Biochem. J. (1997) 325, 707–710 (Printed in Great Britain)
`
`707
`
`Perturbation of the antigen-binding site and staphylococcal Protein
`A-binding site of IgG before significant changes in global conformation
`during denaturation: an equilibrium study
`Xi-De WANG*, Jie LUO*, Zhen-Quan GUO(cid:139), Jun-Mei ZHOU* and Chen-Lu TSOU*(cid:139)(cid:140)
`*National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China, and (cid:139)Department of Biology, Beijing University,
`Beijing 100871, China
`
`Although conformational perturbation of the active sites of
`many enzymes has been reported to precede global molecular
`conformational changes [Tsou (1993) Science 262, 380–381],
`little effort has been made to compare the susceptibility of the
`ligand-binding site of proteins and the protein molecules as a
`whole to perturbation by denaturants. Immunoglobulin is chosen
`in this study to address this problem. It is found that the variable
`and constant regions (Fv and Fc) of a monoclonal antibody of
`an IgG subclass against adenylate kinase lose their abilities to
`
`bind antigen and staphylococcal Protein A after treatment with
`guanidinium chloride concentrations considerably lower than
`those required to change the global conformation of the antibody
`as a whole, as detected by fluorescence and second-derivative UV
`absorption spectroscopy. These results indicate that both ligand-
`binding sites of the antibody concerned are more fragile than the
`molecule as a whole and that the Fv and Fc regions of the
`antibody molecule unfold sequentially during denaturation.
`
`INTRODUCTION
`Many enzymes had been reported to be inactivated by
`guanidinium chloride (GdmCl), urea or heat before gross mol-
`ecular conformational changes could be detected by conventional
`physicochemical methods [1–5] (for a review, see [5]). It has
`therefore been proposed that the active sites of enzymes are
`much more prone to perturbation during denaturation than the
`rest of the molecule [6]. However, the susceptibility to denaturants
`of the ligand-binding sites of proteins as compared with the
`global structure of the molecule has been little explored and it
`remains to be ascertained whether the functional sites of non-
`enzyme proteins are likewise more susceptible to perturbation
`than the molecules as a whole. Plasma fibronectin loses its
`agglutination activity at a lower temperature than that required
`to induce conformational changes as detected by its immuno-
`reactivity [7]. The inhibition of choline transport of the carrier
`protein in erythrocytes by n-alkanols involves the unfolding of a
`small but essential part of the molecule [8]. The interaction of the
`storage protein legumin from Vicia faba to form micelles was
`perturbed at urea concentrations lower than those required for
`global conformational changes of the molecule [9].
`Immunoglobulin is a Y-shaped molecule containing two
`ligand-binding sites, one for its specific antigen at the N-terminal
`variable region (Fv) and the other for staphylococcal Protein A
`(SpA) at the C-terminal CH2 and CH3 hinge region (Fc) [10]. In
`this investigation, changes in ligand-binding abilities of both the
`Fv and Fc regions of immunoglobulin are compared with global
`molecular conformational changes during denaturation in
`GdmCl of different concentrations.
`
`MATERIALS AND METHODS
`Reagents
`
`described previously [11]. Adenylate kinase was isolated from
`rabbit muscle [12]. Horseradish peroxidase-labelled SpA and
`goat anti-mouse IgG conjugate were purchased from Bio-Rad.
`Tetramethyl benzidine (TMB) was used as the substrate for
`horseradish peroxidase. GdmCl was an ultrapure product of
`Schwartz\Mann. Biotinamidocaproate N-hydroxysuccinimide
`ester (BNHS) and ExtrAvidin Peroxidase were purchased from
`Sigma. Avidin was the product of Serva. Other reagents were
`local products of analytical grade.
`
`Measurement of second-derivative absorption spectra
`Antibody at a final concentration of 175 lg\ml was incubated
`with different concentrations of GdmCl overnight at 4 mC. The
`second-derivative spectra were scanned at 20 mC using a Beckman
`DU-7500 spectrophotometer. The relative changes in the peak-
`to-trough distance between 287 and 283 nm of the second-
`derivative spectra in different concentrations of GdmCl were
`taken to represent exposure of the aromatic residues [13].
`
`Measurement of fluorescence
`Antibody at a final concentration of 16 lg\ml was incubated
`with different concentrations of GdmCl overnight at 4 mC. The
`intrinsic fluorescence emission spectra (excitation wavelength
`280 nm) between 300 and 400 nm were recorded at 20 mC with a
`Perkin–Elmer MPF-66 fluorescence spectrophotometer. The
`bandwidths for both excitation and emission were 10 nm. The
`shifts of maximum emission wavelength from native state were
`taken to represent the relative changes in fluorescence for
`denatured antibodies.
`
`Determination of Fc-binding activity
`
`The monoclonal antibody McAb3D3 of the IgG subclass was
`raised against adenylate kinase (EC 2;7;4;3), and purified as
`
`Binding of antibody McAb3D3 to horseradish peroxidase-
`labelled SpA was taken as a measure of the binding activity of
`
`Abbreviations used : Fc, constant region of IgG (CH2 and CH3 domains) ; Fv, variable region of IgG (VH and VL domains) ; GdmCl, guanidinium
`chloride ; PBST, PBS/Tween ; SpA, staphylococcal Protein A ; TMB, tetramethyl benzidine ; BNHS, biotinamidocaproate N-hydroxysuccinimide ester.
`(cid:140) To whom correspondence should be addressed.
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`X.-D. Wang and others
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`the Fc region. The antibodies (McAb3D3) were first coated on to
`the wells of microplates by overnight incubation in PBS at 4 mC.
`Wells were then further treated with 5 mg\ml gelatin, after
`which GdmCl of different concentrations was added to wells in
`triplicate. The supernatants were decanted after 1 h of incubation
`and the wells thoroughly washed with PBST (0n5 % Tween 20 in
`PBS) to remove denaturants. Labelled SpA was then added and
`incubated for 1 h. After being rinsed three times with PBST,
`TMB solutions were added. Reactions were stopped by the
`addition of 100 ll of 2 M H
`. A
`SO
`of the TMB solution was
`%
`%&!
`#
`measured on a Bio-Rad 3550 microplate reader and found to be
`proportional to the amount of antibody applied per well over the
`range 0–0n1 lg per well. The amount of antibody that retained
`binding ability after treatment with GdmCl of different concentra-
`tions was quantified by comparison with standard curves.
`
`Determination of Fv-binding activity
`
`Biotinylation of adenylate kinase
`
`Biotinylated adenylate kinase was prepared by the method of
`Bonnard et al.
`[14]. The biotinylation reagent, BNHS, was
`dissolved in dimethylformamide to a final concentration of
`1 mg\ml as a stock solution. One volume of biotinylation reagent
`stock solution was mixed with 30 vol. of adenylate kinase (0n5–
`1n0 mg\ml in 0n1 M Na
`buffer, pH 9n0) and incubated
`HCO
`for 3 h. After overnight dialysis against 500 vol. of PBS at 4 mC,
`$
`an equal volume of glycerol was added to the dialysed solution,
`which was stored at k20 mC.
`
`#
`
`Determination of antibody-binding activity of biotinylated adenylate kinase
`Avidin (0n5 lg\100 ll\well) was adsorbed on to the microplates
`and the wells then blocked with 5 mg\ml gelatin. Biotinylated
`adenylate kinase of different concentrations was added to each
`well and incubated for 1 h. After three rinses with PBST,
`antibodies (McAb3D3) were added to each well and incubated
`for 1 h. After three similar rinses, 100 ll of horseradish
`peroxidase-labelled goat anti-mouse IgG conjugate (1 : 3000
`dilution) was added to each well, and incubated for 1 h. After
`rinsing, 100 ll of TMB solution was added. Reactions were
`stopped by the addition of 100 ll of 2 M H
`. A
`SO
`was
`%
`%&!
`#
`recorded on a Bio-Rad 3550 microplate reader and shown to be
`linear with the amount of biotinylated adenylate kinase over the
`range 0–50 lg\ml.
`
`Determination of Fv-binding activity
`
`Binding of antibody (McAb3D3) to biotin-labelled adenylate
`kinase was taken as a measure of the binding activity of the Fv
`region. The antibodies were first coated on to the microplates.
`The remaining non-specific binding sites on the wells were then
`blocked with 5 % gelatin. GdmCl of different concentrations was
`then added to the wells in triplicate, which were incubated for
`1n5 h, and then rinsed with PBST five times to remove the
`denaturant. Biotinylated adenylate kinase (100 ll) was added.
`After three rinses, 100 ll of ExtrAvidin Peroxidase (1 : 500
`dilution) was added and the mixture incubated for 30 min. After
`three rinses with PBST, 100 ll of TMB\H
`O
`solution was added
`and the reaction stopped after 10 min with 100 ll of 2 M H
`SO
`.
`%
`#
`The change in A
`was recorded on a Bio-Rad 3550 microplate
`%&!
`reader and shown to be linear with immunoglobulin over the
`range 0–0n12 lg\well.
`
`#
`
`#
`
`RESULTS
`Fluorescence emission spectra of native and denatured
`immunoglobulin
`
`To characterize the overall conformational changes in immuno-
`globulin in different concentrations of GdmCl, intrinsic fluores-
`cence and second-derivative absorption spectra were measured.
`The denatured protein shows increased emission intensity
`together with a shift in the emission maximum from 330 to
`350 nm, indicating a change in the microenvironment of the
`aromatic residues of the immunoglobulin molecule with an
`excitation wavelength of 280 nm (results not shown).
`
`Second-derivative spectra of native and denatured
`immunoglobulin
`
`The difference in peak-to-trough distance between 287 and
`283 nm represents a change in the microenvironment of tyrosine
`residues (results not shown).
`
`Conformational changes in immunoglobulin in different
`concentrations of GdmCl
`
`The relative changes in the shift in emission maximum of the
`intrinsic fluorescence spectra and the peak-to-trough distance of
`the second-derivative spectra of immunoglobulin in GdmCl of
`different concentrations are shown in Figure 1. The maximum
`emission wavelength of native immunoglobulin at 330 nm is
`shifted gradually to longer wavelengths during denaturation,
`reaching 350 nm in 5 M GdmCl. Little shift in maximum emission
`wavelength can be observed in GdmCl concentrations lower than
`1n5 M, and significant shifts take place between 1n5 and 2n8 M,
`with the concentration of GdmCl required to bring a 50 % shift
`in emission maximum at approx. 2n3 M. The second-derivative
`spectra of immunoglobulin do not change in GdmCl concentra-
`
`100
`
`,....
`~
`CD
`:::,
`0
`>
`CD
`>
`:;:;
`0
`CD
`0::
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`2
`
`4
`
`[GuHCI] (M)
`
`6
`
`Figure 1 Relative changes in fluorescence, second-derivative spectra and
`antigen-binding activities of the Fc and Fv parts of McAb3D3 in GdmCl
`(GuHCl) of different concentrations
`
`Curve 1, relative shift of fluorescence emission maximum wavelength ; curve 2, relative values
`of peak-to-trough distance between 287 and 283 nm of second-derivative absorption spectra ;
`curve 3, relative activity of Fc region of McAb3D3 ; curve 4, relative activity of Fv region of
`McAb3D3.
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`tions lower than 1 M. The peak-to-trough distance changes
`sharply between 2n2 and 3n1 M GdmCl, with a transition point at
`2n7 M. The immunoglobulin is unfolded almost completely in
`3n5 M GdmCl. The fluorescence and second-derivative spectra
`monitor the structural environment of the aromatic residues in
`the molecule,
`the latter being more sensitive to micro-
`environmental changes in tyrosine residues and the former more
`sensitive to those of tryptophan residues.
`
`Change in activity of Fc region of McAb3D3 in GdmCl
`
`The Fc part of an immunoglobulin comprises the two C-terminal
`constant regions of the heavy chains that interact with the
`complement system and with specific receptors on the surface of
`a variety of cells. Part of the polypeptide chain of SpA is a series
`of four highly homologous regions [15] each of which is able to
`bind to the Fc part of IgG from various species. The structure of
`the complex formed by human Fc fragment and fragment B of
`Protein A has been determined to high resolution by Deisenhofer
`[10]. In this study, changes in binding of the Fc region of
`McAb3D3 to SpA in different concentrations of GdmCl were
`followed by monitoring the binding to horseradish peroxidase-
`labelled SpA. The amount of functionally reactive antibody
`remaining after treatment with GdmCl of different concentra-
`tions was quantified by comparison with standard curves obtained
`in the absence of GdmCl. Figure 1 curve 3 shows the percentage
`of Fc region that remains capable of binding to SpA after
`denaturation. It is clear that the SpA-binding ability of Fc is
`affected by GdmCl at relatively low concentrations. The con-
`centration of GdmCl required to inactivate the Fc part of
`immunoglobulin by 50 % is about 1n0 M, and the ability of the
`Fc region to bind to SpA is completely destroyed at GdmCl
`concentrations higher than 2 M.
`
`Change in the activity of the Fv part of McAb3D3 in GdmCl
`
`The Fv part of an immunoglobulin contains its specific antigen-
`binding site. Several structures of the antigen-bound Fab frag-
`ment have been determined [16]. For measurement of the binding
`ability of McAb3D3 to adenylate kinase as its antigen by indirect
`ELISA, biotinylated adenylate kinase was prepared and tested
`by ELISA to ensure the immunoreactivity of the conjugates (see
`the Materials and methods section). The results show that the
`binding of biotinylated adenylate kinase to McAb3D3 is pro-
`portional to the amount of biotinylated adenylate kinase. In this
`study, changes in binding of the Fv region of McAb3D3 to
`adenylate kinase after treatment with different concentrations of
`GdmCl were followed by monitoring binding to biotinylated
`adenylate kinase. Figure 1 curve 4 shows the relative percentage
`of Fv region that retains its ability to bind to adenylate kinase.
`It is evident that the Fv region loses its ability to bind to
`adenylate kinase by 50 % at a GdmCl concentration of 0n18 M,
`which is much lower than that required to bring about conforma-
`tional changes in both the Fc region (Figure 1 curve 3) and the
`molecule as a whole, as detected by fluorescence and second-
`derivative spectra (Figure 1 curves 1 and 2). Although it
`could be objected that some refolding of the denatured antibody
`might have occurred with partial recovery of antigen-binding
`activity after GdmCl was washed out, if some re-activation had
`occurred, this could only result in, if anything, an overestimation
`of the antigen-binding activity remaining, making the difference
`in GdmCl concentration required to bring about conformational
`changes and inactivation of the antibody even greater.
`
`Denaturation of IgG
`
`709
`
`DISCUSSION
`Sequential conformational changes during unfolding
`
`The two-state model has been the dominant theory of protein
`folding for several decades. This is based on the experimental
`results of identical relative changes in conformation as measured
`by different methods during the folding–unfolding transitions
`[17]. In recent years, technical progress in both kinetic and
`equilibrium studies of protein folding and unfolding has revealed
`the existence of relatively stable intermediates [18]. Several folding
`intermediates of Fab and Fabalk have been recognized by Lilie et
`al. [19], and the denaturation kinetics of its antigen-binding
`activity for both Fab and Fabalk have also been compared [20].
`These authors found that the rate of loss of its antigen-binding
`activity, as measured by ELISA, is much faster than that of its
`secondary structure, as measured by CD, and concluded that the
`correct quaternary structure of Fab disappeared first, followed
`by a loss of secondary structure. In the present study, the
`activities of the Fv and Fc regions of immunoglobulin are
`separately compared with global conformational changes of the
`molecule in GdmCl of different concentrations. The transition
`points of the overall conformational change in immunoglobulin
`McAb3D3 with increasing concentrations of GdmCl differ
`significantly as detected by second-derivative spectroscopy and
`by fluorescence. The difference suggests that the unfolding of
`immunoglobulin does not follow an ‘ all or none ’ model. More-
`over, the Fc and Fv regions are inactivated at much lower
`GdmCl concentrations, 1n0 and 0n18 M respectively, than required
`for the overall conformational change, suggesting that the
`perturbation of the antigen-binding site and SpA-binding site
`precedes overall changes in conformation during GdmCl de-
`naturation. With increasing GdmCl concentrations, the antigen-
`binding site is affected first, followed by the SpA-binding site,
`and the overall conformational change takes place last. This
`indicates a non-parallel sequential unfolding between sub-
`domains of the immunoglobulin molecule.
`
`The Fv and Fc regions are more fragile than the immunoglobulin
`molecule as a whole
`
`Although previous studies in this laboratory have shown that the
`active sites of many enzymes are more fragile than the molecules
`as a whole [6],
`little effort has been made to compare the
`susceptibility of the ligand-binding site of proteins and the
`overall structure of protein molecules. From the results presented
`above, it appears that, like many enzymes, the ligand-binding
`sites of immunoglobulin are also more fragile than the molecule
`as a whole. The substrate-binding sites of many enzymes are
`situated in the cleft region between domains [21], whereas
`in immunoglobulins, antigens bind to the interfaces composed of
`the N-terminal domains of the light and heavy chains, and the
`SpA-binding site consists of residues from the CH2 and CH3
`domains of immunoglobulin heavy chain [8]. The resemblance of
`ligand-binding sites of enzyme and non-enzyme proteins is further
`illustrated in the structure of the catalytic sites of abzymes by the
`transformation of the non-catalytic binding site of immuno-
`globulin to the catalytic site of an enzyme without marked
`alterations in other regions of the same molecule [22].
`The conformation of the protein macromolecule is held
`together by a large number of weak bonds either of different
`nature or of similar nature but with different environments and
`consequently different in stability ; protein unfolding is of necess-
`ity a complicated process. At low concentrations of some
`denaturants, the disruption of some particularly susceptible
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`bonds, essential to keep the enzyme or protein in an active state,
`inactivates the enzyme without conspicuous structural changes.
`Results presented in the present study indicate that the unfolding
`of a non-enzyme protein, immunoglobulin, is likewise a sequen-
`tial or stepwise process, and its Fv and Fc regions are more easily
`perturbed than the molecule as a whole.
`
`We are grateful to Dr. H. J. Zhang for providing adenylate kinase. This work was
`supported in part by the Pandeng Project of the China National Commission of
`Science and Technology.
`
`1
`2
`3
`4
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`5
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`Received 23 September 1996/21 March 1997 ; accepted 25 March 1997
`
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`Lilie, H., Jaenicke, R. and Buchner, J. (1995) Protein Sci. 4, 917–924
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`22
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