Comparative conformational studies on the tryptic digestion fragments of human immunoglobulins M and G

IgM¹ immunoglobulins were cleaved into Fabμ and (Fc)5μ fragments by tryptic digestion. Comparative circular dichroism studies with the corresponding IgG fragments show that the Fab portions of IgG and IgM proteins have very similar CD spectral features, although the same is not true for their Fc fragments. These studies indicate the presence of higher amount of beta-structured regions in Fcμ than in Fcγ. Also, there are considerable differences in their pH-dependent structural transitions as measured by CD spectral changes. The conformational differences between IgG and IgM immunoglobulins are more pronounced in their Fc portions, which carry out class specific biological functions, rather than in Fab portions, which contain antigen combining sites.     

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.     

Conformational changes induced in a homogeneous anti-type III pneumococcal antibody by oligosaccharides of increasing size.

The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.     

Study of rabbit IgG, modified with carboxycarbonic anhydride of diethylenetriaminopentacetic acid

The affinity-purified by chromatography on immobilized antigen rabbit IgG was modified with mixed carboxycarbonic anhydride of DTPA which markedly alters the interaction of charged residues in the protein molecule. To study the correlation between the antigen binding activity and the conformational mobility of IgG, the reactivity of modified IgG towards conformational probes targeted at variable and constant IgG domains, was investigated. The antibody against CH2 domains of IgG, staphylococcal protein A and protein antigen ferritin were used as conformational probes. It was found that modification of IgG amino groups entails the global increase in conformational mobility involving the Fab fragments, CH2 and, probably, the CH3 domains of the Fc portion of IgG. Taking advantage of Fab fragments modification it was shown that two processes contribute to the global increase in the conformational mobility of IgG. These processes are: i) stimulation of segmental flexibility and, ii) increase in the mobility within the Fv domains of the Fab fragments.     

Idiotypic interactions in type II mixed cryoglobulins

An analysis of interactions between immunoglobulin molecules within cryoglobulins has been carried out in 18 patients with type II mixed cryoglobulinemia. In this series, there was a prevalence of VH I and VK I variable regions subgroups in the monoclonal IgM component. Our analysis of the IgG component indicated a particular selection of IgG molecules during cryoprecipitation. There was a prevalence of IgG3 and of the VH I subgroup and the isoelectrofocusing pattern revealed a very restricted spectrotype in two thirds of these IgG. These results which suggested a restricted reactivity between cryo-IgM and IgG fractions were confirmed by the analysis of the interaction between each IgG and each IgM from the cryoprecipitates. All IgM reacted with intact IgG or Fc fragments but another reaction was observed between cryo-IgM and Fab fragments from a limited number of cryo-IgG, with a pattern suggestive of idiotypic specificity. Results of the absorption of each cryo-IgM on Fc or on Fab fragments from the corresponding cryo-IgG also suggested the existence of a reaction between IgM and IgG Fab in addition to that involving IgM Fab and IgG Fc. The coexistence of the 2 reactions should confer a higher stability to the IgM-IgG complex. Therefore, it is possible that the proliferation of one clone of IgM-RF producing B cells would be followed in certain cases by a relatively restricted anti-idiotypic IgG response. The IgM-RF would preferentially react with these anti-idiotypic IgG.     

Conformational properties of human immunoglobulin G subclasses analysis by temperature-perturbation and solvent-perturbation spectroscopy

Conformational properties of human myeloma immunoglobulins G belonging to four subclasses (IgG1 Van, IgG2 Kom, IgG3 Pla, IgG4 Ang), and also Fab, Fc and pFc′ fragments derived from IgG1 Van, IgG2 Kom and IgG3 Pla have been studied by temperature-perturbation and solvent-perturbation spectroscopy. It has been shown that the immunoglobulins studied practically do not differ in the number of tyrosine and tryptophan residues exposed to different solvent perturbants (saccharose, glycerol, dimethylsulfoxide). The same regularity is observed for isolated Fab and Fc fragments. At the same time, the immunoglobulins compared and their proteolytic fragments significantly differ in the number of aromatic chromophores perturbed by temperature. These data indicate that immunoglobulins of different subclasses and their subunits have a different rigidity of structure in relation to thermal perturbation. The Fc subunits of IgG1 are characterized by the lowest rigidity of structure of internal hydrophobic cores of domains (characterized by the rigidity of the microenvironment of tryptophan residues), as compared with the Fc subunits of IgG2 and IgG3. In the case of IgG1 and IgG2, these differences seem to be brought about by a different rigidity of structure of C_H2 domains, since thermal-perturbation spectra of the pFc′ fragments of these subclasses practically coincide. The total number of chromophores exposed to different solvent perturbants in the isolated Fab and Fc fragments practically coincides with the number of exposed chromophores in intact immunoglobulins. Similar coincidence is observed for the tryptophan residues perturbed by temperature. These data indicate that neither the conformation of surface sites nor the conformation of internal hydrophobic cores of domains significantly changes on isolation of Fab and Fc fragments. At the same time, many more tyrosine residues are perturbed by temperature in the intact immunoglobulin G1 Van than in the corresponding sum of isolated Fab and Fc fragments, while for IgG2 Kom, which has the same length of hinge region, these values practically coincide. This fact can be explained by the greater temperature dependence of motions of subunits in IgG1 Van as compared with IgG2 Kom, and as a result of this by the higher mutual temperature-dependent influence of subunits on their internal structure (on interdomain interactions).     

Elbow‐ and hinge‐bending motions of IgG: Dielectric response and dynamic feature

Immunoglobulin G (IgG) is a Y‐shaped globular protein consisting of two Fab segments connecting to an Fc segment with a flexible hinge region, in which the Fab segments show secondary flexibility at an “elbow” region. In the present work, the hinge‐bending and elbow‐bending motions of aqueous solutions of IgG by microwave dielectric measurements below the freezing point of bulk water was observed. The presence of unfreezable water around the macromolecules reduced the effects of steric hindrance normally generated by ice and enabled the intramolecular motions of IgG. At the same time, the overall IgG molecule rotation was restricted by ice. Papain digestion and reduction of the disulfide linkage at the hinge region was used to generate Fab and Fc fragments. In solutions of these fragments, the dielectric relaxation process of the hinge‐bending motion was absent, although the elbow‐bending motion remained. Three relaxation processes were observed for papain‐digested IgG. The high, middle, and low frequency processes were attributed to unfrozen water, local peptide motions cooperating with bound water, and the elbow‐bending motion, respectively. In the case of the intact IgG, an additional relaxation process due to the hinge‐bending motion was observed at frequencies lower than that of the elbow‐bending motion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 626–632, 2016.     

Solution conformation of wild-type and mutant IgG3 and IgG4 immunoglobulins using crystallohydrodynamics: possible implications for complement activation

We have employed the recently described crystallohydrodynamic approach to compare the time-averaged domain orientation of human chimeric IgG3wt (wild-type) and IgG4wt as well as two hinge mutants of IgG3 and an IgG4S331P (mutation from serine to proline at position 331, EU numbering) mutant of IgG4. The approach involves combination of the known shape of the Fab and Fc regions from crystallography with hydrodynamic data for the Fab and Fc fragments and hydrodynamic and small angle x-ray scattering data for the intact IgG structures. In this way, ad hoc assumptions over hydration can be avoided and model degeneracy (uniqueness problems) can be minimized. The best fit model for the solution structure of IgG3wt demonstrated that the Fab regions are directed away from the plane of the Fc region and with a long extended hinge region in between. The best fit model of the IgG3m15 mutant with a short hinge (and enhanced complement activation activity) showed a more open, but asymmetric structure. The IgG3HM5 mutant devoid of a hinge region (and also devoid of complement-activation activity) could not be distinguished at the low-resolution level from the structure of the enhanced complement-activating mutant IgG3m15. The lack of inter-heavy-chain disulphide bond rather than a significantly different domain orientation may be the reason for the lack of complement-activating activity of the IgG3HM5 mutant. With IgG4, there are significant and interesting conformational differences between the wild-type IgG4, which shows a symmetric structure, and the IgG4S331P mutant, which shows a highly asymmetric structure. This structural difference may explain the ability of the IgG4S331P mutant to activate complement in stark contrast to the wild-type IgG4 molecule which is devoid of this activity.     

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60- 90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.     

Interaction of the complex between cloacin and its immunity protein and of cloacin with the outer and cytoplasmic membranes of sensitive cells.

Longitudinal and transverse proton relaxation rates for water in the hydration spheres of Gd(III) bound to the non-immune rabbit IgG fragments Fc (C-terminal half of heavy-chain dimer), pFc' (C-terminal quarter of heavy-chain dimer) and Fab (N-terminal half of heavy and light chain) have been measured at a number of frequencies and temperatures using pulsed nuclear magnetic resonance spectrometry. For the fragments Fc and pFc', a full computer analysis showed that the results could be fitted by parameters of similar magnitude to those found previously for IgG. In contrast to the results for the other complexes the Fab -Gd(III) complex showed no slow exchange contribution to the relaxation rates. Under these circumstances it was found possible to obtain an accurate value for the hydration number (q) from measurements of the longitudinal and transverse relaxation rates at a chosen frequency such that the product of the nuclear Larmor frequency (omega1) and the correlation time for the dipolar relaxation processes (tauc) was approximately unity. Water-proton relaxation rates were also determined for the complex of Gd(III) with the Fv fragment of the mouse myeloma protein MOPC 315. A computer analysis of the results revealed a slow exchange contribution to the rates and this gave errors in the variable parameters similar to those observed previously for IgG, Fc and pFc'. The conclusions drawn from the different systems are discussed in terms of the present state of application of the proton relaxation enhancement technique in biology.     

Conformational studies on the tryptic digestion fragments of human immunoglobulin G

Conformational studies on Fab(t) and Fc(t) (normal and myeloma) have been carried out by circular dichroism, difference spectra, sedimentation, and viscosity measurements. Both Fab(t) and Fc(t) were largely unfolded in acid (pH 2.1) and alkali (pH 11.7), and the unfolding was greater in alkali than in acid for Fab(t). However, for Fc(t) the circular dichroism spectral change was slightly higher in acid (pH 2.1) than in alkali (pH 11.7). It was also noted that the disorganizations were not complete under these conditions. Similar types of disorganizations were observed in heat denatured Fab(t) and Fc(t). Difference spectral studies demonstrated the presence of a significant number of buried tyrosine and tryptophan residues in both fragments, and a large proportion of the residues became exposed on acid denaturation. The results led us to conclude that both Fab(t) and Fc(t) may be characterized by structures of different stabilities. There seem to be small stable structured regions containing some of the β structures while the other portions are more susceptible to denaturation. Also, there are regions in these proteins which are rich in hydrophobic interactions. Sodium dodecyl sulfate refolded both Fab(t) and Fc(t) into partial helical structures, although the accessibility and rotational freedom of the side chain aromatic chromophores were increased. In 6 m GuHCl containing 0.1 m 2-mercaptoethanol, complete disorganization of Fab(t) was demonstrated despite the fact that its circular dichroism spectra around 217 nm differed from the spectra of random coil polyamino acids.     

Stepwise cleavage of rabbit immunoglobin G by papain and isolation of four types of biologically active Fc fragments

Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ;hinge' region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ;hinge' region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5.0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4.5. Only the C-terminal antigenic determinants were retained by stFc fragment.     

Human monoclonal cryoimmunoglobulins. I. Molecular properties of IgG3 kappa (Jir protein) and the cryo-coprecipitability of its molecular fragments by papain

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.     

Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment

The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.     

Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments

Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively).     

The role of interchain disulphide bridges in the conformational stability of human immunoglobulin G1 subclass. Hydrogen-deuterium exchange studies.

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.     

A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies.

A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross-reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc-gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti-CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis.     

Fractionation of IgG fragments using reversed micellar extraction

Reversed micellar extraction was applied for the fractionation of IgG fragments. With isooctane solution containing 50 mM AOT, Fab, Fe and IgG were extracted to the micellar phase. Each protein had an optimum pH range in the extraction. Fab and Fc were separated from the mixture at pH 7.0, with Fab being extracted to the micellar phase and Fe remaining in the aqueous phase. Extracted Fab fragments were recovered by bringing them into contact with 6M guanidine/HCl followed by dilution with PBS. Fab and Fc fragments were separated and recovered by reversed micellar extraction from IgG lysate digested with papain. Since the procedure is simple and rapid compared with column chromatography, mass preparation of the fragments can be expected by this method.     

The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation‐exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size‐exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co‐incubating Fab and Fc fragments with their respective full‐length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX‐based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein–protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb‐based therapeutics.     

The use of gadolinium as a probe in the Fc region of a homogeneous anti-(type-III pneumococcal polysaccharide) antibody.

The binding of gadolinium [Gd(III)] to a homogeneous rabbit anti-(type-III pneumococcal polysaccharide) IgG (immunoglobulin G) and its Fab (N-terminal half of heavy and light chain) and Fc (C-terminal half of heavy-chain dimer) fragments was demonstrated by measurements of solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 the binding of Gd(III) to the Fc fragment is much tighter (KD approx. 5 micronM) than binding to the Fab fragment (KD approx. 250 micronM). The binding of Gd(III) to the whole IgG molecule (KD approx. 4 micronM) is very similar to that for the Fc fragment alone. This specificity of binding to the Fc region allows the use of Gd(III) as a probe of the Fc conformation. The environment of the Gd(III) in the Fc region of whole IgG is not affected by the presence of octasaccharide derived by hydrolysis of type-III pneumococcal polysaccharide, but the corresponding 28-unit saccharide does cause detectable changes. The addition of 16-unit saccharide to anti-(SIII polysaccharide) IgG in the presence of Gd(III) does not change the solvent water proton relaxation rate, although aggregation does occur. The effects of the 28-unit saccharide may be explained therefore by a change in the tumbling time of the IgG. From a study of the effect of various antigen/antibody ratios, it is concluded that the 28-unit-saccharide-induced changes in the Gd(III) environment in the Fc region are caused by the specific geometrical structure of the antigen-antibody complexes formed, and not simply by occupancy of the combining sites on the antibody.