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Subunits of normal and pathological γ1A-globulins. (β2A-globulins)
ARCHIVES
OF
Subunits
Frmr
BIOCHEMISTRY
AND
of Normal
Medical
BIOPHYSICS
and
Depurtwcnt
102,18i-143
Pathological
-1, Cliniques Received
(1963)
TlA-Globulins.
Unizwsitaires November
St. Pierre,
(&-Globulins)’
Louvain,
Belgicrtrl
27, 19ti2
Yormnl human rl,,-(a2,,-),r,nr-(19S,-) and 1,..-(is,-)globulins, as well as four different., electrophoreticaIly isolated rla-paraproteins were analyzed by electzophoresis in starch gels made in pH 3.2 formnte buffer containing 8 JI urea. The proteins had been exposed t,o 8 M urea, used either as such or associated with mercaptoethanol. In the latter case the reduction of disulfide bonds was made irreversible by means of iodoncetamide. The three mumal y components showed fundamentally comparable patterns, indicating the existence of two types of subunits which corresponded to the light and heavy polvpeptide chains described by Edelman for r.$-globulin. The three y conponents resembled each other wit,11 respect to the subunits corresponding t,o t,he light chains and derived their individuality mainl>- from their heavy chains. The four ?,a-paraproteins presented the normal *,,A pattern, except for the much greater homogeneit?; and individual characteristics of their light chains. The findings arc felt to agree with Edelman’s and with Porter’s concepts of the struct,ure of y components.
abundantly produced under the form of a molecularly homogeneous [“monoclonal” One of t’he present writers has put forward (7)] paraprotein. Recently the idea of ylathe concept (1) that the antibody activities globulin as representing the t,hird class of of human serum are carried by three classes of related proteins: the well-known yss-? immune globulins has gained support from the finding that’ it was synthesized in the (rp-,7S,-)globulins and yl.M- (19S,-, &-) globulins, and the recently isolated (2) lymph nodes and spleen (8) and that fluorescent antibodies could trace it back to the Y1.4-(/3zA-)globulins. This view was at first cytoplasm of plasma cells and to the germibased 011 mere circumst’antial evidence, nal centers (9) in precisely the same manner which included the antigenic cross reactions as has been observed for yss- and for Y~,,~between the three types of y components globulins and for antibodies. Confirmation (3)) their associated deficiency in congenital has come from t.he demonstration of specific sgammaglobulinemia (4, 5, 3), their simulantibody act,ivities in purified sampIes of taneous increase in diseases associat’ed with isolated from human serum hypergammaglobulinemia (1, 3)) and the rl,-globulin w. occurrence of three corresponding types of Two different approaches have been paraproteinemias in which any of the t’hree y components could be observed to be over- devised in order to gain insight into the structure of y components One method, 1 This investigation was supported by a Public initiated by Porter (ll), consists in the use Health Service research grant, No. A-1263 (Cl) of prot’eolyt,ic enzymes. Such studies have from t.he U. S. Department of Health, Education, succeeded in localizing the subunits of yssand Welfare, and hy Grant So. 270 from the Fonds globulin t’hat are responsible for its antide la Recherche Scientifique NICdicale, Brussels, genie cross reactions with Y~.~- and Ye,,Belgium. 2 ss = sense stricto. globulins in that fragment of the molecule INTROl>UCTION
137
which is also the carrier of ant’ibody act#ivit’ies (1, 12, 13). The other approach, which consists in cleavage of disulfide bonds in t’hc A pool of normal sera from several blood donors presence of concentrated urea, has been pro- was used to prepare ?I*-globulin according to a posed by Edelman and I’oulik (14). A fractionation method worked out hy one of us relat#ed method was developed by E’leisch- (2, 3, 18). The material proved to he antigenicslly man, Pain, and Porter (15). This type of homogeneous when tested against multivalent antisera and against antisera specific for each of study has provided the base for a structural model of r,,-globulin (IG), which would also tl(e three immune globulins, as well in Ouchterlony plate analyses as in immunoelectrophoresis. account for the cross react’ions between ysS-, yld-, and rlatl-globulins. Confirmatory cvi2. 1’ATHOLOGI(‘AL ~~.&LOBl:LIX\rS dence favoring this model has been prcscntcd C;nrnIlla-lA-type paraproteinemias m-erc diagby Cohen (17), who used the method of nosed by means of immunoclectrophoresis, using a Fleischman and co-workers for a study of set of monospecific and of specifically absorbed paraproteins of t’hc ysS-, ~l.~-, and ylM t#ypes. antisera, as described elsewhrrr (19). All ?,,YThe present paper describes an applicaparaproteins here studied reacted with anti-y,.i t,ion of Edelman and l’oulik’s method of antisera t,hat had been cross-absorbed w-ith m,_..and entirely failed to precipicleavage with mcrcaptoethanol and urea to and ,lar-globulins, tate wit11 anti-r,,or nnti-rlnl-nntiser:l crossand y,q,-globulirw isolated from Y1a-, Yn-, They were thercforc normal hmuan serum, as well as to several ahsorhed with :I,\-globulin. considered to satisfy tllc criteria set forward (19) different YIA-paraproteins. The results will for the definition of I,,\-paraproteins. be interpreted wit’11 respect to their bearing FOUl scra thus classified as ,I<-parnproon the structure of y component,s. t.einemins were submitted to pwparative clectro-
FIG. 1. Electroplloreses isolated rl,4-glotmlins.
of three
rl.\-p:~ra~)roteirIPrrlia
ser:t and of the corresponding
phoresis in agnr gel. The paraprotein bands were cut out, and their proteins were eluted by frcezing, tllawing, crushing, and centrifuging the gels. Ikrt her purification was acllieved 1)~ salting out with 2 dl amnwniwn sulfatr. The isolated lwr:tpr~~tcins were found to be rlect,ropltorrtic:tll~ I~r~n~ogenrous (Fig. 1) when tested l)y higil-volt~:lge elcctroplloresis in apar gel CM). 13). inrrnunorlectroplioretic st.nnd:lrds. tiny amounts of :rssoci:tted ilitpuritics of the san,(: rlect,roplroretic niobilit> r~~1~1tl I)e sl~~wn. ‘I’liey :Lppcared to correspond Illostly to residual normal -, ,.- glol)ulin underlying t II? p:lr:lprotcin fractions.
ra
U-
7 ss
Chro~~~:~t,ogral~l~~ on 1)ICA&~!:-cellulose with elw tion in a gradient of increasing molarity and decreasing pH, xcording to a procedure described by Fahcy M), was used to prepare y-,-globulin from nornlxl Iinm:u~ serum, A pool of the initial fractions of thp effluent proved tu consist of sntigenitally pure 7,;glol)ulin of slow electrophorctic rnol)ility, wlicn test,cti 1.)~ irilnlrlnoclectro~)tl~)r~sis. 4.
SoHM.ZJ~
y~.~-(;L0uI’1Js
A euglol)ulin fr:&ion of norrn:tl Ilunmn serum was srllmlit ted to prPp:tr:ttivc llltr:rcrntrifug:lt,il)n
U-
ra 7 1A
u-
ra 7 llvr
140
CARBONARA
in a sucrose density gradient (22). A pool of the bottom fractions, which were immunoelectrophoresis to consist of globulin. I>etails of this procedure are elsewhere (23).
was made shown by pure y,Mdescribed
5. C~mvAm
ob- PROTEINS WITH &~ERCAPTOXTHANOL AND ITHEA, ANALYSIS OF THEIR SUBUMTS
The procedures here used were essentially those indicated by Edelman and Poulik (14). One per cent (w/v) solutions of proteins were made in 0.1 M borate bufl’er of pH 8.5 containing 8 moles urea/l. The preparations were analyzed either as such or after addition of 0.02 mole mercaptoethanol/l. In the latter case the reaction was stopped by addition of 0.05 mole/l. iodoacetamide after 1 hr. exposure to the reducing agent. The products of cleavage were examined by electrophoresis in starch gels made in formate buffer of pH 3.1 which contained 8 moles urea/l. RESULTS
1. PATTERNS AND
OF SORMAL -yl~-GLOBULINS
y1.4-, yss-
Reduced and nonreduced samples of YW, yss-, and rl.W-glohulins isolated from normal serum were compared on the same starch gel (Fig. 2).
AND
HEREMANS
The pattern obtained for reduced-alkylated normal r,,-globulin is essentially t,hc same as the one described by Edelman anp I’oulik (14). It consists of two major and rather sharp bands of comparatively slow mobility, and of a much lighter diffuse smear of faster moving material showing some signs of partition into two zones. rll compliance with the nomenclature late1 introduced by Edelman and Gaily (24), the fast’er material will here be called “L-chains” (I, = light) and the slower material “FIchains” (H = heavy), on the basis of the molecular weight estimations made by these authors. Whereas the unreduced T1,,.-globulin hardly penetrated into the urea-starch gel, presumably on account of its molecular weight, the same material after reduction and alkylation appeared to be broken down into two major components. The faster component, which was also the less abundant, consisted of electrophoretically heterogeneous material of a mobility and general appearance much the same as those of the L-chains of r,,q-globulin. The slower and more abundant component was more homo-
FIG. 3. Starch-gel electrophoretic analyses of normal rl.k-globulin proteins of ylA-type, as such and reduced-alkylated. Same conditions treated proteins; ~a: reduced and alkylat,ed proteins.
and of four paraas in Fig. 2. U: un-
aud of 13cncr-Jours proteins which would srcni to account for most of the prcsrnt knowledge on the intcrrelatiouship and im niunological functions of these prot,eiiis. I II their model, r,,,-glohuliu is pi&wed as cousisting of ali as yet uusprcified uunibrr of light polypeptidc chaitls (mol. wt. about, 20,000) and all equallv undecided number of hcavirr chaiiis (mol. A. het,weeu .iCj,OOOaud ‘iO,OOO),whicll would hc linked toget~her by disulfide bridges and would bc srparahlt: aft,tr rrductioii by nielcaptt)rt.hailol in the preseucc of urea. The L-chains would, either by thenwtves or in couucctioll wit’h adjoim iug srgnieuts of the H-chains, br rwpousihlr for autibody activitirs and would, iu accordallcc, show a great, tlcat of 1ictrrogerir~it.y when one considers the wtiolc spectrum of Y,~,~nwlrt:ules prweiit ifi ilornlaJ serum. l’araprot.eilw of the ySSclass, whirl1 represrnt siliglc niolrcular species, would therefore iu starct I display honiogrneous IAalds gels, after rrductiou aijd alkylatioll. J+nce.Joncs protrills wonId cousist, of L-chaiils only aud tw the products of iucornpl&e syuthrsis. (‘hains of similar tlatmc would be present ill yw- aud y,.,&ob~dills and would 2. I’.\‘I’1’F:RSS OF ylr-l’IIIII’RC)TEISS account for t,hc observed antigeuic cross Ttrr folw yl,,-paraprot,eins showed siniilai reactious and ant,ihod.v activitirs iu t’he t,hwt: pattcrtw (Vig. 3). coinpoiieut~s of the y systeni. On t,hc: ottrcl So~~wduwd y14-paraproteins migrated as halld, t,tlr H-chains would t.w distinct fol inult~iplc batA in urea-starch gel, although each of tjlw ttuw irrimuiw globulins aud he t twv provrd to br honiogenrous upoil etec- t,tic bcawrs of their autigrtiic ilidividua1it.y. t rophowsis iu pH 8.6 barbital buffer (Fig. 1). hu rswlitially similar ~nodct for the strucI~cdllctiotl-alkylatioll wsult,rd ill the ap- ture of ~,C,-gtot~ulilis has brcll. dwcritwl t)y pc’aralwc of patterns entirely similar t,o t,liosc I’ortrr (25), who tlesigtlatrd the subrurits of tlescritwd by ZCdelrnau aud I’oulik (I 4) fol large alid small niolrcldar sizr as &1 aild M, similarly trrated paraproteins of the yNL Icsprct,ivcly. class! mid by (‘ohen (17) for paraprotciiis of Jt has also hrrl~ foluld that, tlw rlrxynlit I-w~1.~class. Tile major slow fraction showed callJ7 obtained fragnlrut~ of y..%-globutill wtlirh 1it.t.k: diffcwncr in appearawe and mobility carrirs t tw aiitibody activity [called I, rI wit 11 wsprct, to the corrcspoudiug fraction (ll), .I, C (2(i), 01’ S (%)I coiisists of the Lof t~ornml ~l.,-glot~uIiii. In cont.rast, the fast, cliailis plus sonw part, of t#hr H-ctinilis (17, fractiolr now coilsisted of our, two, or three 28). This finding agwrs wrll wits11 the fact vc~y sharp bands with mohilitirs lying iu that the autigcnic cross rractiorw t)et\vc,eii t tw ra~lgr wvrwd by thr fast subunit of tlir thwr y coiupoiirlits, and brtn-rril tlwni ~mw:it y,.,-globulill. alld t.lw 12c~lce-,Jours prot,eills, are dw t.o soiw coin1n01~ st.iwture which is also lol~~tlt~lniau alid Heiiacerraf (16) havct pro- calized on t*lir alltit)ody-act.ivt~ fragnictlt, of ~,,V-glotmliii ot~t.airirtl by cllzyniic nirt~ltotls posed a model for the structurr of uorrnal (1, I”, I:$). a11c1p:~tl~ological ysS-, y1 ,-, alld ~lu-glotxdills g~rwous and migrated distinctly slower than tlir Kchaiiis of rS,-globulin. Soureduced normal -yl.4-glot~ulit~ showrd souw sigiis of being int~omoge~~rous~~-a poiiit to tw discussed latei.-alld ou the whole n~igyatrd somrwhat faster thau uureduced ilormal r,,S-glotJuliii. After reduct)ion and nlkplatioll, iwrinal yl.4-glot~ulii~ resenlhlrd ttw sinlilart\T twatrd -yY,$-and rl.w-glotx~lins, it1 that it twarnr dissociated into two conlpoiwilts of corrrspo~~ding appcaraucc. The faster compouflit, ~-as elcct~ropliol~et~icall~ clliitck hrtcrogelwous, and its mobility mrrrspondc~d almost rsactlp to that of t,he slowrr part of thr L-chains obtaiiied from I101~n~a1 ~,q,-glotAu, alld to thr c~omparahlr frac*tioll from norn~~l ~l.,&ohutiu. Tlw slowr~~:LII~ much more ahuudaut~ conlponcut was rno~~r honmgeneous aud migrated betwccll tlw H-chains of r,,-globuliu and the cwn~parat~lc fi~agnzcut of ~l.,&~hulin. A slktalt ainoui~t of inaterial was found iu tile sauw arra as t,hc uiltmreatrd normal Y,.,glohuliil aliti inns possibly rrpresent some ~~twlr:l\~~l wsid~w that, had rrsistrd the 1IVnt.llwlrt.
The present resuhs are entirely consistent with Edelman’s and l’ort’cr’s views. In the first place t,he occurrence in normal -yla- and r,,-globulins of material resembling the L-chains of r,,s-globuliu in clectrophorctic mobility, relative amount, and heterogeneity, has been verified, at least, as far as analyses in urea-&arch gels will allow. Secondly, the existence in normal yIA and Y~,,~of a second type of subunit, which is both more abundant and electrophoretically more homogeneous than the faster material, has also been confirmed. Thirdly, as could be predicted, the normal YL-, ylAv,-, and Y,?,~globulins appear to derive their individualities from the different uature of this slowmoving subunit, which in urea-st,arch gel displays electrophoretic mobilit~irs t,hat are characteristic for each class of immune globulin. A similar observation has been rcported by Cohen (17) for paraprot,eins of and yss classes. lciually, paraYlK, YlW, proteins of the ~1~ type do not’ appear to differ much from the normal yla population, as far as their major slow moving subunits are concerned, but seem to obtain their individualities from t’heir fast migrating subunits. The latter are quite homogeneous and their mobilities vary from case t,o case while still remaining in the range covered by the heterogeneous corresponding mat’erial from the uormal yla population. These patterns were obtained for four out of the five y,,-paraproteins here investigated and are the exact replica of the patterns described by Edelman and Poulik (14) in the case of ysstype paraproteins and by Cohen for t’he three classes of paraproteins (17). One unexpected difficulty lies with the obvious electrophoretic multiplicity of bands obtained with unreduced yI,-paraproteins (Fig. 3). Still, the same samples were electrophoretically homogeneous whet1 analyzed by conventional electrophoresis at pH 8.6 (Fig. 1). As already mentioned, some slight contamination with residual normal Y,$~;globulin of similar electrophoretic mobility had to be taken into account, but this could hardly justify the grossly composite pattern furnished by the yl,-paraproteins in ureastarch gels. Kot only were the quantitative discrepancies between the observed ad-
ventitious bands and the presumed yss impurities far too great, but the posit,ions of the former were entirely at variance with the position occupied by uureduced normal y,v,-globulin ill urea-starch gel (Fig. 2). On the other hand, yI,-paraproteins are usually quite heterogeneous upon ultracentrifugation (29), being represented by a set of components with respective sedimentation rates of about 7, 9, 11, 13, and 15 I\’ units. We presume this type of heterogeneit’y to be reflected in the urea-starch gel patterns. Though the molecular sieve effects of such gels appear to be less obvious than those of convent’ional starch gels made in alkaline buffers, they must nevrrtheless he inferred to exist, on account of the poor penetration of unreduced normal TIM-globulin (Fig. 2). Support for this explanation is found in the fact that after exposure to mercaptoethanol, all four yIA components reverted to a much more simplified pattern (1;ig. 3). This is in accordance with the known conversion of ultracentrifugally heterogeneous yIA-paraproteins to single i S material after reduction with mercaptoethanol (X0, 13). The isolation of normal yI,-globulin in a satisfactory state of purity is hard to achieve. The Fample used for the experiment pictured in Fig. 2 was contaminated with small amounts of other serum proteins, mainly albumin and transferrin. This fact could account for the presence of several minor fractions moving ahead of the main material in the urea-starch gel analysis of the unreduced sample. The sample shown in Icig. 3 was much more homogeneous in this respect and may probably be taken as representative of t#ha pattern of pure normal rla-globulin. The method of preparation used (16) always results in the isolation of ~1.~samples showing one major 7 S peak in the ultracent’i,ifugc~, whereas material sedimenting at) higher rates usually accounts for less than 1.5‘b of the total product. It is not known whether these proportions reflect the composit,ion of t,lie normal -yla populabion. At any rate the predominance of 7 S material in t,he two samples here analyzed probably accounts for the observation that the lmrrduced normal y1A, in contrast with the yla-
STRCCTITRE
paraproteins, migrated almost entirely single fast band.
as a
15. FLEISCHMAS, J. B.. PAIN, R. H., ASI) J'OR'L'ER, It. B., Arch. Hiwhem. Hioph!ls. 98, Suppl. 1, 174 (lW2). 16. I
Natl. J.
1, HEREMANS,
F.,
(‘ljn.
C’him.
Acla
4, 639
(1959). J. F., HEREMASS, RI.-TH., II. 15., Plin. Chim. .lctu
2. HEREMANS, S(*HNLTZE,
AND
4, !Ni
8. &:ClIwKy,
Brp!l.
&I.
:2mstrrrd:tIll, 19. HEREMASS.
20.
(1056).
5. (;HABAR, P.. B~xTIN, P., AND QELI~MANN, &I., lieu. Franc. Etudes (‘lin. Niol. 3, 41 (1058). G. HEREMASS, .J. F., ANI) HEREMASS, &l.-'r~., in Proc. Intern. (dower. SW. Hentntol., 7th G’otzgr., Rome, 1958, p. 7. II Pensiero Scientifico, Rorue, 1960. 7. \VALI)ENSTR(I~~, J., in “Progress in IIemntology,” (1~. M. Towntins, ed.), Vol. 3, p. 266. (irune K: Strzttton, Kew York, 19C2. I).,
AND
'I'HORBEC'KE,
(;.
J.,
J.
Med. 114, 171 (l%il).
9. ('ARRONARA, .&. o., ltot~aa1N, J. ;I.. .4NI) TIEREMANS, J. F., ;V~ttcw (in press). 10. HEREMANS, J. F., VAERMAS, J.-P., AND VAER~\IAN, CL., J. Inuxzcnol. 91, (in press). 11. PORTER, 11. It., Hiochem. J. 73, 119 (1959). 12. FR.~NKI,IN, E. C., FIJDENBERB, H., MEI.TzER, I>. Ii. I’roc. .\-atl. hl., *ND STANW~RTH, Alrxzd. Sci. 48, 914 (1962). 13. FIZANKLIS, 15. C., lVulure 195, 393 (1962). 14. EDELMAN, G. hf., AND J'OULIK, nl. I)., J. Expfl. Med. 113, 861 (19til).
.4su IJENACERRAF,
B.. I'roc.
logical Fluids, I’roc. IOtll. (:oII.. lirugrs, 1962, p. 108. 111. I’PP~~I.s. cd.). ISlsevier,
(1959). J. F., “Les glot)ulines sdriques tiu systbne gmmm.” Arsci:t (Brussels) :tnd 1960. Masson (P). 4. SCHEIDEGGER, J.-J., Sc~mainc~ H8p. Paris 32,
(;. AI.,
.-Icnd. Sci. IT. S. 48, 1035 (1962). P. .VU~U,.~ 197, 253 (1963).
17. COHEN, 18. FlEREMANS. .J. I“.. \'AER&IAS, J.-J'., CARBONARA, A. O., I~ODHAIS, J. .\.. ASI) HEREof tile BioMANS, M-TH., in “Protides
3. I~ERE.MANS,
2119
143
OF -,,A-GLOBCLI~S
21. 22. 2':j.
l%i".
J. F., ASD HEREMANS. 31..TH., :trtn Med. Smnd. 169, Suppl. 367, 27 (1961). WIEME, Ii. J., “Studies c,n Ag:w Gel Electrophoresis”. Arsci:t, Brussels, 1959. FAifEY, J. I,., .I. [
24. EDELMAS,
(;. hr.,
AND (;Al,l.~.
J. .I.,
./. E.@l.
Med. 116, 207 (1962). 25. PORTER, 1~. R., “Ttre structure of (~:unn~t. globulins nntl Antitmtlies, itl S!yr~p. /hit, I’roblcms :l:coplasfir~ Ikecrsc~ (A. (:ellhorn :tnd 15. Hirsctltwrg, cd.), (‘~~tunrl)i:~ I-niv. Press, New York, lM2. (in press) 26. FRANKLIN. 15. C., J. (‘lip. Irtocsf. 39, 1933 (1060). 2i.
J':DELMAN, (:. MANS, M.-TII.,
M., HEREMASS, .SND KIINKEL,
J.
I?.,
HERE-
H. (;.. .I. fi’spll.
Med. 112, 203 (1960). Ct. ,\I., J. Esp//. 28. OLINS, 1). It:.. AND GELMAN, filed. 116, fi36 (1962). 39. I,AIJRELL, A. II. E’., .Acta Ned. Scan~tl. 169, Suppl. 367, ri8 (1961). 30. FAIIEY, J. I,., .I. Exptl. Med. 114, 3X5 (1961).
OF
Subunits
Frmr
BIOCHEMISTRY
AND
of Normal
Medical
BIOPHYSICS
and
Depurtwcnt
102,18i-143
Pathological
-1, Cliniques Received
(1963)
TlA-Globulins.
Unizwsitaires November
St. Pierre,
(&-Globulins)’
Louvain,
Belgicrtrl
27, 19ti2
Yormnl human rl,,-(a2,,-),r,nr-(19S,-) and 1,..-(is,-)globulins, as well as four different., electrophoreticaIly isolated rla-paraproteins were analyzed by electzophoresis in starch gels made in pH 3.2 formnte buffer containing 8 JI urea. The proteins had been exposed t,o 8 M urea, used either as such or associated with mercaptoethanol. In the latter case the reduction of disulfide bonds was made irreversible by means of iodoncetamide. The three mumal y components showed fundamentally comparable patterns, indicating the existence of two types of subunits which corresponded to the light and heavy polvpeptide chains described by Edelman for r.$-globulin. The three y conponents resembled each other wit,11 respect to the subunits corresponding t,o t,he light chains and derived their individuality mainl>- from their heavy chains. The four ?,a-paraproteins presented the normal *,,A pattern, except for the much greater homogeneit?; and individual characteristics of their light chains. The findings arc felt to agree with Edelman’s and with Porter’s concepts of the struct,ure of y components.
abundantly produced under the form of a molecularly homogeneous [“monoclonal” One of t’he present writers has put forward (7)] paraprotein. Recently the idea of ylathe concept (1) that the antibody activities globulin as representing the t,hird class of of human serum are carried by three classes of related proteins: the well-known yss-? immune globulins has gained support from the finding that’ it was synthesized in the (rp-,7S,-)globulins and yl.M- (19S,-, &-) globulins, and the recently isolated (2) lymph nodes and spleen (8) and that fluorescent antibodies could trace it back to the Y1.4-(/3zA-)globulins. This view was at first cytoplasm of plasma cells and to the germibased 011 mere circumst’antial evidence, nal centers (9) in precisely the same manner which included the antigenic cross reactions as has been observed for yss- and for Y~,,~between the three types of y components globulins and for antibodies. Confirmation (3)) their associated deficiency in congenital has come from t.he demonstration of specific sgammaglobulinemia (4, 5, 3), their simulantibody act,ivities in purified sampIes of taneous increase in diseases associat’ed with isolated from human serum hypergammaglobulinemia (1, 3)) and the rl,-globulin w. occurrence of three corresponding types of Two different approaches have been paraproteinemias in which any of the t’hree y components could be observed to be over- devised in order to gain insight into the structure of y components One method, 1 This investigation was supported by a Public initiated by Porter (ll), consists in the use Health Service research grant, No. A-1263 (Cl) of prot’eolyt,ic enzymes. Such studies have from t.he U. S. Department of Health, Education, succeeded in localizing the subunits of yssand Welfare, and hy Grant So. 270 from the Fonds globulin t’hat are responsible for its antide la Recherche Scientifique NICdicale, Brussels, genie cross reactions with Y~.~- and Ye,,Belgium. 2 ss = sense stricto. globulins in that fragment of the molecule INTROl>UCTION
137
which is also the carrier of ant’ibody act#ivit’ies (1, 12, 13). The other approach, which consists in cleavage of disulfide bonds in t’hc A pool of normal sera from several blood donors presence of concentrated urea, has been pro- was used to prepare ?I*-globulin according to a posed by Edelman and I’oulik (14). A fractionation method worked out hy one of us relat#ed method was developed by E’leisch- (2, 3, 18). The material proved to he antigenicslly man, Pain, and Porter (15). This type of homogeneous when tested against multivalent antisera and against antisera specific for each of study has provided the base for a structural model of r,,-globulin (IG), which would also tl(e three immune globulins, as well in Ouchterlony plate analyses as in immunoelectrophoresis. account for the cross react’ions between ysS-, yld-, and rlatl-globulins. Confirmatory cvi2. 1’ATHOLOGI(‘AL ~~.&LOBl:LIX\rS dence favoring this model has been prcscntcd C;nrnIlla-lA-type paraproteinemias m-erc diagby Cohen (17), who used the method of nosed by means of immunoclectrophoresis, using a Fleischman and co-workers for a study of set of monospecific and of specifically absorbed paraproteins of t’hc ysS-, ~l.~-, and ylM t#ypes. antisera, as described elsewhrrr (19). All ?,,YThe present paper describes an applicaparaproteins here studied reacted with anti-y,.i t,ion of Edelman and l’oulik’s method of antisera t,hat had been cross-absorbed w-ith m,_..and entirely failed to precipicleavage with mcrcaptoethanol and urea to and ,lar-globulins, tate wit11 anti-r,,or nnti-rlnl-nntiser:l crossand y,q,-globulirw isolated from Y1a-, Yn-, They were thercforc normal hmuan serum, as well as to several ahsorhed with :I,\-globulin. considered to satisfy tllc criteria set forward (19) different YIA-paraproteins. The results will for the definition of I,,\-paraproteins. be interpreted wit’11 respect to their bearing FOUl scra thus classified as ,I<-parnproon the structure of y component,s. t.einemins were submitted to pwparative clectro-
FIG. 1. Electroplloreses isolated rl,4-glotmlins.
of three
rl.\-p:~ra~)roteirIPrrlia
ser:t and of the corresponding
phoresis in agnr gel. The paraprotein bands were cut out, and their proteins were eluted by frcezing, tllawing, crushing, and centrifuging the gels. Ikrt her purification was acllieved 1)~ salting out with 2 dl amnwniwn sulfatr. The isolated lwr:tpr~~tcins were found to be rlect,ropltorrtic:tll~ I~r~n~ogenrous (Fig. 1) when tested l)y higil-volt~:lge elcctroplloresis in apar gel CM). 13). inrrnunorlectroplioretic st.nnd:lrds. tiny amounts of :rssoci:tted ilitpuritics of the san,(: rlect,roplroretic niobilit> r~~1~1tl I)e sl~~wn. ‘I’liey :Lppcared to correspond Illostly to residual normal -, ,.- glol)ulin underlying t II? p:lr:lprotcin fractions.
ra
U-
7 ss
Chro~~~:~t,ogral~l~~ on 1)ICA&~!:-cellulose with elw tion in a gradient of increasing molarity and decreasing pH, xcording to a procedure described by Fahcy M), was used to prepare y-,-globulin from nornlxl Iinm:u~ serum, A pool of the initial fractions of thp effluent proved tu consist of sntigenitally pure 7,;glol)ulin of slow electrophorctic rnol)ility, wlicn test,cti 1.)~ irilnlrlnoclectro~)tl~)r~sis. 4.
SoHM.ZJ~
y~.~-(;L0uI’1Js
A euglol)ulin fr:&ion of norrn:tl Ilunmn serum was srllmlit ted to prPp:tr:ttivc llltr:rcrntrifug:lt,il)n
U-
ra 7 1A
u-
ra 7 llvr
140
CARBONARA
in a sucrose density gradient (22). A pool of the bottom fractions, which were immunoelectrophoresis to consist of globulin. I>etails of this procedure are elsewhere (23).
was made shown by pure y,Mdescribed
5. C~mvAm
ob- PROTEINS WITH &~ERCAPTOXTHANOL AND ITHEA, ANALYSIS OF THEIR SUBUMTS
The procedures here used were essentially those indicated by Edelman and Poulik (14). One per cent (w/v) solutions of proteins were made in 0.1 M borate bufl’er of pH 8.5 containing 8 moles urea/l. The preparations were analyzed either as such or after addition of 0.02 mole mercaptoethanol/l. In the latter case the reaction was stopped by addition of 0.05 mole/l. iodoacetamide after 1 hr. exposure to the reducing agent. The products of cleavage were examined by electrophoresis in starch gels made in formate buffer of pH 3.1 which contained 8 moles urea/l. RESULTS
1. PATTERNS AND
OF SORMAL -yl~-GLOBULINS
y1.4-, yss-
Reduced and nonreduced samples of YW, yss-, and rl.W-glohulins isolated from normal serum were compared on the same starch gel (Fig. 2).
AND
HEREMANS
The pattern obtained for reduced-alkylated normal r,,-globulin is essentially t,hc same as the one described by Edelman anp I’oulik (14). It consists of two major and rather sharp bands of comparatively slow mobility, and of a much lighter diffuse smear of faster moving material showing some signs of partition into two zones. rll compliance with the nomenclature late1 introduced by Edelman and Gaily (24), the fast’er material will here be called “L-chains” (I, = light) and the slower material “FIchains” (H = heavy), on the basis of the molecular weight estimations made by these authors. Whereas the unreduced T1,,.-globulin hardly penetrated into the urea-starch gel, presumably on account of its molecular weight, the same material after reduction and alkylation appeared to be broken down into two major components. The faster component, which was also the less abundant, consisted of electrophoretically heterogeneous material of a mobility and general appearance much the same as those of the L-chains of r,,q-globulin. The slower and more abundant component was more homo-
FIG. 3. Starch-gel electrophoretic analyses of normal rl.k-globulin proteins of ylA-type, as such and reduced-alkylated. Same conditions treated proteins; ~a: reduced and alkylat,ed proteins.
and of four paraas in Fig. 2. U: un-
aud of 13cncr-Jours proteins which would srcni to account for most of the prcsrnt knowledge on the intcrrelatiouship and im niunological functions of these prot,eiiis. I II their model, r,,,-glohuliu is pi&wed as cousisting of ali as yet uusprcified uunibrr of light polypeptidc chaitls (mol. wt. about, 20,000) and all equallv undecided number of hcavirr chaiiis (mol. A. het,weeu .iCj,OOOaud ‘iO,OOO),whicll would hc linked toget~her by disulfide bridges and would bc srparahlt: aft,tr rrductioii by nielcaptt)rt.hailol in the preseucc of urea. The L-chains would, either by thenwtves or in couucctioll wit’h adjoim iug srgnieuts of the H-chains, br rwpousihlr for autibody activitirs and would, iu accordallcc, show a great, tlcat of 1ictrrogerir~it.y when one considers the wtiolc spectrum of Y,~,~nwlrt:ules prweiit ifi ilornlaJ serum. l’araprot.eilw of the ySSclass, whirl1 represrnt siliglc niolrcular species, would therefore iu starct I display honiogrneous IAalds gels, after rrductiou aijd alkylatioll. J+nce.Joncs protrills wonId cousist, of L-chaiils only aud tw the products of iucornpl&e syuthrsis. (‘hains of similar tlatmc would be present ill yw- aud y,.,&ob~dills and would 2. I’.\‘I’1’F:RSS OF ylr-l’IIIII’RC)TEISS account for t,hc observed antigeuic cross Ttrr folw yl,,-paraprot,eins showed siniilai reactious and ant,ihod.v activitirs iu t’he t,hwt: pattcrtw (Vig. 3). coinpoiieut~s of the y systeni. On t,hc: ottrcl So~~wduwd y14-paraproteins migrated as halld, t,tlr H-chains would t.w distinct fol inult~iplc batA in urea-starch gel, although each of tjlw ttuw irrimuiw globulins aud he t twv provrd to br honiogenrous upoil etec- t,tic bcawrs of their autigrtiic ilidividua1it.y. t rophowsis iu pH 8.6 barbital buffer (Fig. 1). hu rswlitially similar ~nodct for the strucI~cdllctiotl-alkylatioll wsult,rd ill the ap- ture of ~,C,-gtot~ulilis has brcll. dwcritwl t)y pc’aralwc of patterns entirely similar t,o t,liosc I’ortrr (25), who tlesigtlatrd the subrurits of tlescritwd by ZCdelrnau aud I’oulik (I 4) fol large alid small niolrcldar sizr as &1 aild M, similarly trrated paraproteins of the yNL Icsprct,ivcly. class! mid by (‘ohen (17) for paraprotciiis of Jt has also hrrl~ foluld that, tlw rlrxynlit I-w~1.~class. Tile major slow fraction showed callJ7 obtained fragnlrut~ of y..%-globutill wtlirh 1it.t.k: diffcwncr in appearawe and mobility carrirs t tw aiitibody activity [called I, rI wit 11 wsprct, to the corrcspoudiug fraction (ll), .I, C (2(i), 01’ S (%)I coiisists of the Lof t~ornml ~l.,-glot~uIiii. In cont.rast, the fast, cliailis plus sonw part, of t#hr H-ctinilis (17, fractiolr now coilsisted of our, two, or three 28). This finding agwrs wrll wits11 the fact vc~y sharp bands with mohilitirs lying iu that the autigcnic cross rractiorw t)et\vc,eii t tw ra~lgr wvrwd by thr fast subunit of tlir thwr y coiupoiirlits, and brtn-rril tlwni ~mw:it y,.,-globulill. alld t.lw 12c~lce-,Jours prot,eills, are dw t.o soiw coin1n01~ st.iwture which is also lol~~tlt~lniau alid Heiiacerraf (16) havct pro- calized on t*lir alltit)ody-act.ivt~ fragnictlt, of ~,,V-glotmliii ot~t.airirtl by cllzyniic nirt~ltotls posed a model for the structurr of uorrnal (1, I”, I:$). a11c1p:~tl~ological ysS-, y1 ,-, alld ~lu-glotxdills g~rwous and migrated distinctly slower than tlir Kchaiiis of rS,-globulin. Soureduced normal -yl.4-glot~ulit~ showrd souw sigiis of being int~omoge~~rous~~-a poiiit to tw discussed latei.-alld ou the whole n~igyatrd somrwhat faster thau uureduced ilormal r,,S-glotJuliii. After reduct)ion and nlkplatioll, iwrinal yl.4-glot~ulii~ resenlhlrd ttw sinlilart\T twatrd -yY,$-and rl.w-glotx~lins, it1 that it twarnr dissociated into two conlpoiwilts of corrrspo~~ding appcaraucc. The faster compouflit, ~-as elcct~ropliol~et~icall~ clliitck hrtcrogelwous, and its mobility mrrrspondc~d almost rsactlp to that of t,he slowrr part of thr L-chains obtaiiied from I101~n~a1 ~,q,-glotAu, alld to thr c~omparahlr frac*tioll from norn~~l ~l.,&ohutiu. Tlw slowr~~:LII~ much more ahuudaut~ conlponcut was rno~~r honmgeneous aud migrated betwccll tlw H-chains of r,,-globuliu and the cwn~parat~lc fi~agnzcut of ~l.,&~hulin. A slktalt ainoui~t of inaterial was found iu tile sauw arra as t,hc uiltmreatrd normal Y,.,glohuliil aliti inns possibly rrpresent some ~~twlr:l\~~l wsid~w that, had rrsistrd the 1IVnt.llwlrt.
The present resuhs are entirely consistent with Edelman’s and l’ort’cr’s views. In the first place t,he occurrence in normal -yla- and r,,-globulins of material resembling the L-chains of r,,s-globuliu in clectrophorctic mobility, relative amount, and heterogeneity, has been verified, at least, as far as analyses in urea-&arch gels will allow. Secondly, the existence in normal yIA and Y~,,~of a second type of subunit, which is both more abundant and electrophoretically more homogeneous than the faster material, has also been confirmed. Thirdly, as could be predicted, the normal YL-, ylAv,-, and Y,?,~globulins appear to derive their individualities from the different uature of this slowmoving subunit, which in urea-st,arch gel displays electrophoretic mobilit~irs t,hat are characteristic for each class of immune globulin. A similar observation has been rcported by Cohen (17) for paraprot,eins of and yss classes. lciually, paraYlK, YlW, proteins of the ~1~ type do not’ appear to differ much from the normal yla population, as far as their major slow moving subunits are concerned, but seem to obtain their individualities from t’heir fast migrating subunits. The latter are quite homogeneous and their mobilities vary from case t,o case while still remaining in the range covered by the heterogeneous corresponding mat’erial from the uormal yla population. These patterns were obtained for four out of the five y,,-paraproteins here investigated and are the exact replica of the patterns described by Edelman and Poulik (14) in the case of ysstype paraproteins and by Cohen for t’he three classes of paraproteins (17). One unexpected difficulty lies with the obvious electrophoretic multiplicity of bands obtained with unreduced yI,-paraproteins (Fig. 3). Still, the same samples were electrophoretically homogeneous whet1 analyzed by conventional electrophoresis at pH 8.6 (Fig. 1). As already mentioned, some slight contamination with residual normal Y,$~;globulin of similar electrophoretic mobility had to be taken into account, but this could hardly justify the grossly composite pattern furnished by the yl,-paraproteins in ureastarch gels. Kot only were the quantitative discrepancies between the observed ad-
ventitious bands and the presumed yss impurities far too great, but the posit,ions of the former were entirely at variance with the position occupied by uureduced normal y,v,-globulin ill urea-starch gel (Fig. 2). On the other hand, yI,-paraproteins are usually quite heterogeneous upon ultracentrifugation (29), being represented by a set of components with respective sedimentation rates of about 7, 9, 11, 13, and 15 I\’ units. We presume this type of heterogeneit’y to be reflected in the urea-starch gel patterns. Though the molecular sieve effects of such gels appear to be less obvious than those of convent’ional starch gels made in alkaline buffers, they must nevrrtheless he inferred to exist, on account of the poor penetration of unreduced normal TIM-globulin (Fig. 2). Support for this explanation is found in the fact that after exposure to mercaptoethanol, all four yIA components reverted to a much more simplified pattern (1;ig. 3). This is in accordance with the known conversion of ultracentrifugally heterogeneous yIA-paraproteins to single i S material after reduction with mercaptoethanol (X0, 13). The isolation of normal yI,-globulin in a satisfactory state of purity is hard to achieve. The Fample used for the experiment pictured in Fig. 2 was contaminated with small amounts of other serum proteins, mainly albumin and transferrin. This fact could account for the presence of several minor fractions moving ahead of the main material in the urea-starch gel analysis of the unreduced sample. The sample shown in Icig. 3 was much more homogeneous in this respect and may probably be taken as representative of t#ha pattern of pure normal rla-globulin. The method of preparation used (16) always results in the isolation of ~1.~samples showing one major 7 S peak in the ultracent’i,ifugc~, whereas material sedimenting at) higher rates usually accounts for less than 1.5‘b of the total product. It is not known whether these proportions reflect the composit,ion of t,lie normal -yla populabion. At any rate the predominance of 7 S material in t,he two samples here analyzed probably accounts for the observation that the lmrrduced normal y1A, in contrast with the yla-
STRCCTITRE
paraproteins, migrated almost entirely single fast band.
as a
15. FLEISCHMAS, J. B.. PAIN, R. H., ASI) J'OR'L'ER, It. B., Arch. Hiwhem. Hioph!ls. 98, Suppl. 1, 174 (lW2). 16. I
Natl. J.
1, HEREMANS,
F.,
(‘ljn.
C’him.
Acla
4, 639
(1959). J. F., HEREMASS, RI.-TH., II. 15., Plin. Chim. .lctu
2. HEREMANS, S(*HNLTZE,
AND
4, !Ni
8. &:ClIwKy,
Brp!l.
&I.
:2mstrrrd:tIll, 19. HEREMASS.
20.
(1056).
5. (;HABAR, P.. B~xTIN, P., AND QELI~MANN, &I., lieu. Franc. Etudes (‘lin. Niol. 3, 41 (1058). G. HEREMASS, .J. F., ANI) HEREMASS, &l.-'r~., in Proc. Intern. (dower. SW. Hentntol., 7th G’otzgr., Rome, 1958, p. 7. II Pensiero Scientifico, Rorue, 1960. 7. \VALI)ENSTR(I~~, J., in “Progress in IIemntology,” (1~. M. Towntins, ed.), Vol. 3, p. 266. (irune K: Strzttton, Kew York, 19C2. I).,
AND
'I'HORBEC'KE,
(;.
J.,
J.
Med. 114, 171 (l%il).
9. ('ARRONARA, .&. o., ltot~aa1N, J. ;I.. .4NI) TIEREMANS, J. F., ;V~ttcw (in press). 10. HEREMANS, J. F., VAERMAS, J.-P., AND VAER~\IAN, CL., J. Inuxzcnol. 91, (in press). 11. PORTER, 11. It., Hiochem. J. 73, 119 (1959). 12. FR.~NKI,IN, E. C., FIJDENBERB, H., MEI.TzER, I>. Ii. I’roc. .\-atl. hl., *ND STANW~RTH, Alrxzd. Sci. 48, 914 (1962). 13. FIZANKLIS, 15. C., lVulure 195, 393 (1962). 14. EDELMAN, G. hf., AND J'OULIK, nl. I)., J. Expfl. Med. 113, 861 (19til).
.4su IJENACERRAF,
B.. I'roc.
logical Fluids, I’roc. IOtll. (:oII.. lirugrs, 1962, p. 108. 111. I’PP~~I.s. cd.). ISlsevier,
(1959). J. F., “Les glot)ulines sdriques tiu systbne gmmm.” Arsci:t (Brussels) :tnd 1960. Masson (P). 4. SCHEIDEGGER, J.-J., Sc~mainc~ H8p. Paris 32,
(;. AI.,
.-Icnd. Sci. IT. S. 48, 1035 (1962). P. .VU~U,.~ 197, 253 (1963).
17. COHEN, 18. FlEREMANS. .J. I“.. \'AER&IAS, J.-J'., CARBONARA, A. O., I~ODHAIS, J. .\.. ASI) HEREof tile BioMANS, M-TH., in “Protides
3. I~ERE.MANS,
2119
143
OF -,,A-GLOBCLI~S
21. 22. 2':j.
l%i".
J. F., ASD HEREMANS. 31..TH., :trtn Med. Smnd. 169, Suppl. 367, 27 (1961). WIEME, Ii. J., “Studies c,n Ag:w Gel Electrophoresis”. Arsci:t, Brussels, 1959. FAifEY, J. I,., .I. [
24. EDELMAS,
(;. hr.,
AND (;Al,l.~.
J. .I.,
./. E.@l.
Med. 116, 207 (1962). 25. PORTER, 1~. R., “Ttre structure of (~:unn~t. globulins nntl Antitmtlies, itl S!yr~p. /hit, I’roblcms :l:coplasfir~ Ikecrsc~ (A. (:ellhorn :tnd 15. Hirsctltwrg, cd.), (‘~~tunrl)i:~ I-niv. Press, New York, lM2. (in press) 26. FRANKLIN. 15. C., J. (‘lip. Irtocsf. 39, 1933 (1060). 2i.
J':DELMAN, (:. MANS, M.-TII.,
M., HEREMASS, .SND KIINKEL,
J.
I?.,
HERE-
H. (;.. .I. fi’spll.
Med. 112, 203 (1960). Ct. ,\I., J. Esp//. 28. OLINS, 1). It:.. AND GELMAN, filed. 116, fi36 (1962). 39. I,AIJRELL, A. II. E’., .Acta Ned. Scan~tl. 169, Suppl. 367, ri8 (1961). 30. FAIIEY, J. I,., .I. Exptl. Med. 114, 3X5 (1961).