MARCH 1974
`
`LIBRARY
`
`VOLUME 71
`
`NUMBER 3
`
`(fl
`0
`~
`Q
`
`PROCEEDINGS OF THE
`
`National
`Academy of
`Sciences
`
`:> R R l1r11 27 ·{'I
`
`OF THE UNITED STATES OF AMERICA
`
`PFIZER EX. 1092
`Page 1
`
`

`

`THE PROCEEDINGS OF THE
`National
`Academy of
`Sciences
`OF THE UNITED STATES OF AMERICA
`
`Officers
`of the
`Academy
`
`Editorial Board
`of the
`Proceedings
`
`PHILIP HANDLER President
`SAUNDERS :\lAc LANE Vice President
`ALLEN V. ARTIX II ome Secretary
`HARRISON BROWN Foreign Secretary
`E. R. PIORE Treasurer
`
`RoBERT L. SINSHEIMER Chairman
`RoBERT :\1. SoLOw Vice Chairman
`:\1ICHAEL KASHA Vice Chairman
`ALLEN V. AsTIN II ome Secretary
`liARRISOX BROWN Foreign Secretary
`E. R. PIORE Treasurer
`c. B. ANFINSJ<;;\1
`ALEXANDER G. BEARN
`P. D. BoYER
`BJ<;RNARD D. DAVIS
`KINGSLEY DAVIS
`HARRY EAGLJ,
`H I"Rl\IAN EISEN
`:\lARK KAC
`
`.:\IARTIN D. KAMEN
`HE:\RY s. KAPLAX
`SEY!'.WUR S. KETY
`:\1ACLYN :\IcCARTY
`EUGEKE P. 0DUM
`ALEXA!IIDER RICH
`PAUL A. SAMUELRON
`
`.Managing Editor: PATRICIA ZEIS THOMA!:;
`.:\IuRRU: W. BuRGAN
`Senior Associate Jfanaging Editor:
`Assistant .lf anaging Editor: GARY T. CocKs
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`PFIZER EX. 1092
`Page 2
`
`

`

`Nat. Acad. Sci. USA
`71, No. 3, pp. 845-848, March 1974
`
`Region Sequences of Five Human Immunoglobulin Heavy Chains
`the V Hill Subgroup: Definitive Identification of Four Heavy
`H ypervariable Regions
`
`lllerlartlmerlt of Microbiology, Mount Sinai School of Medicine of the City University of New York, 10 East 102 Street, New York, N.Y. i0029
`
`kappa) , and Tur (IgA l kappa) were isolated from serum or
`plasma by zone electrophoresis on polyvinyl copolymer
`("Pevicon") (8). After further riurification by gel filtration
`chromatography, they were reduced with 0.1 M 11-mercapto(cid:173)
`ethanol and alkylated with
`iodoacetamide. The heavy
`and light chains were separated by gel filtration in propionic
`acid (9, 10).
`
`Fragment Preparation: Heavy chains were treated with
`cyanogen bromide (11) and the resulting individual frag(cid:173)
`ments purified by gel filtration chromatogra phy in 5 M guani(cid:173)
`dine · H CL Three proteins (Tei, Za p, and Tur) yielded a
`large N-terminal fragment comprising residues 1-85. Pro(cid:173)
`teins Was and Jon, which contain a methionine residue at
`position 34, gave fragments comprising residues 1-34 arid
`35-85. Since air human IgG myelomas have a methionine at
`position 252, Tei and Was yielded a large fragment comprising
`·residues 86- 252. In protein Jon, however, an additional
`methionine was present at position 111. Consequently two
`distinct fragments comprising residues 86-111 and 112-252
`were obtained from this protein. !gAl proteins contain a
`methionine at residue 426 (12) so proteins Zap and Tur both
`yielded a very large fragment composed of residues 86-426.
`
`Sequencing Procedure. Positions 1-85: On both the intact
`heavy chain as well as on the 1-85 fragm ent, proteins Tei,
`Zap, and Tur were sequenced 60 steps on the automated se(cid:173)
`quencer (13, 14). Tryptic peptides were prepared and sepa(cid:173)
`rated on Dowex 50 X 4 with a pyridine-formate buffer system.
`In proteins Tei and Zap two invariant peptides were aligned
`by homology alone (70- 74 and 75- 78) while in protein Tur,
`
`6 .0
`
`1234 56
`H I H H
`I
`I
`
`7
`t--<
`
`8
`, H
`
`10
`H
`
`The variable regions of five human immu(cid:173)
`ll"'!ovuuuu heavy chains of the VHIII subgroup have been
`se(JU4~n4~ed. Tluee of the heavy chains belonged to
`class and two to the lgA class. Examination of
`sequences, and comparison with additio'nal pub(cid:173)
`heavy chain sequences, showed that a total of four
`.-JIYilf!rva
`regions is characteristic of human heavy
`variable regions.
`The relatively conserved character of large segments of
`heavy chain variable region was very evident in these
`. lllu([ies. The conserved segments, which are those sections
`.
`lloeated outside the hypervariab]e regions, comprise ap(cid:173)
`• prv;uu'"·'·"'Y 65 % of the total heavy chain variable region.
`general structural pattern for antibody
`IIIOioecutes emerges from this and related s tudies: an over(cid:173)
`combining region superstructure is provided by the
`conserved segments while the t·efi netnents of the
`aetive site specificity at·e a function of hypervariable re-
`
`.
`
`The antibody combining site is now believed to reside ex(cid:173)
`• ••h"''vPiv in the variable regions of the heavy and light poly(cid:173)
`peptide chains of the immunoglobulin molecule. Evidence
`is accumulating from several laboratories which indicates
`that hypervariable regions within the variable region are
`directly involved in the antibody combining site as well as
`being responsible, at least in part, for the idiotypic determi(cid:173)
`nants of myeloma proteins and specific antibodies (1-5).
`The existence of three hypervariable regions in the vari(cid:173)
`able region of human immunoglobulin heavy chains has been
`established by previous studies from this laboratory. R esi(cid:173)
`dues 31-37 were described as the first hypervariable region
`of the heavy chain (6), and, after fragmentation of IgG
`heavy chains with cyanogen bromide, two additional hyper(cid:173)
`variable regions were localized between residues 86-91 and
`101-110 (7).
`We have now completed the am ino acid sequence from
`residues 41 to 84 of the three VHIII proteins originally re(cid:173)
`ported (6, 7) as well as the complete V region sequence of
`two IgA proteins with V Hill variable regions. The data
`make apparent an additional area of sequence hypervari(cid:173)
`ability between residues 51 and 68, thus supporting the ob(cid:173)
`servations of Cebra et al. made on pooled guinea-pig immuno(cid:173)
`globulins (5). When these data on VHIII proteins are in(cid:173)
`cluded with that available for proteins of the VHI and VHII
`subgroups and analyzed by the method of Wu and Kabat (3) ,
`four distinct areas of sequence hypervariability are observed.
`
`MATERIALS AND METHODS
`
`Myeloma Proteins. Tei (IgGl kappa, Gm az), Was (IgGl
`kappa, Gm az) , Jon (lgG3 lambda, Gm g), Zap (IgAl
`
`845
`
`w
`~ 5 .0
`w
`u
`::l 4 .0
`"' 0
`~ 3 .0
`...
`~ 2 .0
`~ I .0
`
`a:
`
`·
`
`~ J .
`
`'..-... _j
`..
`70
`80
`
`f
`•
`rf·, , .. i
`0 ~~L-
`10
`0
`
`20
`
`', V \
`'._1 ....._..
`50
`60
`40
`3 0
`TUBE NUMBER
`FIG. 1. Representative ion exchange chromatogram of tryptic
`hydrolysate of the amino terminal (1-85) cyanogen bromide
`fragment of a chain from protein Tur. Peptides were isolated
`from a Dowex 50X4 column and characterized and analyzed as
`described in the text.
`
`PFIZER EX. 1092
`Page 3
`
`

`

`Tei
`
`Was
`
`846
`
`Immunology : Capra and Kehoe
`
`10
`GLU VAL GLN LEU VAL GLU SER GLY GLY GLY LEU VAL GLN PRO GLY GLY SER LEU ARG LEU SER CYS ALA ALA S!R
`
`Proc. Nat. Acad. Sci. USA 71 (1 974)
`20
`
`LEU•---------------------------------------------------------------------------
`
`Jon
`
`ASP ______ ~--------------------------------~LYS. __________________________________________ ___
`
`Zap
`
`Tur
`
`Tei
`
`was
`
`Jon
`
`Zap
`
`------------~LEU• __________________________________________________________________ __
`
`ALA. ____________________________ GLY ________________________ __
`
`30
`GLY PRE THR PHE SER THR SER ALA VAL TYR [
`
`40
`~
`J TRP VAL ARG GLN ALA PRO GLY LYS GLY LEU GLU TRP VAL
`
`------ SER - - - - - ASP ___ MET ___ [
`
`ALA TRP MET L YS
`------------------~
`
`----------------------THR SER ARG PRE
`
`J ----~----------------------------­
`] __ ~------------------------------­
`]~----------------~---------------
`
`Tur - - - - - - - - - - - - - 'ARG VAL LEU SER SER [
`
`Tei
`
`was
`
`70
`60
`GLY TRP ARG TYR GLU GLY SER SER LEU THR HIS TYR ALA VAL SER VAL GLN GLY ARG PRE THR ILE SER ARG ASII
`PRE
`
`ALA
`
`LYS
`
`GLN GLU ALA
`
`ASN SER
`
`ASP THR
`
`ASN ----------------- ---
`
`Jon
`
`VAL -------VAL
`
`GLN VAL VAL GLU LYS ALA PHE
`
`ASN - - - - - ASN - - - - - - - - - - - - - - - ---- - -
`
`Zap
`
`GLU PHE
`
`VAL GLN
`
`ALA ILE SER - - - - - - - - - ASP - - - - - - - - - ALA ------------------ ----
`
`Tur
`
`SER GLY
`
`LEU ASN ALA - - - - - ASN LEU
`
`PHE - - - - - - - ALA - - - - - - - - - - - - - - -- ----
`
`Tei
`
`90
`~00
`80
`ASP SER LYS ASN THR LEU TYR LEU GLN MET LEU SER LEU GLU PRO GLX ASX THR ALA VAL TYR TYR CYS ALA ARG
`
`Was ---~------------------ ASN ARG
`
`ALA - - - - - - - - - - - - - - - - - -- - ----
`
`------------------------------------- ASN THR GLY
`
`ILE
`
`VAL THR ---------------------------------------
`ALA ----------------------~~---~-
`
`------------------------------------------------ GLN ALA - - - - - - - - - - - - LEU - - - - - - - ------
`
`110
`120
`VAL THR PRt> ALA ALA ALA SER LEU THR PRE SER ALA VAL TRP GLY GLN GLY THR LEU VAL THR
`
`PHE ARG GLN PRO PRE VAL GLN
`
`PHE ASP
`
`PHE
`
`VAL VAL SER THR
`
`SER MET ASP
`
`THR ARG
`
`GLY GLY TYR
`
`ASP
`
`Tur
`
`LEU SER VAL THR
`
`VAL
`
`ALA PHE ASP
`
`PRO
`
`LYS
`
`SER
`
`SER
`
`FIG. 2. The amino-acid sequence of the variable regions of five human immunoglobulin heavy chains.
`
`the isolation of chymotryptic peptides established the se(cid:173)
`quence unambiguously. In all cases, tryptic peptides were
`sequenced in the automated sequencer, often using 4-sulfo(cid:173)
`phenylisothiocyanate (Pierce Chemical) on the lysine pep(cid:173)
`tides (15). In proteins Was and Jon, which contained cyano(cid:173)
`gen bromide fragments 1- 34 and 35- 85, the first 60 residues
`were established by automated sequencing of the intact
`heavy chain. Thus,
`in these
`two proteins, sequencing
`cyanogen bromide fragm ent 1-34 was superfluous since its
`composition agreed with the previously determined sequence.
`
`Fragment 35-85 of proteins Was and Jon was sequenced 35
`and 40 residues respectively; this, together with the G(cid:173)
`terminal tryptic peptides mentioned above gave the co~­
`plete sequence for this section. Residues 86- 121: In proteiUS
`Tei, Was, Zap, ahd Tur the sequence was established by a
`continuous automated run of 45 steps from residue· 86 int~
`t he CHI domain. In both Zap and Tur, tryptic digestion an
`isolation of t he resulting peptides confirmed a few ques~ion;
`able posit ions. In protein J on, residues 86- 111 were obtaul~
`disulfide linked to residues 1- 34 after cyanogen brorni e
`
`Jon
`
`Zap
`
`Tur
`
`Tei
`
`Was
`
`Jon
`
`Zap
`
`PFIZER EX. 1092
`Page 4
`
`

`

`Nat. A cad. Sci. UBA 71 (1 974)
`
`Human Immunoglobulin Heavy Chains
`
`847
`
`This sequence was obtained by difference since
`1- 34 were known from the initial study of the
`heavy chain. Jon fragment 112-253 was subjected to a
`sequencer run which definitely established the sequence
`residues 112-121 as well as providing sequence data into
`CHI domain.
`
`Jon Exchange Chromatography . An example of a Dowex
`4 chromatogram is shown in Fig. 1 for a tryptic digest
`the Tur 1-85 fragment; 6.5-ml fractions were collected and
`ml of each fraction analyzed by the fiuorescamine pro(cid:173)
`initially described by Udenfriend et al. (16). Ninhydrin
`was also performed after alkaline digestion of 0.5-ml
`In most analyses, only the fluorescamine procedure
`employed since it was much more sensitive. As shown in
`1, 10 fractions were pooled. Each was subj ected to amino(cid:173)
`analysis and several useful peptides were isolated and
`~~>nlum .r.ert . T-1 (Asn Thr Leu T yr Leu Gin Hsr) (79- 85) , T-3
`Asp Ser Lys) (75-78), T-7 (Gly Leu Gly Trp Val Ser
`Arg) (46- 53) , and T-10 (Phe Thr Ile Ser Arg) (70- 74).
`
`RESULTS AND DISCUSSION
`amino-acid sequences of the variable regions of the
`human myeloma proteins is displayed in Fig. 2. The
`factor values determined by the method of Wu
`Kabat (3) for these as well as all the other human V
`sequences available is shown in Fig. 3. These calcula-
`were based on 25 sequences from residues 1 to 34, 11
`from residues 35 to 85, and 14 sequences from
`86 to 122. Previous to this study there were only six
`"'uu"11"u complete V region sequences, all but one (Nie) of
`(Eu) or VHII (Daw, Cor, He, Ou) subgroup (for
`IW!fPrPni'.P<: see legend to Fig. 3). With five additional VHIII
`........ ~ .. ~~~ the variability within and between subgroups can
`be compared more meaningfully. In addition, with the
`~··•ua. uuH_y of 11 complete sequences and several fragments,
`Wu- Kabat plot becomes more statistically significant.
`A discussion of the sequences can be conveniently divided
`those sections of the V region which are relatively con(cid:173)
`(1- 30, 38- 50, 69-83, 92- 100, and 111- 121), and the
`regions (3 1- 37, 51- 68, 84- 91, and 101- 110).
`About 65% of the variable region of the heavy chain shows
`variation. In fact , there are 17 positions (14%) which
`been absolutely invariant in all human heavy chains
`of their V region subgroup assignment. Certain
`are subgroup specific since at these positions all,
`nearly all, of the members of one subgroup have a particular
`acid, while members of th e other subgroup contain a
`·
`amino acid . Utilizing the four available VHII
`, positions 3, 9,16, 17, 19, 21, 23, 28, 29, 39, 42, 46, 50,
`and 82 appear to be subgroup specific. As noted
`ucev'n'11" ''", no subgroup specific residues are identifiable in
`C terminal portion of the V region (7) . There are thus
`positions (27%) in the V region which are either invariant
`or subgroup specific. A comparison with the published se(cid:173)
`of myeloma proteins (17, 18), pooled immuno(cid:173)
`globulins (5, 19, 20), and specifically purified antibodies (5,
`21-23) from lower species, indicates that the particular
`arnino acids found at t hese positions are characteristic of a
`Wide variety of mammals and have been faithfully conserved
`during evolution. Such residues may have extremely im(cid:173)
`Portant attributes for variable region fun ction such as, for
`
`POS ITIO N
`
`Fm. 3. Variability-factor values for the sequences shown in
`Fig. 1 as well as several other published sequences (36) deter(cid:173)
`mined according to the method of Wu and Kabat (3).
`
`example, the provision of a distinct backbone structure which
`is crucial to antibody function.
`As can be seen on inspection of Figs. 2 and 3, about a third
`of the heavy chain variable region can be considered "hyper(cid:173)
`variable." These regions deserve special consideration be(cid:173)
`cause of their specific implications for the formation of the
`antibody combining site, the nature of idiotypic determinants,
`and various theoretical conceptions of the origin of antibody
`diversity.
`In light chains, affinity labels have been localized near or
`within hypervariable regions (23- 25), thus providing direct
`support for the general concept that hypervariable regions
`participate directly in the antibody-combining site. For the
`heavy chain, recent work has also been consistent with this
`idea. For example, Ray and Cebra localized affinity labels to
`the first (31-37) and the fourth (101-110) heavy chain
`hypervariable regions (26), Haimovich et al. (27) localized
`an affinity label to residue 54 of the mouse myeloma protein
`315 (which has anti-dinitrophenol activity), and Press and
`coworkers have localized affinity labels at or near the fourth
`hypervariable region in rabbit antibodies (28). Therefore,
`although the primary structure and affinity labeling studies
`of these proteins was being carried out independently, and
`even in different laboratories in many instances, there is a gen(cid:173)
`eral implication from the experimental observations that the
`same regions of the molecule which show the highest degree
`of sequence variation are near or part of those particular re(cid:173)
`gions of the heavy chain where affinity labels have been local(cid:173)
`ized.
`A second piece of evidence linking the antibody combining
`site to the hypervariable regions has come from comparisons
`of sequences obtained from pooled immunoglobulin heavy
`chains with those of specifically purified antibody heavy
`chains. Sequence analyses of rabbit (29), guinea pig (5), and
`other mammalian heavy chain pools (19), indicate that a
`definitive sequence cannot be obtained within those regions
`which have been identified as hypervariable on the basis
`of studies with myeloma proteins. However, when specifically
`purified antibodies are studied, a single major sequence can be
`determined, as has been shown most definitively by Cebra and
`his coworkers (5).
`Additional support for the functional significance of hyper(cid:173)
`variable regions has been provided by current notions con(cid:173)
`cerning the tertiary structure of the immunoglobulin mole(cid:173)
`cule. Crystallographic analysis of human immunoglobulins
`has now advanced to the point where it has been possible
`to assign the residues which may line a " pocket" within the
`
`PFIZER EX. 1092
`Page 5
`
`

`

`848
`
`Immunology : Capra and Kehoe
`
`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`immunoglobulin molecule which presumably represents the
`combining site itself (30, 31). In each instance, the major
`residues which line the pocket are associable with hyper(cid:173)
`variable regions.
`In addition, the conformational models
`generated by the nearest neighbor calculations of Kabat and
`Wu (32) place hypervariable regions in close association
`with the putative combining site.
`There is also growing evidence that at least some of the
`hypervariable regions are involved in the idiotypic determi(cid:173)
`nants of myeloma proteins and antibodies. Cross idiotypic
`specificity among the cold agglutinins (33) and the anti(cid:173)
`gamma globulins (34) is believed to be related to the com(cid:173)
`bining site. In at least two distinct anti-gamma globulin
`molecules, the hypervariable regions show striking sequence
`similarities (7, 35).
`The genetic origin of hypervariable regions remains un(cid:173)
`clear. The variability within heavy chain hypervariable
`regions seems more marked than that of light chain hyper(cid:173)
`variable regions. Of the 11 proteins which have now had their
`V regions completely sequenced, if one considers the 43
`hypervariable positions of the heavy chain, there are no two
`proteins which have more than 10 residues in common. It
`seems likely that hundreds, or even thousands, of proteins
`would have to be sequenced in order to find two which are
`identical if no. preselection bias (such as selection by idiotypic
`antisera or for combining specificity) is involved. This im(cid:173)
`plies either that there are a very large number of germ
`line genes or that somatic processes are necessary to explain
`the diversity in the heavy chain hypervariable regions.
`Regardless of their origin, the hypervariable regions clearly
`play a crucial role in the antigen binding function of immuno(cid:173)
`globulin molecules.
`We thank Dr. Henry Kunkel for the subclass and genetic typ(cid:173)
`ing of the myeloma proteins. Bonnie Gerber, Ellen Bogner and
`Donna Atherton rendered invaluable technical assistance. This
`work was aided by grants from the National Science Foundation
`(GB 17046) and the U.S. Public Health Service (AI 09810) and a
`Grant-in-Aid from the New York Heart Association. J .D.C. is the
`recipient of National Institutes of Health Career Development
`Award 6-K4-GM-35, and J .M.K. is an Established Investigator
`of the American Heart Association.
`1. Milstein, C. (1967) Nature 216, 330-332.
`2. Franek, F. (1969) Symposium on Developmental Aspects of
`Antibody Formation and Structure, Prague.
`3. Wu, T. T . & Kabat, E. A. (1970) J . Exp. Med. 132, 221-250.
`4. Capra, J .D., Kehoe, J . M., Winchester, R. & Kunkel, H. G.
`(1971) Ann. N .Y. Acad. Sci. 190, 371-381.
`5. Cebra, J. J ., Ray, A., Benjamin, D. & Birshtein, B. (1971)
`Progr. Immunol. (First International Congress of Immunol(cid:173)
`ogy), 269-284.
`6. Capra, J.D. (1971) Nature New Bioi. 230, 61-63.
`7. Kehoe, J. M. & Capra, J . D. (1971) Proc. Nat. Acad. Sci.
`USA 68, 2019-2021.
`
`15.
`
`8. Kunkel, H. G. (1954) Methods Biochem. Anal. 1, 141- 155.
`9. Fleischman, J. G., Porter, R. R. & Press, E. M. (1963)
`Biochem. J . 88, 220-228.
`10. Capra, J . D. & Kunkel, H. G. (1970) J. Clin. I nvest. 49
`610-621.
`'
`11. Gross, E. & Witkop, B. (1962) J . Bioi. Chem. 237, 1856-
`1863.
`12. Chuang, C. Y., Capra, J.D. & Kehoe, J. M. (1973) N ature
`244, 158-160.
`13. Edman, P. & Begg, F. (1967) Eur. J. Biochem. 1, 80-91.
`14. Capra, ,J.D. & Kunkel, H. G. (1970) Proc. Nat. A cad. Sci.
`USA 67,87-92.
`Inman, J. K., Hannon, J . E. & Appella, E. (1972 ) Biochem.
`Biophys. Res. Commun. 46, 2075-2081.
`16. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leim(cid:173)
`gruber, W. & Wiegele, M. (1972) Science, 178, 881-882.
`17. Kehoe, J . M. & Capra, J . D. (1972) Proc. Nat. A cad. Sci.
`USA 69, 2052-2055.
`18. Bourgois, A., Fougereau, M. & de Preval, C. (1972 ) Eur. J.
`Biochem. 24, 446-455.
`19. Capra, J .D., Wasserman, R. W. & Kehoe, J . M. (1973) J.
`Exp. Med. 138,410-427.
`20. Mole, L. E., Jackson, S. A., Porter, R. R. & Wilkinson, J. M.
`(1971 ) Biochem. J. 124, 301-318.
`21. Fleischman, J . B. (1973) Immunochemistry 10, 401-407.
`22. Strosberg, A. D., Jaton, J . C., Capra, J. D. & Haber, E.
`(1972) Fed. Proc. 31, 771.
`23. Goetzl, E. J. & Metzger, H. (1970) Biochemistry 9, 1267-
`1278.
`24. Franek, F. (1971) Eur. J. Biochem. 19, 176-183.
`25. Chesebro, B. & Metzger, H. (1972) Biochemistry 11 , 766-
`771.
`26. Ray, A. & Cebra, J. J . (1972) Biochemistry 11, 3647-3657.
`27. Haimovich, J., Eisen, H. N., Hurwitz, E. & Givol, D. (1972)
`Biochemistry 11, 2389-2397.
`28. Press, E. M., Fleet, G. W. J . & Fisher, C. E. (1971) in
`Progress in Immunology, ed. Amos, B. (Academy Press,
`New York), p. 233.
`29. Cebra, J . J ., Givol, D. & Porter, R. R. (1968) Biochem. J.
`107, 69-70.
`30. Schiffer, M., Girling, R. L., Ely, K . R. & Edmundson, A. B.,
`personal communication.
`31. Poljak, R. J . (1973) Abstracts of Ninth International Con(cid:173)
`gress of Biochemistry-Stockholm, p. 31.
`32. Kabat, E. A. & Wu, T . T . (1972 ) Proc. Nat. Acad. Sci. USA
`69' 960-964.
`33. Williams, R. C., Kunkel, H. G. & Capra, J. D. (1968)
`Science 161, 379-381.
`34. Kunkel, H. G., Agnello, V., Joslin, F. G., Winchester, R. J.
`& Capra, J .D. (1973) J. Exp. Med. 137,331-342.
`35. Capra, J . D. & Kehoe, J. M., unpublished observations.
`36.
`(a) Edelman, G. M., Cunningham, B. A., Gall, W. E., Gott(cid:173)
`lieb, P. D., Rutishauser, U., & Waxdal, M. J. (1969) Proc.
`Nat. Acad. Sci. USA 63, 78-85; (b) Press, E. M. & Hogg, N.
`M. (1970) Biochem. J. 117, 641-660; (c) Cunningham, B. A.,
`Pflumm, M. N., Rutishauser, U. & Edelman, G. M. (1969)
`Proc. Nat. Acad. Sci. USA 64, 997-1003; (d ) Wikler, M.,
`Kohler, H., Shinoda, T . & Putnam, F. W. (1969) Science 163,
`75-78; (e) Ponstingl, H., Schwarz, J., Reichel, W. & Hilsch(cid:173)
`mann, N.(1970) Hoppe-Seyler's Z. Physiol. Chem. 351, 1591.
`
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