Structure of unfolded and refolded recombinant derived [Ala125]interleukin 2

Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds. The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond. When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive. The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond. Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein. A similar conformation is obtained when [Ala125]interleukin 2 [IL-2(Ala-125)] is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid. The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2. In addition to this form, aggregates, as evidenced from gel filtration, are obtained. The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein. CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS.     

H2O2-induced intermolecular disulfide bond formation between receptor protein-tyrosine phosphatases

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) belongs to the subfamily of receptor-like protein-tyrosine phosphatases that are characterized by two catalytic domains of which only the membrane-proximal one (D1) exhibits appreciable catalytic activity. The C-terminal catalytic domain (D2) regulates RPTPalpha catalytic activity by controlling rotational coupling within RPTPalpha dimers. RPTPalpha-D2 changes conformation and thereby rotational coupling within RPTPalpha dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTPalpha from cells treated with H2O2 on non-reducing SDS-polyacrylamide gels to a position that corresponds to RPTPalpha dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTPalpha and constructs encoding the individual protein-tyrosine phosphatase domains, we located the intermolecular disulfide bond to the catalytic Cys-723 in D2. Disulfide bond formation and dimer stabilization showed similar levels of concentration and time dependence. However, treatment of lysates with dithiothreitol abolished intermolecular disulfide bonds but not stable dimer formation. Intermolecular disulfide bond formation and rotational coupling were also found using a chimera of the extracellular domain of RPTPalpha fused to the transmembrane and intracellular domain of the leukocyte common antigen-related protein (LAR). These results suggest that H2O2 treatment leads to oxidation of the catalytic Cys in D2, which then rapidly forms a disulfide bond with the D2 catalytic Cys of the dyad-related monomer, rendering an inactive RPTP dimer. Recovery from oxidative stress first leads to the reduction of the disulfide bond followed by a slower refolding of the protein to the active conformation.     

Initial steps in assembly of microfibrils. Formation of disulfide-cross-linked multimers containing fibrillin-1

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.     

Identification and role of the homodimerization interface of the glycosylphosphatidylinositol-anchored membrane type 6 matrix metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.     

Effect of the central disulfide bond on the unfolding behavior of elongation factor Ts homodimer from Thermus thermophilus

Functionally active elongation factor Ts (EF-Ts) from Thermus thermophilus forms a homodimer. The dimerization interface of EF-Ts is composed of two antiparallel beta-sheets that can be connected by an intermolecular disulfide bond. The stability of EF-Ts from T. thermophilus in the presence and absence of the intermolecular disulfide bond was studied by differential scanning calorimetry and circular dichroism. The ratio of the van't Hoff and calorimetric enthalpies, delta H(vH)/delta H(cal), indicates that EF-Ts undergoes thermal unfolding as a dimer independently of the presence or absence of the disulfide bond. This can be concluded from (1) the presence of residual secondary structure above the thermal transition temperature, (2) the absence of concentration dependence, which would be expected for dissociation of the dimer prior to unfolding of the monomers, and (3) a relatively low heat capacity change (delta Cp) upon unfolding. The retained dimeric structure of the thermally denatured state allowed for the determination of the effect of the intermolecular disulfide bond on the conformational stability of EF-Ts, which is deltadelta G(S-S,SH HS) = 10.5 kJ/mol per monomer at 72.5 degrees C. The possible physiological implications of the dimeric EF-Ts structure and of the intersubunit disulfide bond for the extreme conformational stability of proteins in thermophiles are discussed.     

A genetically engineered human IgG mutant with enhanced cytolytic activity.

A mutant chimeric anti-5-dimethylaminonaphthalene-1-sulfonyl human Ig gamma that exhibited augmented effector function was constructed. Utilizing directed mutagenesis, a serine residue near the carboxyl terminus of the human IgG1 H chain (Ser444) was replaced by a cysteine. Novel intermolecular disulfide bonds between Cys444 residues linked pairs of Ig "tail-to-tail" to form covalent dimers ((H2L2)2). These dimers were 200-fold more efficient, compared with monomeric human IgG1, at antibody-dependent complement-mediated cytolysis of hapten-bearing erythrocytes. The ability to enhance the cytolytic activity of an mAb by genetic engineering may be of value in immunotherapy.     

Synthesis of sulfhydryl cross-linking poly(ethylene glycol)-peptides and glycopeptides as carriers for gene delivery

Sulfhydryl cross-linking poly(ethylene glycol) (PEG)-peptides and glycopeptides were prepared and tested for spontaneous polymerization by disulfide bond formation when bound to plasmid DNA, resulting in stable PEG-peptide and glycopeptide DNA condensates. A 20 amino acid synthetic peptide possessing a single sulfhydryl group on the N-terminal cysteine, with two or five internal acetamidomethyl (Acm)-protected cysteine residues, was reacted with either PEG vinyl sulfone or iodoacetamide tyrosinamide triantennary N-glycan. Following RP-HPLC purification, Acm groups were removed by silver tetrafluoroborate to generate sulfhydryl cross-linking PEG-peptides and glycopeptide that were characterized by either (1)H NMR or LC-MS. Sulfhydryl cross-linking PEG-peptides and glycopeptides were found to bind to plasmid DNA and undergo disulfide cross-linking resulting in stable DNA condensates with potential utility for in vivo gene delivery.     

Synthesis of a disulfide-linked octameric peptide construct carrying three different antigenic determinants

In an effort to develop peptide vaccines against the influenza virus, we have successfully synthesized a disulfide-linked octameric homodimer that bears four copies of the influenza virus M2 protein ectodomain as well as two copies each of T-helper cell hemagglutinin epitopes, the I-E(d) restricted S1 and the I-A(d) restricted S2 fragments. Peptide attachment was via intermolecular disulfide formation from free sulfhydryl-bearing cysteine derivatives in solution. This reaction was efficient only when the amino-group of the cysteine was Fmoc-protected.     

A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization

Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.     

Using intramolecular disulfide bonds in tau protein to deduce structural features of aggregation-resistant conformations

Because tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence the aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, although there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. While it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are few data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, which contains all four microtubule binding repeats, and isolate the monomeric fraction, which corresponds to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-β-structure. These data further our understanding of the precise structural features that retard tau aggregation.     

The arrangement of intra- and intermolecular disulfide bonds in the carboxyterminal, non-collagenous aggregation and cross-linking domain of basement-membrane type IV collagen

The hexameric complex of globular domains of type IV collagen was isolated after collagenase digestion of human placenta and the different monomers and dimers present were chromatographically separated. The ratio of alpha 1(IV)NC1 to alpha 2(IV)NC1 was 2:1. About 50% of the NC1 domains were connected to dimers. Predominantly alpha 1-alpha 1 dimers were found. Only 12% were alpha 2-alpha 2 dimers and no alpha 1-alpha 2 dimers could be detected. The majority (88%) of the intermolecular bonds was found to be disulfide bridges. The remainder could not be cleaved by reduction. To elucidate the arrangement of the disulfide bonds, the unreduced alpha 1(IV)NC1 monomers were treated with cyanogen bromide, the disulfide-bridged peptides isolated and characterized by Edman degradation. Each of the two homologous subdomains within a monomer is stabilized by an identical set of three disulfide bonds. In subdomain I, cysteines at positions 20 and 53 are connected with the C-terminal cysteine pair 108 and 111. Thus formed, the disulfide knot stabilizes two interconnected loops of 32 and 54 residues, respectively. A smaller loop of five residues occurs due to a disulfide bond between the cysteines 65 and 71. A similar disulfide arrangement is indicated for subdomain II which is separated from subdomain I by a segment of 20 amino acid residues. The same arrangement of disulfide bonds has been strongly suggested for the alpha 2(IV)NC1 monomer by the isolation and characterization of its disulfide-bridged tryptic fragments. Similar investigations on the dimeric alpha 1(IV)NC1 domain established the arrangement of the intermolecular disulfide bonds. They are formed by a complete disulfide exchange between corresponding disulfide knots of two monomeric NC1 domains.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde reductase, aldehyde oxidase and xanthine oxidase from horse tissues

Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AHD), aldehyde reductase (AHR), aldehyde oxidase (AOX) and xanthine oxidase (XOX) extracted from horse tissues were examined. Five ADH isozymes were resolved: three corresponded to the previously reported class I ADHs (EE, ES and SS) (Theorell, 1969); a single form of class II ADH (designated ADH-C2) and of class III ADH (designated ADH-B2) were also observed. The latter isozyme was widely distributed in horse tissues whereas the other enzymes were found predominantly in liver. Four AHD isozymes were differentially distributed in subcellular preparations of horse liver: AHD-1 (large granules); AHD-3 (small granules); and AHD-2, AHD-4 (cytoplasm). AHD-1 was more widely distributed among the horse tissues examined. Liver represented the major source of activity for most AHDs. A single additional form of NADPH-dependent AHR activity (identified as hexonate dehydrogenase), other than the ADHs previously described, was observed in horse liver. Single forms of AOX and XOX were observed in horse tissue extracts, with highest activities in liver.     

Close location of the first loop to the third loop of the mitochondrial ADP/ATP carrier deduced from cross-linking catalyzed by copper-o-phenanthroline of the solubilized carrier with Triton X-100

Effects of the cross-linking catalyst copper-o-phenanthroline [Cu(OP)2] on the bovine heart mitochondrial ADP/ATP carrier solubilized with Triton X-100 were studied under various conditions. Without detergent treatment, Cu(OP)2 specifically catalyzed the formation of intermolecular disulfide bridges in submitochondrial particles between two Cys56 residues in the first loop facing the matrix space of the dimeric carrier [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J. Biol. Chem. 270, 29548-29554]. However, an intramolecular disulfide bridge between Cys56 and Cys256 in the third loop was formed in the solubilized carrier. Proteolytic digestion of the carrier with lysylendopeptidase showed that it first cleaves the Lys42-Gln43 bond and then the Lys48-Gln49 bond of the first loop in the membrane-bound carrier, but it cleaves both sites almost simultaneously in the solubilized carrier. These features were observed only with the m-state carrier; the c-state carrier was not subject to any cross-linking or proteolytic digestion. It is suggested that the protruding first loop is located close to the third loop, which could be exposed to a certain degree.     

Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.     

Isoelectric focusing studies of aldehyde dehydrogenases from mouse tissues: variant phenotypes of liver, stomach and testis isozymes

Isoelectric focusing techniques (IEF) were used to examine the tissue distribution and genetic variability of aldehyde dehydrogenases (AHDs) from inbred strains of mice. Twelve zones of AHD activity were resolved which were differentially distributed between tissues. Liver extracts exhibited highest activity for most enzymes, with the exception of isozymes found in stomach (AHD-4) and testis (AHD-4 and AHD-6). Genetic variants for AHD-1 (liver mitochondrial isozyme) and AHD-4 (stomach isozyme) were examined from inbred strains and F1 hybrid animals. The results were consistent with dimeric subunit structures (designated as A2 and D2 isozymes respectively). IEF patterns for activity variants of testis-specific AHD-6 were identical, with 3-banded phenotypes being observed. pI values for the AHD forms as well as for aldehyde oxidase and xanthine oxidase isozymes, which stain in the absence of coenzyme, were reported.     

Disulfide bonds in homo- and heterodimers of EF-hand subdomains of calbindin D9k: stability, calcium binding, and NMR studies.

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39-->Cys, Ile 73-->Cys, and Pro 43-->Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 degrees C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond. Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive co-operativity, and an average affinity of 10(6) M-1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment Tm increases from 59 +/- 2 without covalent linkage to 91 +/- 2 degrees C with disulfide, and Cm from approximately 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.     

Analysis of the disulfide bond arrangement of the HIV-1 envelope protein CON-S gp140 ΔCFI shows variability in the V1 and V2 regions

Disulfide bonding of cysteines is one of the most important protein modifications, and it plays a key role in establishing/maintaining protein structures in biologically active forms. Therefore, the determination of disulfide bond arrangement is one important aspect to understanding the chemical structure of a protein and defining its functional domains. Herein, aiming to understand how the HIV-1 envelope protein's structure influences its immunogenicity, we used an MS-based approach, liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance (LC/ESI-FTICR) mass spectrometry, to determine the disulfide linkages on an oligomeric form of the group M consensus HIV-1 envelope protein (Env), CON-S gp140 ΔCFI. This protein has marked improvement in its immunogenicity compared to monomeric gp120 and wild-type forms of gp140 Envs. Our results demonstrate that the disulfide connectivity in the N-terminal region of CON-S gp140 ΔCFI is different from the disulfide bonding previously reported in the monomeric form of gp120 HIV-1 Env. Additionally, heterogeneity of the disulfide bonding was detected in this region. These data suggest that the V1/V2 region does not have a single, conserved disulfide bonding pattern and that variability could impact immunogenicity of expressed Envs.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-alpha.

The putative receptor-binding region of human transforming growth factor-alpha (TGF alpha) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)2 on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70% yield.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of reassembly of immunoglobulin G.

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