Synthetic immunogens. Part IV: Conformational studies on gastrin conjugates with the human immunoglobulin G1 hinge peptide 225-232/225'-232'

Hybrids of the double-chain bis-cystinyl fragment 225-232/225'-232' of human immunoglobulin G1 (IgG1) with the human little-gastrin sequence 2-17 were found to induce in animals a strong antigastrin humoral immune response with antibody titers comparable to those raised with conventional gastrin/carrier-protein conjugates. The observed gastrin receptor-like specificity of the polyclonal antibodies led to the assumption that the gastrin component of the hybrids is still capable of folding into its preferred bioactive structure and thus to express a similar conformational epitope in the dynamic process of recognition by the B-cell receptors. CD measurements on these hybrid compounds in aqueous and aqueous organic media confirmed the free conformational space for the antigenic gastrin moiety, which is essentially randomly coiled in aqueous solution but retains its ability to fold into the gastrin-specific ordered structure in aqueous organic media as used to mimic the water-limited environment of peptides while interacting with target cells at receptor level. The absence of reciprocal conformational restriction in such hybrid molecules suggests that a compact, rigid heterodetic cyclic structure as the hinge peptide is well suited for the multiple attachment of antigenic sequences in view of the preparation of fully synthetic immunogens.     

Interconversion of conformational isomers of light chains in the Mcg immunoglobulins.

Previous crystallographic studies in this laboratory demonstrated that immunoglobulin light chains with the same amino acid sequence can have at least two and probably three or more conformations, depending on whether the second member of an interacting pair is a light or heavy chain. If a heavy chain is not available in the assembly medium, a second light chain plays the structural role of the heavy chain in the formation of a dimer. In the present work, the lambda-type light chains were dissociated from the heavy chains of a serum IgG1 immunoglobulin from the patient Mcg and reassembled noncovalently into a dimer. The reassembly process was completed by allowing the penultimate half-cystine residues to form an interchain disulfide bond. The covalently linked dimer was compared with the Mcg urinary Bence-Jones dimer, for which an atomic model has been fitted to a 2.3-A electron density map. The assembled dimer and the native Bence-Jones protein were indistinguishable in their chromatographic and electrophoretic properties, as well as in their activity in the binding of bis(dinitrophenyl)lysine. These results indicate that the light chains can be converted into the two types of Bence-Jones conformational isomers. The procedure was also reversed: the two Bence-Jones isomers were dissociated and reassembled as the single type of isomer associating with each of two heavy chains in the IgG1 protein. The change in activity occurring when a light chain associates with a heavy chain instead of a second light chain is illustrated by the fact that the Mcg IgG1 immunoglobulin does not bind dis(dinitrophenyl)lysine in measurable amounts.     

Fully synthetic immunogens. Part II. Studies on parallel dimerization of the human IgG1 hinge-fragment 225-232 with N- or C-terminal gastrin related extensions

The bis-cysteinyl hinge-fragment 225-232 of human IgG1 has been extended at the N- or C-terminus with Nle15-desamido-human-little-gastrin-[5-17] and Nle15-human-little-gastrin-[5-17]-NH2, respectively. Thermodynamically controlled air oxidation of the resulting bis-cysteinyl-peptides led to the predominant formation of the corresponding dimers in parallel alignment despite the incorporation of the immunoglobulin-unrelated gastrin-sequences. These surprising results confirm the high degree of structural information inherent in the hinge-sequence and its intrinsic tendency to fold into the correct structure in terms of cysteine pairings. This protein subdomain-the hinge-peptide-is therefore well suited as core molecule for the design of fully synthetic immunogens with multiple attachment of antigenic determinants.     

Antiparallel alignment of the two protomers of the regulatory subunit dimer of cAMP-dependent protein kinase I

The purified type I regulatory subunit of cAMP-dependent protein kinase is a dimeric protein, and the two protomers of the dimer are linked by two interchain disulfide bonds. The disulfide linkages that join these two polypeptide chains have been identified in order to provide a structural basis for the orientation of the two chains in the asymmetric dimer. Disulfide bonds were found to exist exclusively between Cys-16 and Cys-37, and this assignment, thus, establishes a general antiparallel alignment of the two chains. Two other homologous proteins, the type II regulatory subunit and the cGMP-dependent protein kinase also are dimeric proteins. In all three proteins, a relatively small, nonhomologous, amino-terminal segment of the polypeptide chain is essential for maintaining the dimeric aggregation state.     

Fully synthetic immunogens. Part III. Synthesis of hinge-peptide/gastrin conjugates and their immunological properties.

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.     

Polar residues in membrane domains of proteins: molecular basis for helix-helix association in a mutant CFTR transmembrane segment

Polar side chains constitute over 20% of residues in the transmembrane (TM) helices of membrane proteins, where they may serve as hydrogen bond interaction sites for phenotypic polar mutations that arise in membrane protein-related diseases. To systematically explore the structural consequences of H-bonds between TM helices, we focused on TM4 of the cystic fibrosis conductance regulator (CFTR) and its cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model system. Synthetic peptides corresponding to wild-type (TM4-wt) (residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE arising from disulfide bond formation via wild-type residue Cys-225. However, the TM4-V232D peptide additionally forms a ladder of noncovalent oligomers, including tetramers, hexamers, and octamers, mediated by a hydrogen bond network involving Asp-Gln side chain-side chain interactions. Ala-scanning mutagenesis of the TM4 sequence indicated that ladder formation minimally required the simultaneous presence of the Cys-225, Asp-232, and Gln-237 residues. As random hydrophobic sequences containing these three residues at TM4 equivalent positions did not oligomerize, specific van der Waals packing interactions between helix side chains were also shown to play a crucial role. Overall, the results suggest that polar mutations in membrane domains, in conjunction with critically positioned polar partner residues, potentially constitute a source of aberrant helix interactions that could contribute to loss of function when they arise in protein transmembrane domains.     

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.     

The amino acid sequence of the light chain of human high-molecular-mass kininogen

The complete amino acid sequence of the light chain of human high-molecular-mass kininogen has been determined. The peptide chain contains 255 amino acid residues. The half-cystine, which forms the disulfide bridge to the heavy chain, was identified in position 225. Nine carbohydrate attachment sites were found. All carbohydrate side chains are O-glycosidically linked. Alignment of the present sequence with the bovine kininogen light chain sequence shows a high degree of homology, except for an extension of 22 amino acids within the histidine-rich part of the sequence. The histidine-rich region may have arisen by gene multiplication during evolution.     

Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation

Human fibrinogen is a dimer composed of two identical halves. Each dimeric half contains three peptide chains (alpha, beta, and gamma) linked by disulfide bonds. The two half-molecules are joined by three disulfide bonds, one between the two alpha-chains (residue alpha-28) and two between the two gamma-chains (residues gamma-8 and gamma-9). In the absence of any difinitive experimental evidence, it has been presumed that the joined halves were aligned in a parallel orientation similar to the situation found in immunoglobulins. We have now determined that the two gamma-chains--hence, the dimeric halves--are connected in an antiparallel manner. A tryptic peptide containing gamma-chain residues 6-14 was isolated as a disulfide-linked dimer from CNBr-treated fragment E. Synthetic peptides corresponding to this sequence were prepared, from which parallel and antiparallel dimers were constructed. During the syntheses, cysteine thiol groups were protected as p-methoxybenzyl and acetamidomethyl sulfides; the peptides were dimerized by selective deprotection and disulfide bond formation. First, the p-methoxybenzyl groups were removed by liquid hydrogen fluoride and the newly exposed thiols oxidized in the presence of potassium ferricyanide. Then the monocystine compound was converted to the double-cystine product by iodolytic cleavage of the acetamidomethyl group with concomitant disulfide bond formation. This selectivity was used to prepare peptide dimers which modeled both parallel and antiparallel arrangements. The antiparallel-oriented synthetic peptide was indistinguishable from the native tryptic peptide as judged by elution from reverse-phase high-performance liquid chromatography and circular dichroism spectroscopy. The parallel-oriented synthetic peptide differed from the native material by both criteria.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Design and characterization of the anion-sensitive coiled-coil peptide.

As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides.     

Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein

A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s). A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein. The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end. Five potential N-glycosylation sites were found in each of the two chains of factor XI. The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI. Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide. Each repeat probably forms a separate domain containing three internal disulfide bonds. The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases. The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein.     

Formation of intermolecular disulfide bonds on nascent immunoglobulin polypeptides.

The initial step of intermolecular covalent assembly of immunoglobulins molecules involves formation of heavy chain-light chain or heavy chain-heavy chain disulfide bonds. Using QAE-Sephadex chromatography to isolate microsomal nascent polypeptides, we have shown that this initial step of intermolecular covalent assembly occurs, to a substantial extent, on nascent heavy chains, as well as on completed heavy chains as previously demonstrated by others. In MPC 11 mouse myeloma cells, completed light chains are assembled covalently to nascent heavy chains, whereas in MOPC 21 mouse myeloma cells, completed heavy chains are assembled covalently to nascent heavy chains. These results are consisted with the heavy-light half-molecule being the major initial intermediate in the assembly of MPC 11 IgG2b and heavy-heavy dimer being the major initial intermediate formed in assembly of MOPC 21 IgG1. The nascent MPC 11 heavy chain must be at least 38,000 daltons in size before assembly with the light chain occurs, even though the heavy chain cysteine involved in this disulfide bond is 131 residues (approximately 15,000 daltons) from the NH2 terminus. In addition, pulse-chase labeling studies of MPC 11 cells have shown that the assembly of completed light chains with the nascent heavy chain must occur within a few minutes of the synthesis of the light chain even though a large excess of unassembled MPC 11 light chains remain inside the cell for an average time of 2 h before being secreted.     

Binding of secretory component to dimers of immunoglobulin A in vitro. Mechanism of the covalent bond formation.

A complex between secretory component and an immunoglobulin A (IgA) myeloma dimer has been studied in vitro as a model to elucidate the mechanism of the formation of disulfide bonds during assembly in vivo of secretory immunoglobin A. A small amount of free thiol groups, totally about 0.4 groups per mole of protein, were shown to be present on both the heavy and light chains of the IgA dimer, but not on its J-chain, while no such groups could be demonstrated on free secretory component. The SH-groups on IgA most likely exist as a result of incomplete oxidation of some intra-or interchain disulfide bonds of the molecule, analogous to what has been suggested for IgG. Several types of evidence indicated that the disulfide bonds between secretory component and IgA are formed after the noncovalent association of the two proteins by a sulfhydryl group-disulfide bond exchange reaction, in which the small amount of free sulfhydryl groups on the IgA dimer initiate the reaction by reducing a reactive disulfide bond on secretory component. This exchange reaction, which thus proceeds by the mechanism of so-called disulfide interchange reactions, requires certain conformational features of one or both of the proteins and leads to the formation of presumably two new interchain disulfide bonds between secretory component and IgA. The reaction does not progress to completion, however, but ends in an equilibrium so that a small proportion of the secretory component molecules always are unattached by disulfide bonds.     

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.     

Alpha chain of HLA-DR transplantation antigens is a member of the same protein superfamily as the immunoglobulins

Four cDNA clones, pDR-α-1, pDR-α-2, pDR-α-3 and pDR-α-4, corresponding to the alpha chain of HLADR antigens, have been sequenced. Restriction maps and sequences suggest that all clones are identical apart from a single-base substitution present in pDR-α-1. Amino acid sequence data, together with the nucleotide sequence data, allowed the complete amino acid sequence to be predicted. The alpha chain is composed of 229 amino acids, of which 191 are exposed on the outside of the plasma membrane. The membrane-embedded portion of the chain consists of 23 hydrophobic amino acids. The succeeding 15 amino acids form the cytoplasmically localized hydrophilic tail. The extracellular portion, with carbohydrate moieties linked to Asn⁷⁸ and Asn¹¹⁸, seems to be organized into two domains. The second domain, which contains the only disulfide bond of the alpha chain, displays amino acid sequence homology to immunoglobulin constant regions, to the second domain of the beta chain of a class II antigen, to the third domain of heavy chains of class I antigens and to β2-microglobulin. Thus the subunits of immunoglobulins, class I antigens and class II antigens are related evolutionarily.     

On the genetic and structural similarities between the squash seeds polypeptide trypsin inhibitors and wheat germ agglutinin

Summary In this report we propose the disulfide bridges alignment in the squash polypeptide trypsin inhibitors. The prediction is based on the extensive homology in the amino acid sequence between these inhibitors and a portion of the wheat germ agglutinin domains for which the position of the disulfide bridges are known.     

Proof of a disulfide bridge accessible to disulfide exchange between the heavy chains of IgG

The paper deals with the direct experimental proof that human immunoglobulin G1 (IgG1) contains a reactive disulfide bond that can be opened by 3,3'-dithiobis(6-nitrobenzoate) (DTNB) within 24 h by a SH-catalysed disulfide exchange reaction. These results were obtained with the purified IgG1 myeloma protein and confirm earlier indirect evidence based on correlation analysis of DTNB reactivity and quantitative IgG1 determination. The reactive disulfide bond is most likely the one between Cys235 of the heavy chains in the "hinge"-region, activated for the disulfide exchange by the protonated amino groups of Lys231 as turned out by analysis of IgG1. As with the whole molecule, one mol of reactive disulfide was found per mol of the Fc-fragment. 0.8 mol of labile S-S bonds was detected per mol of F(ab)2. After separation of the excess of reagent, the sedimentation pattern still corresponded with the dimer. The unaltered antigenic properties as well as the crystallizability speak against any severe conformational changes. Therefrom it was concluded that in approximately 80% of the F(ab)2 molecules one of the two inter heavy chain-bridges was opened. With the isolated F(ab)-fragment a reaction with DTNB was ascertained to an extent of 20%, which is probably due to an altered stability of the heavy-light chain-SS-bridge. However, no influence on the sedimentation pattern was observed. The intrachainar disulfide bonds of neither the heavy nor the light chain reacted with DTNB to a measurable extent.     

Folding and oxidation of the antibody domain C(H)3

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.     

Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A).

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.