Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.     

Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies

Binding of fluorescyl ligand by five IgG anti-fluorescyl hybridoma proteins (4-4-20, 6-10-6, 20-4-4, 20-19-=1, 20-20-3) was examined. Relative reduction in fluorescence of bound fluorescein, deuterium oxide (D2O)-induced enhancement of fluorescence, and the effects of pH on binding kinetics were measured for each clone. Individual hybridoma proteins (all of which bind fluorescein with relatively high affinity) exhibited significant differences in the relative contribution of various forces (hydrophobicity, hydrogen bonding, ionic interactions) to binding and hence, affinity. The extent of such variations in binding mechanisms among monoclonal antibodies binding the same hapten is indicative of the extreme functional diversity of active sites. In addition, ligand binding by clone 20-20-3 was examined in greater detail. ABsorption spectra of ligand bound by purified intact antibody, Fab fragments, and reassociated heavy and light chains indicated that protonation of the fluorescyl ligand by a residue within the active site contributed significantly to the binding free energy. Comparative dissociation rates of fluorescein and a structural analog, rhodamine 110, were used to quantitatively substantiate the contribution of this interaction. Association and dissociation rate studies with fluorescein and antibody indicated that: 1) the active site appeared to undergo a conformational change upon ligand binding, and 2) neither intact disulfides nor intersite cooperativity affected the dissociation rate of bound ligand. Observed mechanisms of ligand binding are discussed in terms of proposed mechanisms of antibody affinity maturation and diversity.     

Structural and antigenic studies of an idiotype-bearing murine antibody to the arsonate hapten.

Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.     

Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.

This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.     

Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten-antibody complex. The extent of covalent labelling was 0.5-0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3-5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29-34 and 95-114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.     

Interaction of antibodies specific towards the 2,4-dinitrophenyl group and of their fragments with hapten

The interaction of hapten (ε-DNP lys) with native and S-sulfonated antibodies specific towards the 2,4-dinitrophenyl group, as well as the interaction with isolated chains and a complex obtained by mixing light, (L) and heavy (H) chains of these antibodies, were followed both by polarography and by equilibrium dialysis. With the S-sulfonated antibodies and with the mixture of H and L chains the binding heterogeneity observed in the original antibodies was much lowered or entirely removed. At the same time, the amount of active proteins in the sample decreased approximately by half. The association constants of modified antibodies were of the same order as the average association constants of the original antibodies. A slow increase of the amounts of hapten bound with proteins was observed on mixing the H and L chains and adding hapten. This slow reactivation was not obtained with the original or S-sulfonated antibodies and with isolated chains. It was shown that the reaction determining the kinetics of this reactivation (the slowest reaction) was not the association of H and L chains but the interaction of complexes of the H and L chains with hapten. It was reported previously that H chains were nonspecifically reactivated by binding L chains. The amount of hapten bound by the complex of H and L chains increased with increasing excess of L chains following a curve resembling the Langmuir isotherm. The limiting value of the amount of hapten bound when using antibody L chains was higher than in the case of nonspecific L chains.     

Monoclonal antibodies to streptococcal group A carbohydrate. II. The VK1GAC light chain is preferentially associated with serum IgG3

A kappa-light chain variable region (V kappa) dominantly employed in the serum antibody response of A/J mice to streptococcal group A carbohydrate (GAC) has been termed VK1GAC. Examination of in vitro recombinants between the isolated heavy and light chains of VK1GAC+ and VK1GAC-anti-GAC hybridomas and non-GAC-binding myeloma proteins indicated that two antisera (anti-Id5 and anti-Id20) recognized the VK1GAC light chain when it was free in solution or paired with several heterologous heavy chains. Screening of a panel of A/J anti-GAC monoclonal antibodies with these antisera showed almost complete concordance between Id5 and Id20 expression and the presence of VK1GAC light chain as detected by its unique isoelectric focusing spectrotype. These antisera were used to examine serum expression of the VK1GAC light chain in normal and hyperimmune serum of A/J mice. Normal A/J serum contained from 20 to 100 micrograms Id5/ml serum, whereas only 1 to 10 micrograms Id20/ml serum was detected. The levels of both VK1GAC idiotypes increased dramatically 10- to 20-fold after hyperimmunization of mice with group A vaccine. When serum IgG from normal and immune mice was fractionated into the IgG subclasses (IgG1, IgG2a, and IgG3), it was found that the VK1GAC light chain does not pair randomly with heavy chains of the IgG subclasses, but rather is associated preferentially with heavy chains of the IgG3 subclass whether or not it is associated with antibodies to GAC. These results suggest that the heavy chain pairing exhibited by this VK product may not be random.     

The binding of haptens by the polypeptide chains of rabbit antibody molecules

1. The binding of haptens by the polypeptide chains derived from two rabbit immunoglobulin G antibodies was examined by gel chromatography and equilibrium dialysis. 2. The gamma chains were examined in a dilute sodium acetate buffer, pH5.4, in which they exist as a monodisperse solution of dimers; aggregation of the protein promoted by some haptens had to be avoided. These chains exhibited variable extents of binding, reflecting the specificities of the parent antibody molecules, usually with only small increments above the binding by gamma chains from normal immunoglobulin G. 3. The light chains existed as an interconverting mixture of monomers and dimers in all buffers of near neutral pH that were examined. They bound small amounts of hapten, again broadly reflecting the specificities of the parent antibody molecules. 4. For both the gamma and light chains the dimeric state appeared necessary for appreciable binding of hapten. Apparently in each case the partners in the dimer interact in a manner analogous to the gamma chain-light chain interaction in the parent antibody molecule, to give a site analogous to the antibody site. This implies that the binding of antigens by isolated chains has a large fortuitous element, providing no reliable indication of their contributions to the original antibody sites.     

Three-dimensional structure of a fluorescein-Fab complex crystallized in 2-methyl-2,4-pentanediol

The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.     

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Biochemical and immunological studies on clathrin light chains and their binding sites on clathrin triskelions

Clathrin light chains from bovine brain tissue (LC alpha and LC beta) are monomeric proteins with an average mol. wt. of approximately 33,000, as determined by sedimentation equilibrium. Solution studies on purified light chains indicate a large Stokes radius (Re = 3.3 nm) and little defined secondary structure. Both light chains bind specifically and with high affinity (KA approximately 5 x 10(7)/M) to overlapping sites on clathrin heavy chains. These binding sites are contained within a 125,000 dalton heavy chain fragment that forms truncated triskelions with legs, 15 nm shorter than those of intact triskelions. As judged by immuno-electron microscopy, light chain-specific IgG molecules bind mostly to the center of triskelions, but there are also sites that are scattered some 16 nm along the proximal part of triskelion legs. From heterologous binding experiments using human placenta light chains and heavy chain fragments from bovine brain clathrin, it is concluded that the domains of light and heavy chains that are involved in the interaction are conserved across tissue and species boundaries.     

Influence of the immunoglobulin heavy chain locus on expression of the VK1GAC light chain

The VK1GAC light chain represents the dominant V kappa structure employed in the antibody response of A/J mice to streptococcal group A carbohydrate ( GAC ). Two anti-idiotypic antisera, anti- Id5 and anti- Id20 , with specificity for the VK1GAC light chain were used to examine anti- GAC antibody responses in a series of inbred mouse strains that differ at the heavy chain constant region ( IgCH ) allotype locus. Both idiotypes were expressed in normal and immune sera from mice of most IgCH allotypes, except IgCHb (C57BL/6J) and IgCHf (CE/J). C57BL/6J mice expressed Id5 , but not Id20 , whereas CE/J mice did not express either idiotype. Testing of recombinant inbred strains between BALB/c and C57BL/6 indicated that the pattern of idiotype expression did not correlate with IgCH allotype. The C X B recombinants expressed all three idiotype patterns that were observed in the panel of inbred strains. Testing of allotype congenic mice between BALB/c and C57BL/6 showed that CB.20 and BC.8 mice were Id20 -, whereas BAB-14 mice were Id20 +, indicating that both VH and background (V kappa or regulatory) loci must be derived from BALB/c to obtain Id20 expression. The difference in the frequency of idiotype expression observed between BALB/c and BAB-14 mice indicates that the IgCH locus may exert a quantitative influence on the expression of this light chain. To examine the Id20 -, Id5 + antibodies of C57BL/6 mice, anti- GAC hybridomas were prepared. Of 16 C57BL/6-derived anti- GAC monoclonal antibodies, six were reactive with anti- Id5 and not with anti- Id20 . Isoelectric focusing of the purified kappa light chains from three of these antibodies revealed two distinct spectrotypes that co-migrated with the two known VK1GAC spectrotypes observed with A/J anti- GAC light chains. Idiotypic analysis of in vitro recombinants between the heavy and light chains of A/J and C57BL/6 monoclonal antibodies demonstrated that the C57BL/6 light chains were idiotypically similar to A/J light chains when they were free in solution or paired with A/J heavy chains. These results demonstrate that C57BL/6 mice can express a light chain that is very similar, if not identical, to the VK1GAC light chain, although the light chain is expressed in lower frequency and is paired with a distinct VH structure, which can mask expression of one of the VK1GAC idiotypes. These effects on V kappa expression map to at least three genetic loci: VH, CH, and an unlinked locus.     

Vasoactive intestinal peptide hydrolysis by antibody light chains

This paper describes evidence for hydrolysis of a neuropeptide, vasoactive intestinal peptide (VIP), by light chains purified from the IgG of a human subject positive for VIP binding antibodies. Purified IgG was digested with papain, resultant fragment antigen binding (Fab) fragments were reduced with 2-mercaptoethanol and alkylated with iodoacetamide, and light chains were purified by chromatography on immobilized antibodies to light chains and immobilized antibodies to heavy chains. Non-immunoglobulin components were undetectable in the light chain preparation, judged by sodium dodecyl sulfate-electrophoresis and Western blotting with anti-heavy and anti-light chain antibodies. The light chains hydrolyzed VIP with specific activity 32-fold greater than that of Fab, the pH optimum for light chain-mediated VIP hydrolysis was 7.0-7.5, and the hydrolytic activity was saturable (Vmax, 0.19 pmol/min/microgram light chains; substrate concentration at Vmax/2,380 nM).     

Structural transitions in bovine factor X associated with metal binding and zymogen activation. Studies using conformation-specific antibodies

Conformation-specific antibodies against distinct regions of Factor X were employed to locate antigenic determinants which are altered during zymogen activation or by metal binding. Anti-Factor X antibodies, raised in rabbits against Factor X, were purified by affinity chromatography using Factor X covalently bound to Sepharose. Quantitative equilibrium and kinetic measurements of precipitation of Factor X and Factor Xa by antibodies indicated differences in the antigenic structure of the zymogen and the enzyme form of factor X. The factor X antibodies were further fractionated by sequential immunoabsorption using fragments of Factor X and Factor Xa. With conformation-specific antibodies directed against the heavy chain and the light chain of Factor X, zymogen activation was shown to involve a structural transition in the heavy chain but not the light chain. Antibodies directed against the activation peptide domain 1-51 of the heavy chain, the trypsin-like region of the heavy chain 52-290, and the substrate-binding site suggest a generalized conformational transition in the heavy chain. Antibodies were isolated which are specific for the Factor X:Ca(II) complex and bind to Factor X only in the presence of metal ions. Subfractions were directed against either the heavy chain or the light chain, indicating that both the heavy chain and the light chain of Factor X undergo a metal-induced conformational transition. Half-maximal antibody-factor X interaction was observed at 0.13 mM CaCl2 for the light chain and 0.7 mM CaCl2 for the heavy chain. These results indicate that zymogen activation is limited to structural changes in the heavy chain, but metal binding is associated with changes in the structure of both the heavy and light chains. Metal-dependent binding of Factor X to the platelet Factor Xa receptor after activation may involve surfaces of the heavy as well as the light chains.     

Regulation of immunoglobulin biosynthesis in the murine plasmacytoma MOPC 315.

We have examined certain aspects of IgG biosynthesis by constructing hybrids between MPC11 (gamma2b, kappa) and MOPC 315 (alpha,lambda2) that have lost the ability to synthesize one or the other heavy chain. Cells express the three chains in a stable fashion, and both autologous (parental) and heterologous (nonparental) H and L chain pairs form and are secreted. The alpha H chain was found in polymeric form when associated with the heterologous kappa L chain. The lambda2 L chain covalently assembled to the heterologous gamma2b H chain. Surprisingly, autologous pairing was always favored over heterologous pairing in vivo by 5 to 10:1 in terms of rate of assembly. Similar ratios were maintained in the secreted protein. These results suggest that co-expression of particular H and L chain pairs is predetermined. Evolution presumably operates to improve antigen recognition as well as rate of assembly of active molecules.     

Purification of messenger ribonucleic acids for fast and slow myosin heavy chains by indirect immunoprecipitation of polysomes from embryonic chick skeletal muscle

Fast and slow myosin heavy chain mRNAs were isolated by indirect immunoprecipitation of polysomes from 14-day-old embryonic chick leg muscle. The antibodies were prepared against myosin heavy chains purified by NaDod-SO4-polyacrylamide gel electrophoresis and were shown to be specific for fast and slow myosin heavy chains. The RNA fractions directed the synthesis of myosin heavy chains in a cell-free translation system from wheat germ. Several smaller peptides were also synthesized in lower concentrations. These probably are partial products of myosin heavy chains, since they are immunoprecipitated with antibodies to myosin heavy chains. Immunoprecipitation of the translation products with the antibodies to fast and slow myosin heavy chains showed the RNA preparations to be approximately 94% enriched for fast myosin heavy chain mRNA and approximately 84% enriched for slow myosin heavy chain mRNA with respect to myosin HC type. Peptides having slightly different mobilities on NaDodSO4-polyacrylamide gels were immunoprecipitated by antibodies to fast and slow myosin heavy chains.     

Mapping of four light chain-associated idiotopes of a human monoclonal rheumatoid factor

In an effort to analyze both IgM rheumatoid factor (RF) repertoire and regulation of RF production in humans, we developed a panel of four mouse monoclonal antibodies (mAb) defining distinct K light chain-associated idiotopes (id) of a human monoclonal IgM RF (Alt). These mAb (A75, AM1, AM2, AM3) had equivalent reactivities with the immunizing RF during classic inhibition of antigen-binding assays. These anti-id reagents were reacting to neither other tested monoclonal IgM RF nor normal polyclonal IgM. It was possible to distinguish the id defined by the mAb from the results of four sets of experiments: dissociation of Alt RF heavy (H) and light (L) chains showed that A75, AM1, and AM2 reacted to id located on the L chain, whereas AM3 defined a conformational RF id; recombination experiments of H and L chains showed that A75 and AM2 reacted well with both homologous (Alt H + Alt L) and heterologous (Alt L + unrelated H) recombinants, whereas AM1 reacted better with the homologous recombinant than with the heterologous one; the relative affinities of the mAb were drawn from their ability to shift already bound labeled Alt RF from solid phase IgG; and radiolabeling of two mAb (A75 and AM3) and experiments of inhibition of id binding with cold anti-id and cold anti-CK showed that A75 recognized a proximal id (close to the K constant region), whereas AM3 defined a more distal id, AM2 and AM1 being located between A75- and AM3-defined sites. This topographic mapping of K light chain-associated id of a human RF with anti-id of known relative affinities could help in studying idiotypic regulation in humans.     

Two-Dimensional nmr studies of the interactions between a peptide of cholera toxin and monoclonal antibodies

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody–peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab–peptide complex (Fab: antibody fragment made of Fv—the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen—the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3). which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by the antibodies, the structure of their combining sites, and relationships between antibodies' primary structure and their interactions with peptide antigens. © 1994 John Wiley & Sons, Inc.     

Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.     

Monoclonal antibodies to human protein C: effects on the biological activity of activated protein C and the thrombin-catalyzed activation of protein C1

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.