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Human immunoglobulin G1 Van was studied by negative staining, freeze drying and high resolution shadow casting. The Fab and Fc subunits of an intact IgG1 molecule were shown to possess limited mobility. It was found that about 70% of molecules in the IgG1 Van specimen are not flat but have a tripod-like shape.

Human myeloma proteins of IgG4 subclass in contrast to myeloma proteins IgG1, IgG2 and IgG3, were capable of blocking PCA reactions in monkeys mediated by human reaginic antibodies of IgE class. In addition to IgE, IgG4 myeloma protein was also capable of sensitizing leukocytes from normal individuals and gave histamine release (HR) upon challenge with anti-human IgG4. Leukocytes from 11 allergic individuals and from 9 normal subjects sensitized with the serum of allergic patients, were capable of releasing histamine with anti-human IgG4, anti-human IgE, and the specific allergen. No response was obtained with anti-human IgG1 and IgG3 sera. Leukocytes from the normal individuals released histamine from 3 to 20% with anti-human IgG4 and from 6 to 30% with anti-human IgE. Moreover, normal leukocytes sensitized with IgG4 myeloma protein or a serum of an allergic patient heated at 56 degrees C for 2 h, released a significant amount of histamine on challenge with anti-human IgG4 whereas no response was obtained with anti-human IgE. The biological role of human IgG4 in immediate hypersensitivity reactions is discussed in relation to human IgE.

The conformational equilibria and the kinetics of the approach to equilibrium of an IgG1 myeloma (Wes) Fab fragment (SSFab) and its mildly reduced and S-carboxyamidomethylated derivative (RAFab) were studied as a function of guanidine hydrochloride concentration. The unimolecular denaturation of SSFab, the bimolecular denaturation of RAFab, and the denaturation of Wes L chain reported previously (Rowe, E. S., and Tanford, C., (1973), Biochemistry 12, 4822) are interpreted in terms of the domain structure and evaluated in terms of the thermodynamic stability of the protein and the covalent and noncovalent interactions among its subunits. The Fd-L interactions are found to be extremely strong and are maintained at concentrations of denaturant sufficient to denature the individual domains. It is shown that all of the data are consistent with a two region structure for Fab, one composed of the vL and vH domains, and the other composed of the cL and cH domains, so that there are two sites of noncovalent Fd-L interactions. One region, identified as the C region, is found to be 10(2)-10(4) times more stable than the other; this difference in stability is attributed largely to a stronger and more extensive interaction between the domains of this region. The kinetics of the approach to equilibrium are found to be extremely slow in the center of the transitions, requiring up to a week for equilibration for RAFab, and several months for SSFab. This unusual kinetic behavior is shown to be due to the strong Fd-L interaction under conditions where the monomeric domains are unstable.

Oligosaccharide patterns obtained from human IgG by hydrazinolysis were fairly constant from sample to sample. In contrast, a variety of oligosaccharide patterns were obtained from IgG myeloma proteins. Structural analysis of each oligosaccharide by sequential exoglycosidase digestion and methylation studied indicated that the different patterns of IgG myeloma proteins are produced by different degrees of incompleteness of the formation of a single sugar chain: Sia alpha 2 leads to Gal beta 1 leads to 4GlcNAc beta 1 leads to 2 Man alpha 1 leads to 6(Sia alpha 2 leads to Gal beta 1 leads to 4GlcNAc beta 1 leads to Man alpha 1 leads to 3)(GlcNAc beta 1 leads to 4)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAcOT.

The amino acid sequence of the heavy-chain variable region of the human immunoglobulin. New has been determined. Since the amino terminus of the heavy chain was blocked, the sequence of residues 1-69 was established by digesting the appropriate CNBr fragment separately with trypsin, chymotrypsin, and thermolysin and sequencing the resulting peptides. The region from residues 70 to 120 was present in another CNBr fragment which was submitted directly to automatic Edman degradation. The result of this experiment extended the sequence to residue 100. The primary structure of the remaining portion of the VH region was determined by automatic Edman degradation of a lysine-blocked tryptic peptide derived from this region which included residues 98-214. The sequence of the VH region of New corresponds most closely to VH sequences of proteins in the VH II subgroup. This primary structure makes it possible to construct a model from the high-resolution electron-density map of protein New.

High field magnetic resonance spectroscopy has been utilized to deduce the primary structure of the glycopeptides from a human myeloma gamma 1-immunoglobulin G (Tem). The major structures found belong to the biantennary complex class of glycopeptides, with a minor (5%) fraction belonging to the bisected biantennary complex class. In the biantennary class, three structures were present with different residues at the termini of the alpha Man(1-6) and alpha Man(1-3) arms: (i) with beta Gal(1-4) and alpha NeuNAc(2-6), respectively (33%); (ii) with beta Gal(1-4) and beta Gal(1-4), respectively (45%); and (iii) beta Gal(1-4) and beta GlcNAc(1-2), respectively (17%). In the bisected biantennary class only the latter termini were found for the two arms. These results suggest that the galactosyl transferase in these cells has a preference for the beta GlcNAc(1-2) of the alpha Man(1-6) arm and that the sialyltransferase has a preference for the beta Gal(1-4) of the alpha Man(1-3) arm.

The effects of KCl and temperature on the kinetics of the recombination reaction of alkylated H and L chains of a human myeloma protein were studied by means of circular dichroism (CD). The recombination rate was considerably reduced by the presence of KCl. The results of the difference spectra showed that tryptophyl residue(s) in L chains are perturbed by KCl. No changes in the CD and absorption spectra with KCl concentration were observed for H chains and recombined H2L2. The change in the rate constant with KCl concentration paralleled the changes in the CD at 235 nm and the difference in molar extinction coefficient at 293 nm. These facts suggest that the reduction of the recombination rate in the presence of KCl is due to a change in the conformation of L chains as a result of specific interaction with chloride ions or by a shift to the dimer form in the monomer-dimer equilibrium of L chains. The rate constant showed a temperature dependence with an activation energy of 17.4 kcal/mole.

Fourteen new VK sequences derived from BALB/c IgG myeloma proteins were determined to the first invariant tryptophan (Trp 35). These partial sequences were compared with 65 other published VK sequences using a computer program. The 79 sequences were organized according to the length of the sequence from the amino terminus to the first invariant tryptophan (Trp 35), into seven groups (33, 34, 35, 36, 39, 40 and 41aa). A distance matrix of all 79 sequences was then computed, i.e. the number of amino acid substitutions necessary to convert one sequence to another was determined. From these data a dendrogram was constructed. Most of the VK sequences fell into clusters or closely related groups. The definition of a sequence group is arbitrary but facilitates the classification of VK proteins. We used 12 substitutions as the basis for defining a sequence group based on the known number of substitutions that are found in the VK21 proteins. By this criterion there were 18 groups in the Trp 35 dendrogram. Twelve of the 14 new sequences fell into one of these sequence groups; two formed new sequence groups. Collective amino acid sequencing is still encountering new VK structures indicating more sequences will be required to attain an accurate estimate of the total number of VK groups. Updated dendrograms can be quickly generated to include newly generated sequences.

The binding of mouse myeloma IgG1, IgG2a, IgG2b, IgG1 Fc, IgG2b Fc and a pepsin produced C-terminal subfragment of IgG1 Fc and IgG2b Fc (provisionally identified as pFc') to mouse peritoneal macrophages was investigated. The high affinity cytophilic antibodies belonged to IgG2 subclasses and the binding site of these antibodies was located in the CH3 homology region.

The denaturation and renaturation by guanidine hydrochloride of Fc(t) fragment whose interchain disulfide bonds are reduced and alkylated (R.A.Fc(t)) and pFc' fragment of a human myeloma protein (IgGl, kappa) were studied using tryptophyl fluorescence. R.A.Fc(t) was found to consist of a slow-unfolding region and a rapid-unfolding region. The denaturation of pFc' was extremely slow. Comparison of the kinetic and equilibrium data of the denaturation of R.A.Fc(t) with those of pFcl indicated that the slow-unfolding region of R.A.Fc(t) corresponds to the CH3 region and the fast-unfolding region the CH2 domain. This was also confirmed by the analysis of the CD and fluorescence spectra for R.A.Fc(t) and pFc' at various concentrations of guanidine hydrochloride. Although the kinetic stability of the CH3 region was much higher than that of the CH2 region, the thermodynamic stabilities of these domains were almost the same; the free energy change of the denaturation in water being about 6 kcal . mol-1. This value is also the same as the value for the CL fragment (Goto, Y. & Hamaguchi, K. (1979) J. Biochem. 86, 1433--1441). It was suggested that the high kinetic stability of the CH3 region in R.A.Fc(t) is due to the strong tendency for the CH3 domains to form a dimer.

Myeloma gamma globulins have been reported to interfere with fibrinogen-fibrin conversion. A patient with multiple myeloma is described with a gamma globulin IgG1lambda concentration of 11 g per dl, prolonged thrombin time and poor clot retraction. Purified gamma globulin from the patient's serum and from normal serum caused prolongation of the thrombin time and reptilase clotting time assays in both normal plasma and in solutions of bovine fibrinogen. In addition, fibrin clots formed during the thrombin time assays were found to be ultrastructurally abnormal. This data suggests that the interaction of gamma globulin in the polymerization of fibrin may, in at least some cases, be due to nonspecific protein interaction.

Multiple myeloma provides a unique model for studying factors affecting IgG isotype distribution in humans. Evaluations were made as to whether monoclonal immunoglobulins (M-proteins) of different IgG isotypes are associated with different extents of hypogammaglobulinemia and whether all residual subclasses are decreased comparably. The isotype patterns were analyzed in the context of the gene order of the constant regions of gamma (gamma) heavy chains on chromosome 14. Using monoclonal antibody-based immunoenzymometric assays, IgG subclasses were quantitated in the sera of 50 patients having IgG M-proteins, 38 with multiple myeloma and 12 with monoclonal gammopathy of undetermined significance. Thirty-three (66 percent) patients had IgG1, nine (18 percent) had IgG2, four (8 percent) had IgG3, and four had IgG4 M-proteins, paralleling the normal IgG subclass distribution. The concentration of residual IgG (sum of the evaluatable polyclonal IgG subclasses) was significantly decreased in patient sera (p less than 0.05). However, in only seven (14 percent) of the patients were all three subclasses below the reference range, suggesting some selectivity of immunosuppression. Patients with M-proteins of different IgG subclasses had markedly different patterns of suppression. Patients with IgG2 M-proteins (78 percent) were more likely to have depressed residual IgG than patients with IgG3 (50 percent), IgG1 (27 percent) or IgG4 (0 percent) M-proteins. Some patients had deficits of only one or two IgG subclasses. When considering all sera together, residual IgG1 was disproportionately reduced, followed by residual IgG2, IgG3, and IgG4. Next it was determined whether or not patterns of suppression were predicted by the gamma heavy-chain gene order (5' to 3'): gamma 3, gamma 1, gamma 2, gamma 4, as seen in some other immunologic disorders. Interestingly, the normal isotypes encoded by genes in juxtaposition to that of the M-protein were most often decreased (p less than 0.05). Thus, the patterns of hypogammaglobulinemia in multiple myeloma are heterogeneous. They may be influenced by the M-protein itself, possibly through interactions with regulatory cells. In addition, factors at the gene rearrangement level may contribute.

The urinary light chain dimer and serum monoclonal IgG1 protein from a patient (Mcg) with multiple myeloma and amyloidosis were systematically tested for their binding activities to peptides presented on solid supports. The system was validated using a series of enkephalins, beta-casomorphins and DNP-lysine derivatives which were known to complex with the dimer. Sets of peptide ligands binding to the proteins were constructed by incremental additions of amino acid residues to minimal binding units [Geysen et al., J. Immun. Meth. 102, 259-274 (1987)]. Both the amino acid sequences and the combinations of optical isomers were optimized at each stage of the syntheses. Binding could be demonstrated for ligands ranging in size from a tethered single amino acid to pentapeptides. At the dipeptide levels, the dimer and the IgG1 protein showed different preferences (Hp versus qf, where lower case letters designate D-amino acid residues). However, in a tetrapeptide ligand (qfHp) for the dimer, both of these initial preferences had converged. With few exceptions, the IgG1 molecule showed binding activity for the ligands developed for the dimer. Two sets of selected peptides, one based on Hp and the other on mW, were synthesized for diffusion into crystals of the dimer. X-ray analyses showed that these peptides bound exclusively in the main binding cavity between the "variable" domains of the dimer. As predicted from the ELISA results with tethered ligands, the relative occupancies in the crystals followed the order of tetrapeptide greater than tripeptide much greater than dipeptide. The crystallographic studies confirmed that peptides with very different sequences can bind in the same cavity.

The conformational changes of antibody structure induced by hapten molecule binding were investigated by means of thermal perturbation difference spectroscopy. The studies of the free rabit anti-dinitrophenyl antibodies show the conformational transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. Binding of the hapten molecules induces a similar transition to that which occurs between the two temperature dependent states of the free antibody. In contrast to our previous results with anti-dansyl rabbit antibodies the dinitrophenyl lysine stabilizes the "low temperature" native state of the protein. The investigation of the MOPC-315 mouse immunoglobulin A myeloma protein possessing anti-dinitrophenyl activity indicates no conformational transition at temperatures between 25 and 35 degrees C and only a small decrease of tyrosine exposure induced by the hapten binding. Our present and previous results indicate that most of the free immunoglobulins exist in two native conformational states which have a small difference in free energy. Hapten binding causes the transition in equilibrium between the two states towards the one of better binding. It is possible that this transition is necessary but not sufficient step for inducing the effector function of antibodies.

The hydrogen-deuterium exchange data of human immunoglobulin G1 (IgG1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10(-3) for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol). In the range of pH 6.2-8.45, at 25 degrees C no gross conformational changes are reflected in the hydrogen-deuterium exchange behaviour of the native, the reduced-nonalkylated-reassociated and the reduced-S-alkylated-reassociated IgG1. No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter-subunit disulphide bridges in S-alkylated-reassociated molecules results in an increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average.

Normal IgG and myeloma proteins of the IgG1, 2, 3,and 4 subclasses were mixed with human Clq and studied in the analytical ultracentrifuge for complex formation. Binding of IgG to Clq is apparent both from the enlargement of area and from the increase in sedimentation rate of the well-separated schlieren peak of Clq. The accurate determination of binding parameters requires that sedimentation rates be corrected for hydrodynamic interaction, and area measurements corrected for the Johnston-Ogston effect. At the highest immunoglobulin concentrations employed in these studies more than ten IgG molecules are bound to each Clq. If we assume that the number of binding sites must be an integral multiple of 6, then the data best support a 12 binding site model, although an 18 site model cannot be rule out. Myeloma IgG proteins of all subclasses bind to Clq, with affinities decreasing in the order G3 greater than G1 greater than G2 greater than G4. No binding of IgA to Clq could be detected.

Recurrent gangrenous leg ulcers were associated with a monoclonal IgGk cryoglobulinaemia, though without overt myeloma. Cryoprecipitation of the serum in vitro gave a reversible gel with microtubular structure, built from the monomer and with a high degree of order. The same structure was found distending the superficial vessels of the ulcer biopsy, as well as extravascularly. There was no vasculitis, and luminal obstruction appeared responsible for the ulceration. Microtubular structures occur in nature among diverse proteins and viruses. The protein studied here is similar to a reported instance of myeloma cryo-IgGk.

A simple theoretical model for the formation of interchain disulfide bonds (covalent assembly) in immunoglobulins has been developed. This model successfully simulates the experimentally determined sequence of disulfide bond formation in vitro for human immunoglobulin G1 and G4 (Petersen, J. G. L., and Dorrington, K. J. (1974) J. Biol. Chem. 249, 5633-5641). The model appears to be generally applicable suggesting that the various pathways of covalent assembly observed in vivo and in vitro reflect structural differences between the immunoglobulin classes and subclasses. The rate of assembly, however, is dependent upon environmental factors.

The observation that reduction of the inter-chain disulfides in rabbit antibody destroys its ability to interact with complement was confirmed and shown to be true also of human meyloma IgG1 subclass proteins. In the latter case a C1-binding assay was used. Further studies indicated that it was the interheavy chain disulfides which were essential for complement-binding activity: Non-covalently reassembled IgG (LHHL) was devoid of C1-fixing activity whereas molecules formed from covalently linked heavy chain dimers, and reduced and alkylated light chains (ie., LH-HL) were as active as the parent intact IgG. Fc fragments from IgG1 bound C1 and this activity was insensitive to the presence or absence of intact interchain disulfides. These bonds therefore are neither directly involved in C1 binding nor essential for the integrity of the binding site. We have also shown that although IgG4 does not bind C1, Fc fragments derived from this subclass fix C1 with an affinity comparable to that of the corresponding fragment from IgG1. These data suggest that quaternary interaction with other regions of the molecule (ie., Fab) may modulate the activity of the C1-binding site.

The difference in the reactivity of tyrosine and histidine residues in mono- and heteroclonal IgG protein toward enzyme catalyzed and chemical iodination has been studied. One heteroclonal and four monoclonal IgG proteins were iodinated using lactoperoxidase or chloramine T. The ratio of the degrees of substitution of the light and the heavy chains varied from IgG to IgG. One of the monoclonal IgG proteins, IgG-Dam, could only be modified in the gamma-chain. The lack of reactivity was attributed to steric hindrance and other local peculiarities. This interpretation is supported by spectrophotometric titration studies.

Amino acid sequence analysis of the pFc' fragment obtained by pepsin digestion of an IgG3; G3m(g) human myeloma protein HER shows it to consist of 112 residues. It starts at position 334 (gamma1 numbering), contains eight residues of the Cgamma2 region, and the whole Cgamma3 domain. Comparison with the sequence of gamma1 shows five differences including an extra Met at 397. Each is accountable by a single base substitution. The sequence is identical to that of a G3m(b0) molecule except for the previously noted allotype related Tyr/Phe exchange at position 436. The high degree of homology (95%) among gamma-chain subclasses suggests a recent diversification.

Intact rabbit IgG antisheep erythrocyte antibodies, and the corresponding F(ab')2 fragments and partially reduced and alkylated IgG were studied for the capacity to induce cytotoxic plaque formation by normal human mononuclear leukocytes in surface monolayers of sheep erythrocytes. The F (ab')2 fragments did not induce plaque formation, whereas partially reduced and alkylated IgG antibodies had a good plaque-inducing capacity compared to untreated IgG anti-SRBC antibodies. The plaque formation was inhibited by human IgG, but not by IgM, IgA, IgD or IgE. Normal and myeloma IgG in aggregated form gave a stronger inhibition than the corresponding proteins. Strong inhibition was observed with IgG1, IgG3, and IgG4, and with IgG2 after aggregation. Both the Fc and pFc' corresponding to the C terminal domain gave a strong inhibition. Thus, the region of the IgG molecule involved in binding to the Fc receptor of the plaque-forming cells appears to be located within the CH3 domain. These observations, therefore, indicate that the plaque-forming cells are of monocytic origin.

The radial immunodiffusion method of Mancini has been applied to quantitative study of IgG subclasses in a normal population. The method was assessed in terms of both reproducibility and antiserum consumption. The distribution of IgG subclasses in a group of normal individuals was studied, and compared with their incidence in a series of monoclonal proteins investigated by previous workers and particularly with other quantitative studies on groups of normal individual's sera.

The amino acid sequences of the first six CNBr fragments (303 residues) of MOPC 21 heavy chain have been determined, using conventional techniques. This completes the determination of the covalent structure of MOPC 21 immunoglobulin (IgG1, K). MOPC 21 gamma1 chain is a unique sequence of 440 amino acid residues. The variable region is homologous with human VHIII sequences. A comparison of the constant region with homologous sequences shows that mouse gamma1 and gamma2 proteins differ much more than the gamma subclasses in man and in some other species and supports earlier suggestions that mouse gamma1 and the four human gamma subclasses have a common ancestor different from the mouse gamma2 ancestor. Unlike some other subclasses, but like those in the guinea pig, mouse gamma1 and gamma2 genes have diverged before speciation. An expansion-contraction mechanism is proposed for the evolution of gamma genes.

The amino acid sequences of most of the CH1, CH2 and CH3 domains of IgG Zie, a myeloma protein belonging to the IgG2 subclass, are presented. These data make possible a comparison of the sequences of residues 253-446 of all four subclasses of immunoglobulins: these residues make up almost the entire Fc regions. A comparison can also be made of the CH1 domain of IgG1 Eu and the CH1 domain of IgG2 Zie. Earlier sequence analyses of the Fc regions of subclass 1 and 3 proteins, and parts of the Fc regions of subclass 2 and 4 proteins showed that about 95% of these sequences were identical. The extended comparisons made possible by the data presented here show that this very high degree of identity is maintained throughout the four subclasses. Similarly, the CH1 domains of gamma 1 and gamma 2 chains were found to have about 93% sequence identity. It is unlikely that the few single amino acid changes within the constant region domains can account for the marked differences between subclasses observed in the region domains can account for the marked differences between subclasses observed in the biological effector functions of immunoglobulin Fc regions, especially since most of the changes are highly conservative. Rather, it seems probable that these functional differences are caused by conformational differences between the subgroups, which result from sequence differences in the hinge regions.

The noncovalent interaction of light (L) chain with heavy (H) chain or Fd isolated from a human myeloma protein Jo (IgG1, kappa) was studied by following circular dichroic (CD) change at 235 nm. The dimerization constants of Jo-L chain determined by measuring the CD change at 293 nm with protein concentration showed that the Jo-L chain exists as the monomeric form under the experimental conditions used for recombination with H chain. The second-order rate constants for the interaction between H and L chains were in good agreement with those for the interaction between Fd and L chain at various pH values. The binding behavior of L chain to Fd could be described by a single association constant. In the interpretation of the binding of L chain to H chain, however, it was necessary to assume that the binding of L chain to one of the two sites on H chain dimer (H2) decreases the affinity of the other site for L chain. The binding constant of the first L chain to H2 was the same as that of L chain to Fd. Renaturation processes of L chain, Fd, Fab(SS) fragment (with intact interchain disulfide bond), and Fab(RA) fragment (in which the interchain disulfide bond had been reduced and alkylated) from the denatured states in 0.5 or 1 M acetic acid on neutralization were studied. The renaturation of Fd occurred very rapidly, while that of L chain consisted of a very rapid process and a slow process which followed first-order kinetics. The renaturation process of Fab(SS) consisted of rapid and slow phases, of which the latter followed first-order kinetics. The renaturation process of Fab(RA) also consisted of rapid and slow phases, but the latter process followed second-order kinetics. The overall rate constant of renaturation of Fab(RA) was the same as that of the reformation of Fab(RA) from isolated Fd and L chain. On the basis of these facts, the kinetic mechanism by which Fd and L chain recombine to yield Fab(RA) can be described in terms of the scheme Fd + L in equilibrium Fd ... L leads to Fab(RA), where Fd ... L is an intermediate, and CD change is only observed in the second unimolecular process and not in the first bimolecular process.

The temperature-dependent precipitability of a monoclonal IgG3 kappa cryoimmunoglobulin (Jir) without known antibody activity is shown to be affected by various physico-chemical factors, such as protein concentration, pH value and NaCl concentration. The molecular properties characterizing this protein (carbohydrate and amino acid compositions, peptide constitutions and susceptibility to enzymatic proteolysis) are described. The cryoprecipitability of the protein was completely lost upon papain hydrolysis, and none of the isolated fragments, Fab-Fc, Fc, and Fab, showed any precipitating activity. In the cryo-coprecipitation assay using the 125I-labeled fragments, it was demonstrated that the association activity with intact Jir protein was still retained on the Fab-Fc and Fc fragments, but not on the Fab fragment. The evidence suggests that a specific interaction may be involved in the primary intermolecular association required to form the cryoprecipitate at temperatures below the critical point, and that one of the pairing sites resides on the Fc portion of the protein molecule.

The evidence of the intramolecular flexibility of human myeloma immunoglobulin G belonging to the second, third and fourth subclasses has been obtained using the impulse NMR method. It has been found that the degree of intramolecular flexibility for the myeloma immunoglobulin G belonging to the first subclass decreases significantly by cooling from 37 to 10 degrees.

The structure of the Fab' fragment of a human myeloma protein (IgG1 (lambda) New) has been determined by X-ray crystallographic analysis to a nominal resolution of 0.2 nm. Each of the structure subunits corresponding to the variable and to the constant homology regions of the light and heavy polypeptide chains contains two irregular beta-sheets which are roughly parallel to each other and surround a tighly packed interior of hydrophobic side chains. The regions of the hypervariable sequences in the light and heavy chains occur in close spatial proximity at one end of the molecule, defining the active site of IgG New. The role of these hypervariable regions in defining the size and shape of the active site of different immunoglobulins is discussed on the basis of the three-dimensional model of Fab' New. Several ligands that bind to the active centre of IgG New have been used to obtain crystalline ligand-Fab' New complexes which were investigated by difference Fourier maps. These studies are analysed in terms of the biological function and specificity of antibodies.