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.     

Separation of complexes containing protein A and IgG or Fcγ fragments by high-performance liquid chromatography

Protein A of Staphylococcus aureus is a bivalent Fc receptor that can form complexes with immunoglobulin G (IgG) or Fcγ fragments that activate humoral (e.g., complement) and cellular (e.g., lymphocyte) components of the immune system both in vitro and in vivo. To obtain complexes formed between protein A of Staphylococcus aureus (SpA) and rabbit IgG or Fcγ fragments for purposes of characterizing their compositions and studying their biological activities, we have used high-performance liquid chromatography to separate complexes in 20 min. Complexes were prepared with trace amounts of ¹²⁵I-SpA and ¹³¹I-IgG or ¹³¹I-Fcγ to simplify the analyses. With excess molar amounts of IgG or Fcγ the complexes have the molecular formulas [(IgG)₂SpA]₂ or [(Fcγ)₂SpA]₂. With excess SpA, complexes corresponding to (IgG)(SpA) or (Fcγ)(SpA) are formed, perhaps with other complexes that have different ratios of components. Since SpA is a rod-shaped molecule it elutes at a molecular weight corresponding to 240,000 rather than the true value of 42,000. This behavior is reflected in the elution of certain complexes at shorter retention times than expected on the basis of actual molecular weights, and facilitates separation of complexes from free IgG or Fcγ. The true molecular weights and molecular formulas of complexes isolated by HPLC were verified by ultracentrifugation. This HPLC method was used to study the interconversion and stability of complexes.     

The products from papain and pepsin hydrolyses of guinea-pig immunoglobulins γ1G and γ2G

1. The products from papain and pepsin hydrolyses of the guinea-pig immunoglobulins gamma(1)G and gamma(2)G were isolated and characterized with regard to molecular weight, amino acid composition, hexose content and antigenic specificity. 2. Fragments Fab and (Fab')(2) from immunoglobulins gamma(1)G and gamma(2)G have similar electrophoretic and antigenic properties, but show some class-specific differences in amino acid composition. 3. Three Fc fragments were obtained after papain digestion of immunoglobulin gamma(2)G, namely, fragment Fc dimer (mol.wt. 58000), fragment Fc monomer (mol.wt. 29000) and fragment Fc' (mol.wt. 8000). A single crystalline fragment, namely fragment Fc' (mol.wt. 11000), was isolated after papain digestion of immunoglobulin gamma(1)G. 4. Peptic digestion of immunoglobulins gamma(1)G and gamma(2)G releases C-terminal fragments, namely, fragments pFc', of similar molecular weight (13000) but different amino acid compositions and distinct antigenic specificities. 5. Digestion-time studies show that immunoglobulin gamma(1)G is far more susceptible to proteolysis than is immunoglobulin gamma(2)G and suggest that at least a proportion of molecules are split primarily at a site that liberates fragment gamma(1)Fc'.     

The controlling effect of carbohydrate in human, rabbit and bovine immunoglobulin G on proteolysis by papain

About two-thirds of the hexose of human and rabbit immunoglobulin G (IgG) was located in the Fc fragment and one-third in the `hinge' region of the γ (heavy) polypeptide chain at the junction of the Fab and Fc fragments. In contrast, bovine IgG contained more hexose in the `hinge' region than in the Fc fragment. The initial cleavage of susceptible IgG molecules into Fab and Fc fragments by papain under the conditions given by Porter (1959) had reached completion after digestion for 2hr., though bovine IgG was digested somewhat more slowly than human or rabbit IgG. The release of `hinge' peptides from human and rabbit IgG had also reached completion by 2hr., but was slower from bovine IgG and continued for several hours longer. Since bovine IgG molecules contained on the average a greater amount of hexose in the `hinge' region, carbohydrate on this part of the γ-chain may influence not only the initial rate of enzymic hydrolysis into Fab and Fc fragments, but also, and to a greater extent, the rate of further limited hydrolysis of the N-terminal regions of the Fc fragment. The presence of carbohydrate in the `hinge' region does not appear to account for the resistance of some IgG molecules to papain digestion and of some Fc fragments to N-terminal degradation.     

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s⁰_20,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier R_G values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.     

Subfragments from the Fc fragment of human immunoglobulin G. Isolation and physicochemical characterization

A fragment termed fragment Fc' and a related fragment termed fragment pFc' produced by the actions of papain and pepsin respectively on human immunoglobulin G have been isolated and characterized. Amino acid analyses and experiments utilizing cyanogen bromide to cleave the methionyl bonds of the Fc' and pFc' fragments make it possible to locate both fragments within the known chain structure of the immunoglobulin G molecule. The pFc' fragment is probably a non-covalently linked dimer situated at the C-terminal end of the molecule, containing about 232 amino acid residues and having a molecular weight of 26000. The Fc' fragment is a similar dimer of about 182 residues extending from near residue 14 to near residue 105 (numbered from the C-terminal end) of the gamma-chain and has a molecular weight of 21000.     

Physiochemical characterization of proteolytic cleavage fragments of bovine colostral immunoglobulin G1 (IgG1).

Normal bovine colostral immunoglobulin G1 was subjected to enzymic digestion (pepsin, papain and trypsin) and the resulting fragments separated by a combination of molecularsieve and phosphocellulose chromatography.Fragments F(ab')2 derived from peptic digestion, fragment Fab from papain digestion and fragment Fab(t) from tryptic digestion showed complete antigenic identity with each other. Although fragment F(ab')2 (peptic digestion) had a sedimentation coefficient (S2o,w) of 5.3S, those for fragments Fab' (peptic digestion), Fab (papain digestion) and Fab(t) (tryptic digestion) were found to be 3.9S, 3.7S and 3.7S respectively. The mol.wts. calculated for the various fragments from the sedimentation equilibrium data were: F(ab')2, 104000 +/-200; Fab', 51900+/-340; Fab, 50900+/-230; Fab(t) 50900+/-300. Fragment Fc' (peptic digestion) had an S20,w of 3.2S and a mol. wt. of 42900+/-650; fragment Fc (papain digestion) had an SI0,w of 3.7S and a mol.wt. of 50800+/-300; fragment Fc(t) had an S20,w of 3.7S and a mol.wt. of 50800+/-300; fragment Fc(t) had an S20,w of 3.7S and a mol.wt. of 50800+/-450.     

Energy transfer distance measurements in immunoglobulins. III. Location of the light-heavy interchain disulfide bond in rabbit immunoglobulin G antibody

The distance between the hapten combining site and the light-heavy interchain disulfide bond in the Fab fragment of rabbit immunoglobulin G has been determined by measuring the efficiency of energy transfer between chromophores specifically attached at these sites on the molecule. The donor chromophore, Dns-Lys⁴, was non-covalently bound in the combining site of the Fab fragment of high-affinity anti-Dns antibody. The acceptor chromophore, fluorescein, was covalently attached by disulfide interchange of racemic DiFlCys with specific sulfhydryls generated by reduction. The presence of acceptor decreased the donor fluorescence lifetime from 23.6 nanoseconds to 21.6 nanoseconds. From the transfer efficiency of 8.4%, an average separation distance of 76 ± 10 Å was calculated. However, a statistical analysis of the molar concentrations of donor and acceptor on Fab fragments showed that approximately equal numbers of Fab probably contained donor but no acceptor on the one hand, and both donor and acceptor on the other hand. The presence of the former subpopulation would result in an average measured efficiency of energy transfer that would be too low. Treatment of the decay data by a double-exponential analysis which took account of these two populations of Fab fragments, led to a transfer efficiency of 20% and a correspondingly shorter separation distance of 64 ± 10 Å. The latter value is to be preferred. From the results presented here, and those reported previously on the location of the combining site at the tip of the Fab fragment and of the interheavy chain disulfide bond (Bunting &; Cathou, 1973), a general summary of the dimensions of rabbit immunoglobulin G Fab is given.     

Adsorption of IgG onto hydrophobic teflon. Differences between the Fab and Fc domains

The effect of differences in the degree of hydrophobicity of protein patches/fragments on the adsorption behaviour of the protein is investigated. The adsorption isotherm of a monoclonal mouse anti-human immunoglobulin G (isotype 2b) onto hydrophobic Teflon particles is measured using a depletion method. The adsorption-induced denaturation of the immunoglobulin as a function of the adsorbed amount is studied by differential scanning calorimetry, and the corresponding rearrangements in the secondary structure of the whole IgG molecule and its F_ab and F_c fragments are determined by circular dichroism spectroscopy. The effects of adsorption on the F_ab and F_c fragments in the intact IgG molecule occur independently. Adsorption of the whole IgG molecule leads to denaturation of the F_ab fragments, whereas the F_c fragment remains unperturbed; adsorption of the isolated fragments results in structural changes in both F_ab and F_c. The surface hydrophobicity of the isolated fragments was studied by HPLC. These experiments support the hypothesis that differences in the degree of denaturation between F_ab and F_c are due to the higher degree of hydrophobicity of the F_ab fragment. The adsorption-induced changes in the secondary structure are more prominent for the isolated fragments as compared to intact IgG. This is ascribed to the higher flexibility of the isolated fragment, as compared to the fragment in the whole molecule.     

Fc and Fab Fragments from IgG<Subscript>2</Subscript> Human Immunoglobulins Characterized

Papain digestion of 7S immunoglobulin G (IgG) produces two 3.5S Fab fragments and one 3.5S Fc fragment^1–8. The Fab fragment contains one light chain and one Fd fragment and is still able to combine specifically univalently with antigen. The Fc fragment is a dimer of the carboxyl terminal half of the heavy chain. Pepsin splits 7S IgG into some small peptides derived from Fc and one 5S F(ab′)₂ fragment, which contains both antigen-binding sites. Based on this information, some investigators^6,7 have postulated that pepsin splits the γ chains at the C-terminal side of the inter-heavy chain disulphide bridges, whereas papain splits at the N-terminal side of the inter-heavy chain disulphide bridges. We report here evidence that this model does not apply to all IgG subclasses. In the case of human IgG₂ subclass myeloma proteins, papain splits initially at the C-terminal side of inter-heavy chain disulphide bridges. We also show that the amino-acid sequence of the Fc fragment of human IgG₂ subclass so far determined has approximately 95% homology with that of human IgG₁ and IgG₄ subclasses reported by others^9–15.     

Extended flexible linker structures in the complement chimaeric conjugate CR2-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.     

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering,analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.     

Semi-extended solution structure of human myeloma immunoglobulin D determined by constrained X-ray scattering

Human immunoglobulin D (IgD) occurs most abundantly as a membrane-bound antibody on the surface of mature B cells (mIgD). IgD possesses the longest hinge sequence of all the human antibody isotypes, with 64 residues connecting the Fab and Fc fragments. A novel rapid purification scheme of secreted IgD from the serum of an IgD myeloma patient using thiophilic (T-gel) and lectin affinity chromatography gave a stable, homogeneous IgD preparation. Synchrotron X-ray scattering and analytical ultracentrifugation of IgD identified the solution arrangement of its Fab and Fc fragments, and thereby its hinge structure. The Guinier X-ray radius of gyration R(G) of 6.9(+/-0.1)nm showed that IgD is more extended in solution than the immunoglobulin subclass IgA1 (R(G) of 6.1-6.2nm). Its distance distribution function P(r) showed a single peak at 4.7nm and a maximum dimension of 23nm. Velocity experiments gave a sedimentation coefficient of 6.3S, which is similar to that for IgA1 at 6.2S. The complete IgD structure was modelled using molecular dynamics to generate IgD hinge structures, to which homology models for the Fab and Fc fragments were connected. Good scattering curve fits were obtained with 18 semi-extended best fit IgD models that were filtered from 8500 trial models. These best-fit models showed that the IgD hinge does not correspond to an extended polypeptide structure. The averaged solution structure arrangement of the Fab and Fc fragments in IgD is principally T-shaped and flexible, with contribution from Y-shaped and inverted Y-shaped structures. Although the linear sequence of the IgD hinge is much longer, comparison with previous scattering modelling of IgA1 and IgA2(m)1 suggests that the hinge of IgA1 and IgD are more similar than might have been expected, Both possess flexible T-shaped solution structures, probably reflecting the presence of restraining O-linked sugars.     

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.     

Differential reduction of interchain disulphide bonds of mouse immunoglobulin G

The relative lability of the interchain disulphide bonds of mouse G_2a-myeloma protein 5563 was studied as a function of 2-mercaptoethanol concentration. Analysis of partial-reduction mixtures by polyacrylamide-gel electrophoresis and microdensitometry showed that the disulphide bonds between light and heavy chains are much more susceptible to reduction than the bonds between heavy chains. At a low concentration of 2-mercaptoethanol (10mm) the major dissociable products of mouse immunoglobulin G are heavy-chain dimers and free light chains. These findings contrast with the reported behaviour of rabbit immunoglobulin G, for which the lability of inter-heavy-chain bonds was found to exceed that of the bonds linking light and heavy chains (Hong & Nisonoff, 1965); the relative stability of rabbit immunoglobulin G interchain bonds was confirmed in the present study. Examination of human immunoglobulin G and an immunoglobulin G (γ2) of guinea pig showed that at least in the majority of molecules, as with mouse immunoglobulin G, the disulphide bonds between light and heavy chains are more susceptible to reduction than the inter-heavy-chain bonds.     

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 Å, compared to a value of 81 Å for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1–2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a M_r 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of M_r 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.     

The subunit structure of thymus leukemia antigens.

EDTA-containing buffer solubilizes thymus leukemia antigens (TLa) from crude thymocyte membrane fractions. The TL antigens consist mainly of molecules of a size similar to immunoglobulin G when gel chromatography analyses were performed under physiological conditions. A single component of TLa was apparent on sucrose density gradient ultracentrifugation of solubilized thymocyte membrane macromolecules as monitored by indirect immunoprecipitation. The sedimentation constant for the TL antigens (5.8 S) was considerably less than that for immunoglobulin G. The gel chromatography and ultracentrifugation data suggest an apparent molecular weight for TLa of about 120000. TLa isolated by indirect immunoprecipitation is composed of two types of polypeptide chains. The smaller subunit was identified as beta2-microglobulin. The larger polypeptide chain carried the alloantigenic determinants and displayed a molecular weight of about 50000 after reduction and alkylation. TLa subjected to molecular weight determination under denaturing conditions was composed of two components. The smaller component was beta2-microglobulin which evidently is linked to the larger polypeptide chain by noncovalent interactions only. The larger component had a size greater than reduced and alkylated immunoglobulin G heavy chains. Upon reduction and alkylation of the latter component its size was reduced and it appeared to have a molecular weight of about 50000. Consequently, TLa is composed of two disulfide linked heavy polypeptide chains and two beta2-microglobulin molecules. TLa solubilized by papain digestion comprises two polypeptide chains, one of which is beta2-microglobulin. The larger 37000-dalton subunit is a fragment of the heavy polypeptide chain. This was demonstrated by digesting solubilized 120000-dalton TLa with papain. The proteolytic fragments obtained were indistinguishable from those directly released from the cell surface by proteolysis. The papain-derived TLa fragment exhibited most if not all the alloantigenic determinants.     

Solution structure of human and mouse immunoglobulin M by synchrotron X-ray scattering and molecular graphics modelling. A possible mechanism for complement activation

The pentameric 71-domain structure of human and mouse immunoglobulin M (IgM) was investigated by synchrotron X-ray solution scattering and molecular graphics modelling. The radii of gyration RG of human IgM Quaife and its Fc5, IgM-S, Fab'2 and Fab fragments were determined as 12.2 nm, 6.1 nm, 6.1 nm, 4.9 nm and 2.9 nm in that order. The RG values were similar for mouse IgM P8 and its Fab'2 and Fab fragments, despite the presence of an additional carbohydrate site. The IgM scattering curves, to a nominal resolution of 5 nm, were compared with molecular graphics models based on published crystallographic alpha-carbon co-ordinates for the Fab and Fc structures of IgG. Good curve fits for Fab were obtained based on the crystal structure of Fab from IgG. A good curve fit was obtained for Fab'2, if the two Fab arms were positioned close together at their contact with the C mu 2 domains. The addition of the Fc fragment close to the C mu 2 domains of this Fab'2 model, to give a planar structure, accounted for the scattering curve of IgM-S. The Fc5 fragment was best modelled by a ring of five Fc monomers, constrained by packing considerations and disulphide bridge formation. A position for the J chain between two C mu 4 domains rather than at the centre of Fc5 was preferred. The intact IgM structure was best modelled using a planar arrangement of these Fab'2 and Fc5 models, with the side-to-side displacement of the Fab'2 arms in the plane of the IgM structure. All these models were consistent with hydrodynamic simulations of sedimentation data. The solution structure of IgM can therefore be reproduced quantitatively in terms of crystallographic structures for the fragments of IgG. Putative Clq binding sites have been identified on the C mu 3 domain. These would become accessible for interaction with Clq when the Fab'2 arms move out of the plane of the Fc5 disc in IgM, that is, a steric mechanism exposing pre-existing Clq sites. Comparison with a solution structure for Clq by neutron scattering shows that two or more of the six globular Clq heads in the hexameric head-and-stalk structure are readily able to make contacts with the putative Clq sites in the C mu 3 domains of free IgM if if the Clq arm-axis angle in solution is reduced from 40 degrees-45 degrees to 28 degrees. This could be the trigger for Cl activation.     

Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1

Immunoglobulin A (IgA), the most abundant human immunoglobulin, mediates immune protection at mucosal surfaces as well as in plasma. It exists as two subclasses IgA1 and IgA2, and IgA2 is found in at least two allotypic forms, IgA2m(1) or IgA2m(2). Compared to IgA1, IgA2 has a much shorter hinge region, which joins the two Fab and one Fc fragments. In order to assess its solution structure, monomeric recombinant IgA2m(1) was studied by X-ray and neutron scattering. Its Guinier X-ray radius of gyration R(G) is 5.18 nm and its neutron R(G) is 5.03 nm, both of which are significantly smaller than those for monomeric IgA1 at 6.1-6.2 nm. The distance distribution function P(r)for IgA2m(1) showed a broad peak with a subpeak and gave a maximum dimension of 17 nm, in contrast to the P(r) curve for IgA1, which showed two distinct peaks and a maximum dimension of 21 nm. The sedimentation coefficients of IgA1 and IgA2m(1) were 6.2S and 6.4S, respectively. These data show that the solution structure of IgA2m(1) is significantly more compact than IgA1. The complete monomeric IgA2m(1) structure was modelled using molecular dynamics to generate random IgA2 hinge structures, to which homology models for the Fab and Fc fragments were connected to generate 10,000 full models. A total of 104 compact best-fit IgA2m(1) models gave good curve fits. These best-fit models were modified by linking the two Fab light chains with a disulphide bridge that is found in IgA2m(1), and subjecting these to energy refinement to optimise this linkage. The averaged solution structure of the arrangement of the Fab and Fc fragments in IgA2m(1) was found to be predominantly T-shaped and flexible, but also included Y-shaped structures. The IgA2 models show full steric access to the two FcalphaRI-binding sites at the Calpha2-Calpha3 interdomain region in the Fc fragment. Since previous scattering modelling had shown that IgA1 also possessed a flexible T-shaped solution structure, such a T-shape may be common to both IgA1 and IgA2. The final models suggest that the combination of the more compact IgA2m(1) and the more extended IgA1 structures will enable human IgA to access a broader range of antigens than either acting alone. The hinges of both IgA subclasses appear to show reduced flexibility when compared to their equivalents in IgG, and this may be important for maintaining an extended IgA structure.     

The flexibility of low molecular weight double-stranded dna as a function of length: I. Isolation and physical characterization of seven fractions

Sonicated calf thymus DNA was fractionated by rate zonal centrifugation into seven fractions with weight average molecular weights ranging from 0.28 to 1.3 × 10⁶ daltons, as determined by sedimentation equilibrium and light scattering measurements (the latter are described in the accompanying paper). Electron microscopy and sedimentation equilibrium analysis revealed these fractions to be narrowly disperse with M_w/M_n ratios averaging about 1.06. Intrinsic viscosities and sedimentation rates were measured and found to vary linearly with molecular weight in double-logarithmic plots in fair agreement with previously published functions relating these parameters for low molecular weight DNA. The average value for β from the Mandelkern— Flory equation was 2.59 × l0⁶, also agreeing with reported estimates of this parameter for short DNA. These data will be used in the second paper of this series to calculate the persistence length of the DNA fragments in each of the seven fractions by light scattering and hydrodynamic theories for the Kratky—Porod worm-like coil.