivo

should no longer react with its homologous antibody.  The polysaccharide

was thoreforo treated with an ethereal solution of diazo methane, and the

methyl estor separated from the reaction mixture.  Solutions of the methyl

ester of the polysaccharide gave no sorolot:ical reaction when added to

homologous antibody.  lhen the methyl groups nere removed by gentle

alkaline hydrolysis the carbohydrate again reacted specifically with anti-

body in dilutions as high as 1 part in 4 million. Quantitative detormina-

tions of the methoxyl content of the carbohydrate before and after

hydrolysis shonod that tho mothoxyl derivative contained ono methoxyl group

per carboxyl group.  The reversible changes in immo.nological specificity

brought about by rovcrsible alterations in chemical structure indicate

that at least two of the molecular groupings involvod in antigen-antibody

union aro tho olectro..positive amino group of the immune protein and the

olectro-negative carboxyl group of the specific polysaccharide.

This concept alone does not explain the specificity of the reaction.

There is, honcvor, certain evidonco to support tho vien that the

specificity of such serological reactions is t:overned by the arrangement

in space of the polar groups of the reactive carbohydrate.  It hns been


shown that anti(;cns prepared from the diazophonol glycosidee of glucose

and galactose react only in thoir homolo;:ous antisera.  In respect to the

numbor and nature of thoir polar groups, those tvo ~:lycosides are identical.

Since the reactive groups are in each instance tho SOIJC, the mechanism

underlying tho union of both glycosidos with their honologous antibodies

rmst likcwisc bo the sane.  The specificity of this reaction therefore can

bo accounted for only by known difforcncos in the spatial arrangcnont of

the polar groups of the fourth carbon atom of each glycosic?e.

    Since tho spatial arrangenent of identical polar groups in a carbo-

hydrato suffices to detomine specificity, it therefore aeons justifiable

to assuue in the case of the antibody that the spatial arrangenent of polar

groups in immno protein Roy likcniso dotcmine its specific capacity to

react nith CL given hnpten.

In view of these considerations it is believed that the general

nechanisn underlying the union of antibody and carbohydrate involves the

interaction of polar groups of opposite charge.  fn the case of the Pneuuo-

coccus Type I hapten and its specific antibody,  it is A certain axin0 group

of the latter, and tho carbowl group of the polysaccharide which interact

to f om tho 1-e procipitato.  It is sum:, ested, f'urthemore, that the      '

specificity of this reaction-is dotemined.by the storeocheuical relation-

ship of the douinant polar groups in tho reacting molecules, whether they

be antigen or antibody.  If the spatial pattorn of the polar (;roups of

both anticen and antibody is of czactly the correct order, then union occurs.

If, however, this relationship is disturbed 3y artificial IJeans, as has been

experinentally Cenonstrated by covering the doninant polar ,=;roup of either

anti@n or antibody with a chonical rAdiC81, tho pattern is destroyod, and

union between antigen and antibody fails to take place.  When the original
constitution of the acting substances is restored, honevor, serological
specificity is onto rlore roga1ncd,


Publications.

Goebel, Walther F. and dabers, Frank H., Derivatives of glucuronic acid.
       I, The preparation of glucuronic acid..from glucuron and a
       conparison of their reducing values. J.Biol.Chem.,
        1933, loo, 573,

Goobel, Walthcr F. and dabcrs, Frank H., Derivatives of glucuronic acid.
        II. The acetylation of glucuron. J.Biol.Chem., 1933,
        100, 734,

Goebol, Walthcr F. and babora, Frank H., Derivatives of glucuronic acid.
          III.  Tho synthesis of diacetylchloroglucuron,
        J.Biol.Chom., 1933, m, 173.

Avery, Oswald T. and Goebel, Walther F., Cnemo-imnmnological studies on
       the soluble epecific substance of Pneumococcus. Isolation
       and propertiee of tho acetyl polysaccharide of Pneumo-
       coccus Type I, J.Exp.Med., 1333, 2, 731.

Goebel, Walther F. and Avery, Oswald T., Chemo-imumnological studies on
        conjugated carbohydrate-protein antigene.  VIII. Influence
        oE the acetyl group on specificity of hexoside-protein
        antigens. J.Exp.Med., 1934, -

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