THE COVALENT STRUCTURE OF
`AN ENTIRE TG IMMUNOGLOBULIN MOLECULE*
`BY GERALD M. EDELMAN, BRUCE A. CUNNINGHAM, W. EINAR GALL,
`PAUL D. GOTTLIEB, URS RUTISHAUSER, AND MYRON J. WAXDAL
`
`THE ROCKEFELLER UNIVERSITY
`Communicated by Theodore Shedlovsky, March 21, 1969
`Abstract.-The complete amino acid sequence of a human yG1 immunoglobu-
`lin (Eu) has been determined and the arrangement of all of the disulfide bonds
`Comparison of the sequence with that of another myeloma
`has been established.
`protein (He) suggests that the variable regions of heavy and light chains are
`homologous and similar in length. The constant portion of the heavy chain con-
`tains three homology regions each of which is similar in size and homologous to
`the constant region of the light chain. Each variable region and each constant
`The half-cystines
`homology region contains one intrachain disulfide bond.
`participating in the interchain bonds are all clustered within a stretch of ten
`residues at the middle of the heavy chains.
`These data support the hypothesis that immunoglobulins evolved by gene
`duplication after early divergence of V genes, which specified antigen-binding
`functions, and C genes, which specified other functions of antibody molecules.
`Each polypeptide chain may therefore be specified by two genes, V and C, which
`The internal homol-
`are fused to form a single gene (translocation hypothesis).
`ogies and symmetry of the molecule suggest that homology regions may have
`similar three-dimensional structures each consisting of a compact domain which
`contributes to at least one active site (domain hypothesis).
`Both hypotheses
`are in accord with the linear regional differentiation of function in antibody
`molecules.
`
`Antibodies or immunoglobulins can interact with a wide range of different
`antigenic determinants and, after specific binding to an antigen, they play a
`The
`fundamental part in physiological functions of the immune response.
`specificity of antigen binding depends ultimately upon amino acid sequences of
`It is the diversity of these
`the variable or V regions of antibody molecules.
`sequences which results in the range of specificities required for a selective im-
`In contrast, other regions of the antibody molecule have rela-
`mune response.
`Like
`tively constant sequences and are responsible for physiological functions.
`enzymes, these C regions appear to have evolved for a restricted set of interac-
`This unusual picture of intramolecular differentiation has emerged from
`tions.
`studies of the structure of immunoglobulins from different animal species.' To
`date, only portions of immunoglobulin molecules have been subjected to amino
`acid sequence determination.
`We now report the amino acid sequence of an entire human 'yG1 immuno-
`globulin (molecular weight 150,000), the location of all disulfide bonds, the
`arrangement of light and heavy chains, and the length of the heavy chain V
`region.
`
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`79
`
`Materials and Methods.-The isolation of the myeloma protein Eu2 and the preparation
`Similar methods were used for the isola-
`of its CNBr fragments3' 4have been described.
`tion of the -yG1 myeloma p)rotein He and for the preparation of its CNBr fragments.
`We have previously described the methods used for enzymatic digestion with trypsin,
`chymotrypsin, and pepsin, gel filtration, ion exchange chromatography, high voltage paper
`electrophoresis, determination of NH2-terminal and COOH-terminal residues, amino acid
`analysis, and determination of amino acid sequences by the dansyl-Edman procedure.4-8
`The positions of glutamine and asparagine were assigned9 by determining the electro-
`phoretic mobility of peptides and by amino acid analysis of the peptides after enzymatic
`hydrolysis. The half-cystinyl residues contributing to each intrachain disulfide bond
`were determined10 using the diagonal electrophoresis method.1'
`Results.-The organization of the whole molecule is shown in Figure 1; an un-
`equivocal proof of the arrangement of the two identical light chains and two
`identical heavy chains has already been given.4 Each light chain is linked to its
`neighboring heavy chain by a disulfide bond between corresponding half-
`
`HayPCA
`chain
`
`I
`
`Light
`
`CHI
`~S
`S S
`S
`S
`III
`IV___
`v
`V
`S
`S
`S
`NH2 VL,-|L
`
`4i
`
`Fab (t)
`
`Fab ()
`
`--
`_S-S II
`~S
`S
`S
`VII
`sI'
`Vi
`S
`S S
`
`½.S PCA
`~S
`S
`N H2
`
`S
`S
`
`III
`
`HOC HO
`ix
`
`XI
`
`Fc t)
`
`FIG. 1.-Over-all arrangement of chains and disulfide bonds of -yGl immunoglobulin
`Half-cystinyl residues are numbered I-XI; numbers I-V designate corresponding
`Eu.
`residues in light and heavy chains. PCA: pyrollidonecarboxylic acid. CHO: carbo-
`"Fab(t)" and "Fc(t)" refer to fragments produced by trypsin, which cleaves
`hydrate.
`the heavy chain as indicated by dashed lines above haif-cystinyl residues VI. VH, VL:
`CHL,
`constant region of light chain.
`variable regions of heavy and light chains, CL:
`CH2, Cn3: homology regions comprising CH or constant region of heavy chain.
`
`Half-cystines VI and VII form bonds linking the half-molecules via
`cystines V.
`Trypsin cleaves the molecule at lysyl residue 222 to form two
`the heavy chains.
`Fab(t) and one Fc(t) fragments.2' 5
`There are several strikingly linear arrangements in the primary structure.
`From their amino termini to half-cystines V, the light and heavy chains can be
`The intrachain disulfide bonds are linearly and period-
`aligned or put in register.
`ically disposed.'2' 13
`In accord with the alignment of light and heavy chains,
`corresponding intrachain disulfide bonds are in similar positions and the disulfide
`loops are of approximately the same size.
`Previous studies7 have suggested that V regions of light and heavy chains have
`similar lengths and begin at the NH2-termini; this will be confirmed below.
`The CL region of the light chain has the same length as VL, but the CH region of
`the heavy chain is about three times as long. CH may be divided into three
`
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`BIOCHEMISTRY: EDELMAN ET AL.
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`PROC. N. A. S.
`
`homologous regions of approximately equal length:
`CH1, CH2, and CH3 (Fig. 1).
`We have already reported the amino acid sequence of the first 877 and the last
`224 residues8 of the heavy chain as well as the partial sequence of the entire light
`chain.6 The complete amino acid sequence of the light chain (214 residues) is
`shown in Figure 2.
`Positions of the half-cystinyl residues may be compared
`with Figure 1 and the methionyl residues may be correlated with previous studies
`on the CNBr fragments of Eu.3' 4 The variable region extends through residue
`In accord with other studies,1 valine 191 is related to the Inv specificity.2
`108.
`The complete sequence of the heavy chain (446 residues) is presented in Figure
`3 which may be compared with Figure 2 for alignment with the light chain
`Isolation of a single glycopeptide8 indicated that the polysaccharide
`sequence.
`
`10
`20
`1
`ASP- ILE-GLN-MET-THR-GLN- SER-PRO- SER-THR-LEU- SER-ALA- SER-VAL-GLY-ASP-ARG-VAL-THR-
`
`30
`40
`ILE-THR-FCYS-ARG-ALA-SER-GLN-SER- ILE-ASN-THR-TRP-LEU-ALA-TRP-TYR-GLN-GLN-LYS-PRO-
`50
`60
`GLY-LYS-ALA-PRO-LYS-LEU-LEU-MET-TYR-LYS-ALA-SER-SER-LEU-GLU-SER-GLY-VAL-PRO-SER-
`
`70
`80
`ARG-PHE- ILE-GLY-SER-GLY-SER-GLY-THR-GLU-PHE-THR-LEU-THR- ILE-SER-SER-LEU-GLN-PRO-
`
`100
`90
`ASP-ASP-PHE-ALA-THR-TYR-TYR-CYS-GLN-GLN-TYR-ASN- SER-ASP- SER-LYS-MET-PHE-GLY-GLN-
`
`120
`110
`GLY-THR-LYS-VAL-GLU-VAL-LYS-GLY-THR-VAL-ALA-ALA-PRO-SER-VAL-PHE- ILE-PHE-PRO-PRO-
`
`140
`130
`SER-ASP-GLU-GLN-LEU-LYS- SER -GLY-THR-ALA- SER-VAL-VAL-FCYS-LEU EU-ASN-ASN- PHE-TYR-
`160
`150
`PRO-ARG-GLU-ALA-LYS-VAL-GLN-TRP-LYS-VAL-ASP-ASN-ALA-LEU-GLN-SER-GLY-ASN-SER-GLN-
`
`180
`170
`GLU-SER-VAL-THR-GLU-GLN-ASP-SER-LYS-ASP-SER-THR-TYR-SER-LEU-SER-SER-THR-LEU-THR-
`
`190
`200
`LEU-SER-LYS-ALA-ASP-TYR-GLU-LYS-HIS-LYS-VAL-TYR-ALA-CYS-GLU-VAL-THR-HIS-GLN-GLY-
`
`214
`210
`LEU-SER-SER-PRO-VAL-THR-LYS-SER-PHE-ASN-ARG-GLY-GLU-CYS
`
`FIG. 2.-Complete amino acid sequence of the Eu light chain.
`Half-cystinyl residues are in
`boxes and methionyl residues are underlined.
`
`portion of the molecule is attached at Asx residue 297.14
`In a previous study8 we
`have suggested that glutamyl residue 356 and methionyl residue 358 may be
`associated with Gm 1 specificities.
`The sequence of Eu (Gm 4+) between
`residues 211-252 can be compared with the partial sequence of immunoglobulin
`Daw'7 (Gm 4-). The presence of arginine in position 214 of Eu and lysine in a
`comparable position of Daw may be associated with their Gm 4 specificities.'8
`Of particular significance is the determination of the point at which VH ends
`and CH begins. A CNBr fragment comparable to fragment H4 was isolated
`from myeloma protein He which has the same Gm specificity as protein Eu. The
`sequence of the amino terminal portion of the CNBr fragment from He differed
`from that of the H4 fragment from Eu.2'
`
`PETITIONER'S EXHIBITS
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`Exhibit 1091 Page 3 of 8
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`
`BIOCHEMISTRY: EDELMAN ET AL.
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`81
`
`1
`10
`20
`PCA- VAL - GLN- LEU- VAL-GLN- SER -GLY- ALA-GLU- VAL -LYS -LYS -PR0-GLY- SER- SER- VAL -LYS -VAL -
`
`30
`40
`SER-FY-LYS-ALA-SER-GLY-CLY-THR-PHE-SER-ARG-SER-ALA-ILE-ILE-TRP-VAL-ARG-GLN-ALA-
`50
`60
`PR0 - GLY - GLN - GLY - LEU - GLU - TRP - MET - GLY - GLY - I LE - VAL - PR0- MET - PHE - GLY - PR0 - PR0- ASN - TYR -
`
`70
`80
`ALA-GLN-LYS-PHE-GLN-GLY-ARG-VAL-THR- ILE-THR-ALA-ASP-GLU-SER-THR-ASN-THR-ALA-TYR-
`
`90
`100
`MET-GLU-LEU-SER- SER-LEU-ARG-SER-GLU-ASP-THR-ALA-PHE-TYR-PHE-CYS ALA - GLY - GLY - TYR -
`
`110
`120
`GLY- ILE-TYR- SER-PRO-GLU-GLU-TYR-ASN-GLY-GLY-LEU-VAL-THR-VAL-SER- SER-ALA- SER-THR-
`
`130
`140
`LYS-GLY-PR0-SER-VAL-PHE-PR0-LE L-ALA-PR0-SER-SER-LYS-SER-THR-SER-GLY-GLY-THR-ALA-
`
`150
`160
`ALA-LEU-GLY-GY[SLEU-VAL-LYS-ASP-TYR- PHE-PRO-GLU-PRO-VAL-THR-VAL-SER-TRP-ASN-SER-
`
`170
`130
`GLY -ALA -LE U -THR -SER -GLY -VAL- HI S -THR -PHE -PR0- ALA -VAL -LEU -GLN -SER -SER -GLY -LE U -TYR -
`
`19020
`SER -LE U -SER -SER -VAL -VAL -THR -VAL -PR0- SER -SER -SER -LEU -GLY -THR -GLN -THR -TYR - LE -
`
`210
`220
`ASN-VAL-ASN-Hi S-LYS-PR0- SER -ASN-THR-LYS-VAL-ASP-LYS-ARG-VAL-GLU-PRO-LYS- SER-LS
`Cy230
`240
`s-B-PR0- PR0 -S -PR0- ALA -PR0- GLU -LEU -LEU -GLY -GLY -PR0- SER -VAL -
`250
`260
`PHE -LEU -PHE -PR0- PR0- LYS -PR0- LYS -ASP- THR -LEU- MET- ILE -SER -ARG -THR -PR0- GLU-VAL -THR-
`
`ASP -LYS -THR -HSI S- THRF
`
`2U00
`270
`EaVAL-VAL-VAL-ASP-VAL-SER-HIS-GLU-ASP-PRO-GLN-VAL-LYS-PHE-ASN-TRP-TYR-VAL-ASP-
`300
`290
`GLY -VAL -GL N- VAL -H S -ASN -ALA -LYS -THR -LYS -PR0- ARG -GL U -GLN -GLN -TYR -ASX -SER -THR -TYR -
`
`320
`310
`ARG- VAL -VAL -SER -VAL -LEU -THR -VAL -LEU- HI S -GLN -ASN -TRP-LEU -ASP- GLY- LYS- GLU- TYR -LYS-
`
`340
`330
`LYSVAL- SR -SN-LYS -AA-LEU- PR0O-ALA- PR0- ILE-GLU-LYS-THR -I LE -SER-LYS-AL-LYS-
`
`360
`3 50
`GLY- GLN -PR0-ARG- GLU- PR0- GLN -VAL -TYR -THR-LEU- PR0- PR0-SER -ARG-GLU- GLU-MET-THR-LYS-
`
`380
`370
`ASN -GLN -VAL -SER -LEU -THR -cYSLEU -VAL -LYS -GLY -PHE -TYR -PR0- SER -ASP- ILE -ALA-VAL -GLU -
`
`400
`390
`TRP-GLU- SER-ASN-ASP-GLY-GLU-PR0-GLU-ASN-TYR-LYS-THR-THR-PR0-PR0-VAL-LEU-ASP-SER-
`
`420
`410
`ASP- GLY -SER -PHE -PHE -LEU -TYR -SER -LYS- LEU -THR -VAL -ASP-LYS --SER -ARG- TRP- GLN- GLU- GLY -
`
`440
`43 0
`ASN -VAL - PHE - SER -FC-~SER -VAL -MET -H IS-GLU-ALA-LEU-HI S-ASN-H IS-TYR- THR-GLN-LYS- SER-
`446
`LEU- SER - LEU - SER - PRO - GLY
`
`FIa. 3.-Complete amino acid sequence of the Eu heavy chain.
`Half-cystinyl residues are in
`boxes and methionyl residues are underlined.
`A comparison of the sequence of the two fragments from residues 101 to 121
`is given in Figure 4. The sequences become identical at residue 115 (Eu num-
`bering).
`Further studieS21 confirmed that from residue 115 to residue 252 the
`In addition,
`sequence of the He fragment was identical to that of Eu H4.
`tryptic fingerprints of the Fe fragments from Eu and He were identical.
`These
`
`PETITIONER'S EXHIBITS
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`E L
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`HE
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`BIOCHEMISTRY: EDELMAN ET AL.
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`PROC. N. A. S.
`
`120
`115
`110
`105
`101
`-GLY- ILE -TYR - SER -PRO - GLU-GLU- TYR-ASN-GLY-GLY - LEU- VAL - THR- VAL - SER - SER- ALA - SER-THR- LYS -
`
`- THR -LEU- ALA - PHE -ASX- VAL -TRP-GLY-GLX -GLY- THR- LYS -VAL -ALA -VAL - SER - SER -ALA - SER- THR- LYS -
`
`Fin(. 4.-Comparison of the amino acid sequence of the Eu heavy chain from residue 101-121
`with the corresponding sequence of the heavy chain of myeloma protein He.
`
`data suggest that the transition between VH and CH is located in the vicinity of
`Studies on a number of additional proteins and a
`residue 114 (Eu numbering).
`search for VH region subgroups7 will be required to locate this point definitively.
`Discussion.-The present studies provide proof of the covalent structure and
`The half-molecule of Eu is the
`arrangement of chains in -yG1 immunoglobulin.
`largest protein unit (446 + 214 residues) for which a complete amino acid
`In a protein of this size, one cannot neglect the
`sequence has been determined.
`possibility of small errors in sequence assignment; for this reason we are carrying
`out a number of further checks using various methods of peptide cleavage and
`fractionation.
`One of the most striking features of the immunoglobulin molecule that emerges
`from the completed sequence is the sharp demarcation of its polypeptide chains
`into linearly connected regions that are associated with different functions.
`Variations in the sequences of paired VH and VL regions for the function of
`antigen binding in the selective immune response, and at the same time, conser-
`vation of sequence in CH and CL regions for other immunological functions appear
`to require special genetic and evolutionary mechanisms.22
`The amino acid sequence of Eu provides convincing evidence that the immuno-
`globulin molecule evolved by successive duplication of precursor genes.23 24 Our
`analysis of a complete heavy chain has revealed an additional homology region
`Earlier comparisons7' 8
`(CH 1), the structure of which was not previously known.
`of polynucleotide sequences corresponding to both chains of Eu showed evidence
`of homology between VH and VL and homologies among CL, CH2, and CH3. A
`complete comparison of the amino acid sequences of CL, CH1, CH2, and C113 is
`In a stretch of 100 residues, any two regions are identical in
`given in Figure 5.
`It is noteworthy that the stretch in the heavy chain from
`29 to 34 positions.
`residue 221 to 233 which contains the interchain disulfide bonds5 has no homol-
`ogous counterpart in other portions of heavy or light chains.
`In the data accumulated so far, little or no homology has been found between
`This prompts the speculation that V genes and C genes
`V and C regions.
`diverged early in the evolution of antibodies to serve two major groups of func-
`antigen recognition functions (ARF) and effector functions (EF) such as
`tions:
`interaction with cells and complement. The order of emergence of CL genes or
`CH genes from a precursor gene is not apparent from the data. A comparison
`with sequences of ju, a, 6, or e chains may indicate similarities in their VH regions25
`and may show whether any of the homology regions are conserved in constant
`regions of heavy chains of these classes.26
`Early evolutionary divergence of V and C genes is consistent with the evi-
`dence27 28 that each chain is specified by two genes, V and C, and the hypothe-
`sis7, 22, 29 that V gene episomes are translocated to C genes to form a single VC
`
`PETITIONER'S EXHIBITS
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`\OL. 63, 1969
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`IBIOCHEMISTRY: EDELMAN ET AL.
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`83
`
`EU CL
`(RESIDUES 109-214)
`EU CH1 ;RESIDUES 119-220)
`EU CH2 (RESIDUES 234-341)
`EU CH3 (RESIDUES 342-446:
`
`110
`THR VAL ALA ALA
`
`120
`LE PHE PRO PRO '-SE
`SER THR LYS GLY PRO SEA VAL PtK PRO -LEU ALA PRO
`LEU LEU GLY MY PRO SEW
`PHE LEU PHE PR0 PO LYS
`VAt
`CLN PRO ARG GLU PRO GLN VAL TYR THR LEU PRO PRO SER
`
`RO
`
`ASP GLU CLN
`SER LYS SER
`PRO LYS ASP
`ARC GLU CLU
`
`LEU LYS SER GLY
`-
`-
`THR SER GLY GLY
`-
`-
`THR LEU MET
`LE SER ARG
`MET THR LYS ASN
`-
`-
`
`130
`THR ALA
`THR ALA
`THR PRO
`GLN VAL
`
`SER VAL VAL CYS LEU LEU ASN ASN PiEi
`ALA LEU GLY CYS LEU VAL LYS ASP TYR
`GLU VAL THR CYS VAL VAL VAL ASP VAL
`SER LEU THR CYS LEU VAL LYS GLY PHE
`
`140
`,TYR
`PHE
`SER
`WR
`
`PRO ARG GLU ALA LYS VAL
`PRO GLU PRO VAL THR VAL
`HIS GLU ASP PRO GLN VAL LYS PHE
`PRO SER ASP I LE ALA VAL
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`150
`GLN TRP LYS VAL
`SER TRP ASN SER
`ASN TRP TYR VAL
`GLU TRP GLU SER
`
`-
`
`ASP ASN ALA LEU G'N'f4S9 ..(t
`GLY ALA LEU TH R seRL Y
`ASP GLY
`VAL GLN VAL H I S
`ASN ASP
`CLY GLU PRO GLU
`
`-
`
`-
`
`-
`
`160
`tN SER GLN GLU
`VAL HISSTR
`ASN ALA LYS THR
`..SN TYR LYS THR
`
`SER VAL THR GLU GLN ASP SER
`PHE PRO ALA VAL LEU GLN SER
`LYS PRO ARC7 GLU GLN GLN TYR
`THR PRO PRO VAL LEU ASP SER
`
`1 70
`LYS ASP
`SER
`ASP
`ASP
`
`-
`
`-
`
`-
`
`SEQ THR
`GLY LEU
`SER THR
`GLY SER
`
`TYR SER LEU SER SER.
`TYR SER LEU SER SER
`TYR ARG VAL VAL SER
`PHE PHE LEU TYR SER
`
`180
`THR LEU THR LEU SER LYS ALA ASP TYR GLU LYS
`VLVAL TSR VAL PRO SER SER SER LEU GLY THR
`rIEUJ T1lR VAL LEU HIS GLN ASN TRP LEU ASP
`LYS Utll T'R VAL ASP LYS SER ARG TRP GLN
`CLU
`
`190
`HIS LYS VAL
`GLN
`THR
`GLY LYS GLU
`GLY ASN VAL
`
`-
`
`TYR ALA CYS GLU VAL THR
`TYR ILE CYS ASN VAL ASN
`TYR LYS CYS LYS VAL SER
`PHE SER CYS SER VAL MET
`
`;
`
`GLN GLY l EU
`LYS PRO SER
`ASN LYS ALA LEU
`CSGLU ALA LEU
`
`SER SER PRO VAL TSR
`LYS SER PHE
`ASN THR LYS VAL
`ASP LYS ARC
`VAL
`GLU LYS THR
`ILE SER
`PRO ALA PRO ILE
`HIS ASN HIS TYR TSR GLN LYS SER LEU SER
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`210
`GLY GLU CYS
`ASN ARC
`GLU PRO' LYS SER CYS
`LYS ALA LYS GLY
`LEU SER PRO GLY
`
`FIG. 5.-Comparison of the amino acid sequence of CL, C.IA, CH2, and CH3. Deletions indi-
`cated by dashes have been introduced to maximize the homology. Identical residues are darkly
`shaded; both dark and light shading are used to indicate identities which occur in pairs in the
`same positions.
`
`gene in lymphoid cell precursors.
`Dreyer and Bennett30 have previously sug-
`gested a translocation of C genes and, more recently, this has been abandoned in
`favor of a detailed "copy-splice" mechanism.3'
`Translocation of genes may be the
`basis of the phenomena of clonal expression and allelic exclusion in antibody
`production.
`Irreversible differentiation and commitment of a lymphoid pre-
`cursor cell may thus occur at the time of gene translocation.
`The alignment of disulfide bonds, the arrangement of symmetry axes, and the
`fact that proteolytic enzymes cleave the molecule to produce Fab, Fc and Fc'
`fragments32 suggest that each homology region may be folded in a compact
`domain29 stabilized by a single intrachain disulfide bond and linked to neighbor-
`ing regions by less tightly folded stretches of polypeptide chain.
`Such domains
`would have similar but not identical tertiary structures, and each domain would
`
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`BIOCHEMISTRY: EDELMAN ET AL.
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`PROC. N. A. S.
`P
`
`contribute to at least one active site mediating a function of that class of im-
`This domain hypothesis is consistent with the hypothesis that
`munoglobulin.
`the molecule evolved by gene duplication as well as with the translocation
`As mentioned above, comparison of the structure and function of
`hypothesis.
`CH regions in different immunoglobulin classes should reveal whether addition or
`deletion of homology regions and corresponding domains is a major mechanism
`in the evolution of these classes.
`Support for the domain hypothesis would come from finding that limited
`proteolysis of Fab fragments yields fragments containing halves of the Fd frag-
`Similar treatment of Bence-Jones proteins may produce VL and CL
`ments.
`fragments.33
`Additional evidence may come from location in CH of sites for
`complement fixation and skin fixation.
`Final proof or disproof of this hypothesis
`obviously rests on the results of X-ray crystallographic analysis.
`It is clear
`that a rotation axis passes through the disulfide bonds linking the heavy chains
`and there may be an axis of pseudosymmetry between the light and heavy chains.
`The locations of the interchain and intrachain disulfide bonds, the extensive
`homologies, and the alignment of the light and heavy chains with each other
`suggest that the overall relationships described above will be conserved in the
`three-dimensional structure regardless of the details of folding.
`The exact contribution of the variable regions to the antigen combining site
`must also await analysis of the three-dimensional structure.
`It is known that the
`Fab fragment contains both VL and VH regions, and affinity-labeling experi-
`ments24 indicate that tyrosyl residues34' 3 in these regions are directly involved
`in antigen binding.
`The constancy of the disulfide bonds in VH and VL regions
`and their coordinate location suggest the possibility that the site is fixed by these
`bonds and that variations in the branches of a chain connected by each bond
`may be sterically arranged around the bond as a center. The closely homologous
`CL and CHi regions may serve additionally to stabilize the structure in the face of
`the variation, so that both VL and VH can participate in the site.
`In contrast to the diversity of V regions, the origin of which is still unknown,22
`the C regions appear to be quite stable in various animal species.
`Recent
`studies8 show striking resemblances in the Fc portions of rabbit23 and human
`'yG immunoglobulin.
`In this respect, C regions, like enzymes, may have evolved
`to interact with specific molecules, e.g., those of the complement system. The
`presence of genetic differences in C regions has not so far been related to their
`function, but there is no reason to expect that the origin of variations in C regions
`will differ from other genetic polymorphisms.
`We are deeply indebted to Dr. Jack Brook for supplying Eu plasma. We are grateful
`to Miss Joan Low, Miss Catherine Volin, and Mrs. Helvi Hjelt for their expert technical
`assistance.
`* Supported by grant GB 6546 from the National Science Foundation and by grant AM
`04256 from the National Institutes of Health.
`' For a general review, see Cold Spring Harbor Symposia on Quantitative Biology, vol. 32
`(1967), and Nobel Symposium 3: Gamma Globulins, Structure and Control of Biosynthesis, ed.
`J. Killander (Stockholm: Almqvist and Wiksell, 1967).
`2 Edelman, G. M., W. E. Gall, M. J. Waxdal, and W. H. Konigsberg, Biochemistry, 7, 1950
`(1968).
`
`PETITIONER'S EXHIBITS
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`Exhibit 1091 Page 7 of 8
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`

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`VOL. 63, 1969
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`BIOCHEMISTRY: EDELMAN ET AL.
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`85
`
`3 Waxdal, M. J., W. H. Konigsberg, W. L. Henley, and (G. M. Edelman, Biochemistry, 7, 1959
`(1968).
`4Waxdal, M. J., W. H. Kotiigsberg, aid (G. M. Edelman, Biochemistry, 7, 1967 (1968).
`5 Gall, W. E., B. A. Ctinningham, i\. J. Waxdal, W. H. Konigsberg, and (X. M. Edelonan,
`Biochemistry, 7, 1973 (1968).
`6 Cunningham, B. A., P. D. Gottlieb, W. H. Konigsberg, and G. M. Edelman, Biochemistry,
`7, 1983 (1968).
`7 Gottlieb, P. D., B. A. Cunningham, M. J. Waxdal, W. H. Konigsberg, and G. M. Edelman,
`these PROCEEDINGS, 61, 168 (1968).
`8 Rutishauser, U., B. A. Cunningham, C. Bennett, W. H. Konigsberg, and G. M. Edelman,
`these PROCEEDINGS, 61, 1414 (1969).
`9 Bennett, C., W. H. Konigsberg, and G. M. Edelman, manuscript in preparation.
`10 Gall, W. E., and G. M. Edelman, manuscript in preparation.
`11 Brown, J. R., and B. S. Hartley, Biochem. J., 101, 214 (1966).
`12 Waxdal, M. J., W. H. Konigsberg, and G. M. Edelman, in Cold Spring Harbor Symposia on
`Quantitative Biology, vol. 32, 53 (1967).
`13 Pink, J. R. L., and C. Milstein, Nature, 216, 941 (1967).
`14 Inasmuch as the mechanism of attachment of the carbohydrate is unknown, we have not
`specified whether residue 297 is Asp or Asn.
`This residue begins the tripeptide sequence Asx-
`Ser-Thr (residues 297-299), which is the same as the Asx-X-Thr sequence found at the point
`of attachment of the carbohydrate portion of several other glycoproteins.'15. 16
`16 Eylar, E. H., J. Theoret. Biol., 10, 89 (1966).
`16 Catley, B. J., S. Moore, and W. H. Stein, J. Biol. Chem., 244, 933 (1969).
`17 Steiner, L. A., and R. R. Porter, Biochemistry, 6, 3957 (1967).
`18 The association with Gm 1 specificities is based on definitive studies.19. 20
`Additional
`evidence is required for the correlation of sequence variation with Gm 4 specificities.
`19 Thorpe, N. O., and H. F. Deutsch, Immunochemistry, 3, 329 (1966).
`20 Frangione, B., C. Milstein, and J. R. L. Pink, Nature, 221, 145 (1969).
`21 Cunningham, B. A., M. Pflumm, U. Rutishauser, and G. M. Edelman, manuscript in
`preparation.
`22 Edelman, G. M., and J. A. Gally, in Brookhaven Symposia in Biology, No. 21, 328 (1968).
`23 Hill, R. L., R. Delaney, R. E. Fellows, Jr., and H. E. Lebovitz, these PROCEEDINGS, 56,
`1762 (1966).
`24 Singer, S. J., and R. F. Doolittle, Science, 153, 13 (1966).
`25Wikler, M., H. Kchler, T. Shinoda, and F. W. Putnam, Science, 163, 75 (1969).
`26 For generality, we have used CL and CH1, CH2, etc., to designate homology regions.
`If
`such regions are found in other classes, a more specific nomenclature, e.g. C,, Cy1, Cy2, etc., and
`perhaps Cj1, etc., may be required. The same obviously holds for V regions.
`27 Hood, L., and D. Ein, Nature, 220, 764 (1968).
`28 Milstein, C., C. P. Milstein, and A. Feinstein, Nature, 221, 151 (1969).
`29Edelman, G. M., and W. E. Gall, Ann. Rev. Biochem., 38, (1969), in press.
`30 Dreyer, W. J., and J. C. Bennett, these PROCEEDINGS, 54, 864 (1965).
`31 Dreyer, W. J., W. R. Gray, and L. Hood, in Cold Spring Harbor Symposia on Quantitative
`Biology, vol. 32, p. 353 (1967).
`32 Turner, M. W., and H. Bennich, Biochem. J., 107, 171 (1968).
`33 BerggArd, I., and P. Peterson, in Nobel Symposium 3: Gamma Globulins, Structure and
`Control of Biosynthesis, ed. J. Killander (Stockholm: Almqvist and Wiksell 1967), p. 71.
`34 Singer, S. J., and N. 0. Thorpe, these PROCEEDINGS, 60, 1371 (1968).
`35 Although there are no direct data on affinity labeling of human antibodies, by analogy (see
`ref. 24), we would suggest that tyrosyl residues 86 of the light chain and 94 of the heavy chain
`may have a function in the active site.
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`Exhibit 1091 Page 8 of 8