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INTRODUCTION |
Investigations on the mechanisms of protein folding have for many
years emphasized the role of short range interactions in the formation
of secondary structure elements, i.e.
-helices and
-turns/
-sheets, at early stages of the folding process (1-4). Although the necessity of a coupling between secondary structure and
some long range interactions, such as specific disulfide bonds, was
pointed out long ago (5), the possibility that specific long range
interactions may drive the folding from the very beginning of the
folding process was not taken seriously until the application to
proteins of the notion of "energy landscape" (6). Yet experimental evidence was already available that indicated the need for such long
range interactions in promoting or stabilizing secondary structure
elements at early stages of the folding of some proteins. Thus, the
native secondary structure of hen lysozyme was shown to be recovered in
less than 4 ms of folding when the native disulfide bonds were kept
intact in the denatured state (7), whereas no detectable secondary
structure was present after 4 ms of folding when the denatured protein
was reduced (8). Recent studies even showed that the regain of
secondary structure during the renaturation/oxidation of reduced
lysozyme is very slow and likely to depend strictly on the formation of
at least some native disulfide bonds (9).
Similarly, horse cytochrome c folds efficiently only when it
carries its cofactor. Indeed, the apoprotein behaves essentially as an
unfolded polypeptide chain (10, 11), indicating that the interactions
between the polypeptide chain and the heme moiety are needed to achieve
a folded state. This was confirmed by the observation that long range
interactions between the heme and specific side chains of apocytochrome
c are formed during the initial phases of the folding of
holocytochrome c (12), suggesting that these interactions
contribute to either initiating the folding process or stabilizing very
early intermediates, rather than only to the stabilization of the
folded state. Moreover, the noncovalent binding of heme to
apocytochrome c promotes a structured conformation of the
polypeptide chain (13). However, although clearly shown to be
definitely folded and compact, this conformation certainly differs from
that of native cytochrome c. In particular, the far UV CD
and the visible absorption spectra of the noncovalent complex differ
from those of the native protein (13). In order to gain some insight to
the respective contributions of covalent and noncovalent heme/protein
interactions in structuring the polypeptide chain, it seemed to us of
interest to find out whether or not the noncovalent complex shares some
conformational features with native holocytochrome c. We
therefore undertook investigations on the conformation of the
noncovalent complex between heme and apocytochrome c.
In the present paper, we describe an easy, large scale purification
procedure of apocytochrome c and the characterization by
circular dichroism spectroscopy and analytical ultracentrifugation of
the resulting apoprotein and of its noncovalent complex with heme. We
also report structural investigations of the noncovalent complex,
involving measurements by
ELISA1 and isothermal
titration microcalorimetry of the affinities of two
"conformation-specific" monoclonal antibodies for holocytochrome c, apocytochrome c, and the noncovalent
heme-apocytochrome c complex.
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MATERIALS AND METHODS |
Chemicals and Buffers--
Heme was obtained as ferriporphyrin
hydroxide (hematin) from Aldrich. Heme stock solutions were prepared in
0.1 M sodium hydroxide, and their concentration was
determined spectrophotometrically (
385 = 5.84 × 104 M
1 cm
1) All
chemicals used were reagent grade. Unless otherwise stated, the buffer
was 50 mM sodium phosphate, pH 7.5, containing 5 mM DTT.
Proteins--
Horse heart holocytochrome c was
purchased from Sigma and used without further purification.
Apocytochrome c was obtained as described previously (10).
100 mg of holocytochrome c were dissolved in 2 ml of
distilled water and supplemented first with 160 mg of silver sulfate
dissolved in 18 ml of water and second with 1.6 ml of pure acetic acid.
The mixture was incubated at 40 °C for 4 h and centrifuged. The
purification of the apocytochrome c thus obtained was
achieved as follows. The supernatant (21 ml) was dialyzed overnight at
4 °C against 2 liters of 0.1 N acetic acid and then for
24 h against 2 liters of 50 mM ammonium acetate adjusted at pH 5; the dialysis buffer was changed after the first 12 h. The dialysate was centrifuged at 8000 rpm for 30 min. in a
Sorvall SS 34 rotor. The resulting supernatant (25 ml) was slowly supplemented under gentle mixing with 0.25 ml of a saturated solution of ammonium sulfate. The mixture was incubated at room temperature without agitation for 1 h and centrifuged as above. The
supernatant was discarded, and the pellet was redissolved with 20 ml of
6 M guanidinium chloride in 50 mM ammonium
acetate, pH 5, and 0.5 M DTT. The solution was dialyzed at
4 °C against 2 liters of ammonium acetate, pH 5, 5 mM
DTT for 3 h, the buffer was changed and the dialysis carried on
overnight. The dialysate was centrifuged as above to remove a fine,
yellowish precipitate. The supernatant (29 ml) was separated in 1.5-ml
aliquots, lyophilyzed, and stored at
20 °C in a dessicator with
silica gel.
The
subunit of Escherichia coli tryptophan synthase was
prepared as described previously (14).
Monoclonal antibodies 5F8 and 2B5 were obtained, starting from
hybridoma cell lines kindly provided by Dr. Barry Nall (Department of
Biochemistry, University of Texas Health Center, San Antonio, TX).
Hybridoma cells were cultured, injected intraperitoneally into
pristane-primed BALB/C mice, and antibodies were purified from the
resulting ascitic fluids by ammonium sulfate precipitation of the
immunoglobulins followed by ion exchange chromatography on DEAE,
following previously described protocols (15). The characterization of
these antibodies has been reported previously (16). The preparation and
characterization of antibody mAb164 was described previously (17).
Protein concentrations were determined spectrophotometrically, using as
extinction coefficients
280 = 0.92 ml/mg·cm for
apocytochrome c (10),
280 = 1.5 ml/mg·cm
for the monoclonal antibodies (18),
280 = 0.46 ml/mg·cm for the
subunit of tryptophan synthase (19) and
410 = 8.58 ml/mg·cm for holocytochrome c
(20).
Spectroscopic Measurements--
Absorption spectra were recorded
in a Lambda 2 double beam spectrophotometer (Perkin-Elmer) and
fluorescence spectra in a LS-5 double monochromator spectrofluorometer
(Perkin-Elmer), both thermostatted at 20 °C. For quantitative heme
binding studies, the quenching of the intrinsic fluorescence
(excitation at 295 nm, emission at 348 nm) was measured immediately
after addition of the heme. The excitation and emission bandwidth were
adjusted to 5 nm. When low apocytochrome c concentrations
were used (0.4 µM) the solution was supplemented with 100 µg/ml of pure
subunit from Escherichia coli tryptophan
synthase in order to prevent adsorption of the apocytochrome onto the
walls of the cuvette; because this protein contains no tryptophan, the
fluorescence background thus introduced was only moderate. The inner
filter and dilution effects on the measured fluorescence due to heme addition was determined in a control experiment with a mixture of
lysozyme (25 µg/ml) and tryptophan synthase
subunit (100 µg/ml), and the correction factor was applied to the fluorescence measured with apocytochrome c.
Circular dichroism spectra were recorded in a CD6 spectropolarimeter
(Jobin-Yvon, Longjumeau, France) equipped with a thermostatted (20 °C) cell holder. The optical path of the cells was 0.2 mm for
the far UV CD spectra and 1 cm for the near UV/visible CD spectra. The
scanning interval was 0.2 nm, the integration time was 1 s, and
the bandwidth was kept constant at 2 nm. Five successive scans of each
solution were averaged. The spectrum of the buffer alone, obtained
under the same scanning conditions, was substracted from that of the
sample solution to obtain the CD contribution of the protein to the spectrum.
Amino Acid Analysis--
Amino acid analysis of apo- and
holocytochrome c was achieved in duplicate on a Beckman 6300 amino acid analyzer after acid hydrolysis of 0.2-mg protein samples in
sealed tubes for 20 h at 110 °C in 6 N HCl and
0.2% phenol.
Mass Spectrometry--
For molecular weight measurements by mass
spectrometry, samples of lyophilized apo or holocytochrome c
were dissolved in water:methanol:formic acid (50:50:5) and introduced
into an API 365 (Perkin-Elmer-Sciex, Thornhill, Canada)
triple-quadrupole mass spectrometer at 5 µl/min by means of a syringe
pump (Harvard Apparatus, South Natick, MA). The device was equipped
with an atmospheric pressure ion source used to sample positive ions
produced from a pneumatically assisted electrospray interface. The
ionspray probe tip was held at 4.5 kV, and the orifice voltage was set
at 45 V. The mass spectrum was scanned continuously from m/z
700-1800 with a scan step of 0.1 and a dwell time per step of 2.0 ms
resulting in a scan duration of 22.0 s. Ten scans were averaged
for each analysis. Mass calibration of the instrument was accomplished
by matching ions of polypropylene glycol to known reference masses
stored in the mass calibration table of the instrument. Data were
collected on a Power Macintosh 8600/200 and processed through the
Biotoolbox 2.2 software from Sciex.
Analytical Ultracentrifugation--
Ultracentrifugations were
performed in a Beckman Optima-XLA analytical centrifuge, using standard
double sector cells with 1.2-mm-thick aluminum centerpieces. The
temperature was 20 °C. The cells were scanned at the wavelengths
indicated in the text. For sedimentation velocity runs, single scans
were performed at constant time intervals. The sedimentation profiles
were analyzed using the XLAVEL program (Beckman) provided with the
analytical centrifuge. The observed sedimentation coefficients were
corrected for density and viscosity of the solvent to obtain the
standard sedimentation coefficients. For sedimentation-diffusion
equilibrium studies, 0.15 µl of sample solution were laid over 50 µl of fluorochemical FC43 (3 M-Minesotta). Radial scans
were recorded at 1.5-h intervals for at least 18 h to check that
equilibrium was reached. When equilibrium was achieved, 5 or 10 successive scans were averaged to improve the signal to noise ratio in
the recordings. The centrifuge was then accelerated to 59,000 rpm to
clear the meniscus from any residual protein and determine the
absorbance base line. The protein distribution at equilibrium was
analyzed with the Origin-based Optima XL-A Data Analysis Software
(Beckman). The different fitting models (Ideal 1, Ideal 2, and Assoc4)
for single data sets (XLA-Single program) were systematically tested
and the best fitting was retained on the basis of both the
2 value and the lack of systematic deviation of the
residuals. In all cases, the base line was first fixed at the value
observed after meniscus clearing and allowed to float only as an
ultimate fitting refinement, which was accepted only if the resulting
baseline differed by less than 0.02 absorbance unit from that
determined experimentally. The value of the partial specific volume
used for molecular weight calculations and for sedimentation
coefficient corrections was 0.719, as determined (21) from the amino
acid composition of cytochrome c.
Affinity Measurements by ELISA--
The affinities of antibodies
for holocytochrome c and the noncovalent heme-apocytochrome
c complex were measured by the method of Friguet et
al. (22). Briefly, a constant amount of monoclonal antibody was
incubated with a varying amount of the antigen until equilibrium was
reached. The quantity of antibody that remained free at each
concentration of antigen was monitored by a classical indirect ELISA in
which holocytochrome c was coated in the microtitration plate. A nonlinear regression method was used to extract affinity constants from the saturation curves. Each affinity measurement was
done in triplicate.
Isothermal Titration Calorimetry (ITC)--
Experiments were
performed on the MicroCal MCS ultra-sensitive titration calorimeter
(MicroCal Inc., Northampton, MA) using the OBSERVER software provided
by the manufacturer for instrument control and data acquisition (23).
To improve base line stability, the temperature of the adiabatic
jacquet was kept 5 °C below the temperature of the experiment by
setting the circulating water bath at 10 °C and by activating the
jacquet feedback power, and temperature was equilibrated for 12 h.
During a titration experiment, the protein sample was thermostatted at
20.0 ± 0.1 °C in a stirred (410 rpm) reaction cell (1.3514 ml), and aliquots of the titrant solution were injected from a 100-µl
or 250-µl syringe as indicated in the legends of the figures. The
reference cell of the calorimeter contained water plus 0.01% sodium
azide. Data points were averaged and stored at 2-s intervals. All
buffer solutions were thoroughly degassed by stirring under vacuum
before use (buffer compositions are given in the figure legends).
Protein samples were prepared in buffer of the same batch to minimize
artifacts due to any differences in buffer composition. Titration
experiments were performed with protein concentrations in the reaction
cell and in the syringe insuring a final titrant/binding-site molar
ratio of 2:1 in the reaction cell. Raw calorimetric data,
i.e. heats released accompanying the addition of aliquots of
titrant solution into the protein solution in the reaction cell, were
processed using the software package ORIGIN (23, 24). The area under
the resulting peak following each injection is proportional to the heat
of interaction Q. When corrected for the titrant dilution
heat and normalized to the concentration of added titrant, Q
is equal to the binding enthalpy
H'b at that
particular degree of binding. The calorimetric binding isotherm was
fitted by an iterative nonlinear least squares algorithm (the Marquardt
method) to a binding model as indicated in the text. The association
(Ka) or dissociation (Kd) constants, molar binding stoichiometry (N), and molar
binding enthalpy (
H°), were determined directly from
the fitted curve. The Gibbs free energy and molar entropy of binding
were calculated using the equations
G° =
RT ln Ka and
S° = (
H°
G°)/T, respectively,
where R is the gas constant and T the absolute temperature in Kelvin.
 |
RESULTS |
Large Scale Purification of Apocytochrome c--
An efficient
procedure for cleaving the covalent bonds linking the heme to two
cysteine residues in cytochrome c has been previously
described (25). Based on this procedure, we devised a new purification
method based on the expected difference in solubility between apo- and
holocytochrome c. Indeed, in the holoprotein a large number
of hydrophobic residues are buried in the core of the molecule, in
contact with the heme (26), whereas in the unfolded apoprotein, these
residues are exposed to the solvent. Thus, the heme and apoprotein were
first resolved by treatment with silver sulfate (10) and, after
centrifugation and dialysis (see under "Materials and Methods"),
the resulting mixture was treated with 1% ammonium sulfate. This was
enough to completely precipitate apocytochrome c, whereas
holocytochrome c remained entirely soluble under these
conditions. After centrifugation, the ammonium sulfate precipitate was
dissolved in 6 M guanidinium chloride and 50 mM
ammonium acetate at pH 5 containing 0.5 M DTT. The
guanidine was then removed by extensive dialysis against 50 mM ammonium acetate, pH 5, supplemented with 5 mM DTT. After dialysis, the apocytochrome c was
obtained in a soluble form. A typical purification based on these steps
is described in Table I.
The absorption spectrum of the resulting protein showed very little
residual absorbance in the 400 nm region (Table I), indicating that no
significant amount of heme remained in the purified apocytochrome c. However, a very small shoulder near 350 nm could still be
observed in some preparations
(A350/A280 < 0.04). No
attempt was made to identify the nature of this trace contaminant.
Physicochemical Properties of the Apo-enzyme--
Purified
apocytochrome c was submitted to amino acid analysis to
verify its chemical integrity. The amino acid composition expected from
the known sequence of cytochrome c was obtained. The two
cysteinyl side chains were already reported to be obtained in the
reduced state (11). The absorption and fluorescence spectra showed that
the tryptophan residue was present and intact in the apocytochrome
preparation. The mass spectrum of the apocytochrome c
preparation showed a largely predominant peak at an estimated mass of
11,743.0 ± 0.7 Da. The excess of 42 Da over the mass calculated from the known amino acid sequence of horse cytochrome c
(11701.6 Da) corresponds exactly to an acetylation, a chemical
modification known to exist at the N-terminal extremity of cytochrome
c. The same excess in mass was observed with holocytochrome
c, indicating that the acetylation indeed existed in
the starting material. These observations lead to the conclusion that
the methionine residues have not been altered by the procedure used to
prepare apocytochrome c.
The far UV CD and the fluorescence emission spectra of apocytochrome
c were recorded (data not shown) and found to be essentially identical to those previously reported (10, 11). The far UV CD spectrum
showed no signal characteristic of secondary structure, and the
fluorescence emission spectrum was that of solvent exposed tryptophan
residues, two features confirming that apocytochrome c
appears devoid of detectable organized secondary and tertiary structure.
The sedimentation coefficient of apocytochrome c (30 µM) in 50 mM potassium phosphate, pH 7.5, and
5 mM DTT was determined by analytical centrifugation at
20 °C and 59,000 rpm. The observed sedimentation coefficient was
1.08 ± 0.05 S. After correction for the density and viscosity of
the solvent, the standard sedimentation coefficient was found to be
s20,w = 1.13 ± 0.05 S. From this value and
from the molecular weight of N-acetylated apocytochrome
c (11,744 Da), one could estimate the Stokes radius of the
apoprotein to be about 25 Å, a value compatible with that determined
by dynamic light scattering under similar solvent conditions (27). The sedimentation profile showed a unique, symmetrical boundary, suggesting that the preparation of apocytochrome c contained no low
molecular weight aggregates. This was confirmed by observing that the
absorptions at 280 nm of a 62 µM solution of
apocytochrome c in the centrifugation cell immediately after
reaching 5000 and 59,000 rpm were, within the experimental precision
(i.e. ± 2%), identical. Moreover, these plateau
absorbances were equal to the absorbance measured in a spectrophotometer before the centrifugation, indicating the absence heavy aggregates.
Physicochemical Properties of the Noncovalent
Complex--
Apo-cytochrome c was saturated with heme as
described previously (13), and its spectral properties were
investigated under reducing conditions (5 mM DTT) in the
absence of cyanide. As previously reported, the absorption spectrum of
the heme-apocytochrome c mixture showed a Soret absorption
band at about 406 nm that, after a rapid initial change, underwent a
slow further increase. The fluorescence spectrum of the mixture showed
a strong quenching of the tryptophan fluorescence emission, which was
used to monitor the formation of the heme-cytochrome c
complex. The results (not shown) from 5 experiments conducted at
apocytochrome c concentrations of 0.4 and 2.1 µM showed simple binding with a dissociation constant of
1.4 ± 0.2 µM and 2 hemes bound per polypeptide
chain, in excellent agreement with previous reports (13). The far UV CD
spectrum of the noncovalent complex showed an increase in the amplitude of the ellipticity in the 220 nm region. This confirmed previous reports indicating that the far UV CD spectrum of the noncovalent complex, although quite different from that of native holocytochrome c, reflects the presence of some organized secondary structure.
The hydrodynamic properties of the noncovalent complex were then
investigated. A solution of apocytochrome c (4 or 8 µM) was partly saturated with a limiting amount of heme
(total concentration, 2 or 4 µM, respectively) to
minimize possible "nonspecific" heme binding and optical problems
due to the very poor solubility of free heme at neutral pH. The
sedimentation of the noncovalent complex was specifically observed by
scanning the centrifugation cell at 420 nm, where apocytochrome shows
no absorption. The standard sedimentation coefficient thus obtained was
s20,w = 1.88 ± 0.06 S, a value identical
to that determined for native holocytochrome c
(s20,w = 1.80 ± 0.04 S). Assuming that the
noncovalent complex is monomeric, this indicates that the noncovalent
complex is as tightly packed as native holocytochrome c,
with a Stokes radius estimated to be about 16.2 Å.
That the noncovalent complex is monomeric was verified by
sedimentation-diffusion equilibrium of the noncovalent complex. A 6.2 µM solution of apocytochrome c was saturated
with 6.2 µM heme and submitted to centrifugation at
40,000 rpm and 20 °C. The centrifugation cell was scanned for
absorbance at 560 nm, a wavelength at which only the heme-cytochrome
c complex absorbed, ensuring that neither unsaturated
apocytochrome c nor free heme that might exist in the
solution would contribute to the absorption. The absorbance
distribution after 18 h of centrifugation was analyzed. The
experimental data were fit using as a model an ideal solution with a
unique molecular species. The best fit provided an estimate of 12,830 Da for the molecular weight of the heme-apocytochrome c
complex, a value reasonably close to those calculated for the monomeric
noncovalent complex between the apoprotein and one or two hemes (12,360 and 12,976 Da, respectively). However, whereas equilibrium seemed at
first to have been reached after 18 h, the protein pattern went on
evolving slowly, with a progressive decrease in the total protein
concentration. This suggested that the protein might undergo a slow
aggregation during the centrifugation.
Slow aggregation was confirmed as follows. Apocytochrome c
and heme (6.2 µM each) were mixed and incubated for
3 h, at which time the absorption spectrum was stable. The
solution was then introduced in the centrifugation cell, accelerated
first to 5,000 rpm and then to 59,000 rpm. The absorbance at 560 nm was
recorded immediately after reaching 5000 and 59,000 rpm. The values
observed were 0.785 and 0.735, respectively, indicating that about 7%
of the noncovalent complex were pelleted during the acceleration at
high speed. This result confirmed that unlike apo- and holocytochrome c, which are stable in solution, the noncovalent complex is
prone to aggregate and must therefore be prepared extemporaneously
before use.
Finally, the CD spectrum of the noncovalent complex was also
investigated in the absorption region of the heme and compared with
that of holocytochrome c. In a control experiment with free heme at the same concentration, no CD signal was detected, indicating that the spectrum observed with the noncovalent complex indeed corresponded to heme bound to apocytochrome c. Comparison of
the two spectra in Fig. 1 clearly
indicated that the environment of the heme in the two species were
largely different.

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Fig. 1.
Near UV and visible CD spectra of
holocytochrome c and of the noncovalent complex.
Holocytochrome c (63 µM) or a mixture of
apocytochrome c and free heme (63 µM each) in
50 mM sodium phosphate, pH 7.5, and 5 mM DTT
were placed in a 5-mm optical path cell thermostatted at 20 °C. The
near UV CD spectra were recorded with a scan step of 1 nm and an
integration time per step of 2 s. Three scans were averaged for
each sample. The base line with buffer alone was recorded under the
same scanning conditions and was substracted to obtain the spectra
shown. Apocytochrome c or free heme alone at 63 µM showed no CD signal in the wavelength range
investigated. Thin line, holocytochrome c;
thick line, apocytochrome c + heme.
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From all of these studies, it appeared that except for its tight
packing and for its far UV CD spectrum suggesting the presence of some
organized secondary structure, the noncovalent complex showed no direct
evidence of sharing structural features with native holocytochrome
c. Hence, the need to analyze in more details the
conformation of the noncovalent complex. Neither x-ray crystallography nor NMR could be envisaged for these studies because of the propensity of the complex to aggregate, a feature that precluded the use of the
high protein concentrations needed for such experiments. We therefore
undertook a conformational study of the noncovalent complex, using two
monoclonal antibodies that had been reported to bind selectively to
native holocytochrome c.
Immunochemical Properties of Apocytochrome c, Holocytochrome c, and
the Noncovalent Complex--
Two monoclonal antibodies, 5F8 and 2B5,
that bind strongly to native holocytochrome c have been
described (16). Their epitopes lie on opposite sides of the native
antigen. Antibody 5F8 recognizes a region comprising lysine 60, near
the N-terminal end of an
-helix. Antibody 2B5 recognizes residues
around proline 44 in a type II
-turn (16). These two antibodies
appeared unable to bind apocytochrome c, and hence as
specific of native holocytochrome c (28). They were
therefore chosen to find out whether or not the "folded" noncovalent heme-apocytochrome c complex shares native-like
epitopes with holocytochrome c.
The affinities of both antibodies for native holocytochrome
c and for the noncovalent heme-apocytochrome c
complex were first determined, using the competition ELISA method (22)
under conditions where the antibody immobilized during the ELISA
represented less than 10% of the free antibody (22) and where
saturation of the antibody with the antigen was sufficiently high for
straightforward interpretations of the data (29). The results of such
measurements are depicted in Fig. 2.
Fitting binding isotherms to the data provided the affinities reported
in Table II, which indicated that the two
antibodies recognized the noncovalent complex with affinities that
differed by a factor of about 10 only from their affinities for native
holocytochrome c. The reduced apparent affinity of the
antibodies for the noncovalent complex as compared with holocytochrome
c could be interpreted in two ways. Either only 10% of the
cytochrome c molecules were recognized by the antibodies with the same affinity as native cytochrome c, or all
molecules were recognized, but with a reduced affinity. To sort this
alternative, a solution containing 4 µM apocytochrome
c, 2 µM heme (so that no significant amount of
free heme would remain in solution), and 6 µM 5F8
antibody (i.e. 12 µM antigen binding sites)
was submitted to analytical centrifugation at 58,000 rpm and 20 °C,
and the cell was scanned at 420 nm at 10-min intervals. The
sedimentation profile showed a unique symmetrical boundary
(s20,w = 7.3 S). No slowly sedimenting material
could be detected at 420 nm. This demonstrated that all the
heme-saturated apocytochrome c molecules were bound to the
antibody. In a control experiment with a nonspecific monoclonal
antibody, all of the hemoprotein migrated as the free noncovalent
complex, indicating that the binding observed with mAb5F8 was indeed
specific. This indicated that all, rather than only a small fraction,
of the heme-saturated molecules were recognized by the specific
antibodies.

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Fig. 2.
Determination of the affinity of mAb2B5 for
the noncovalent apocytochrome c-heme complex by
competition ELISA. Aliquots of a mAb2B5 solution at a fixed
concentration were mixed with equal volumes of a stoichiometric mixture
of apocytochrome c and heme at various concentrations. After
a 2-h incubation at room temperature (20 ± 2 °C), the amount
of unsaturated antibody in the solution was measured by ELISA as
indicated under "Materials and Methods." The results were plotted
in the Klotz representation as suggested by Friguet et al.
(22) and fit by linear regression. The slope of the straight line thus
obtained equals the dissociation constant (i.e. reciprocal
of the affinity).
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The two epitopes thus appeared to be present in a nearly native
conformation in the noncovalent complex. This interpretation relied on
the assumption that the monoclonal antibodies could not recognize the
unfolded cytochrome c molecule. It seemed important to check
the validity of this assumption by determining the affinities of both
antibodies for apocytochrome c. Because apocytochrome c has a strong tendency to stick on the ELISA plates, a
prohibitively high background of binding of the free antibody to this
"over-adsorbed" antigen precluded the use of the ELISA competition
test at the high antigen concentrations needed to measure low
affinities. None of our attempts to reduce this nonspecific adsorption
was successful. Similarly, the BIACORE method could not be used because of a strong adsorption of apocytochrome c onto the chips. We
therefore turned to isothermal titration microcalorimetry, a method
already used previously (30) to study the binding of mAbs 2B5 and 5F8 to holocytochrome c.
In a first set of experiments, mAb5F8 (10 µM) was placed
in the calorimeter cell, and apocytochrome c (250 µM) was injected from the syringe in 10 µL aliquots.
The diagram obtained (Fig. 3A)
was analyzed assuming a unique set of independent binding sites. The
best fit to the experimental data was obtained with an affinity of
8.7 × 105 ± 0.7 × 105
M
1 and 2.02 ± 0.03 binding sites per
IgG molecule. In a symmetrical experiment, apocytochrome c
(4 µM) was placed in the calorimeter cell and mAb5F8
(18.5 µM) in the syringe. Using a unique set of independent binding sites as a model, the best fit was obtained with an
affinity of 3.0 × 106 M
1
and 2.2 binding sites per IgG molecule. The difference by a factor of 3 in the affinities obtained under the two sets of experimental conditions may be explained by the large difference in the
apocytochrome c concentration (250 and 4 µM
for the antigen in the cell or in the syringe, respectively). Whereas
apocytochrome c was monomeric in analytical centrifugation
experiments performed at about 4 µM (see above),
significant aggregation might occur at 250 µM. This
assumption was supported by the observation that when kept at high
concentration for several hours, apocytochrome c showed complex titration patterns that could be best explained by the dissociation of aggregated material (data not shown). For this reason,
only experiments in which apocytochrome c solutions were prepared extemporaneously were taken into consideration. In a control
experiment with the nonspecific antibody mAb164 (directed against
tryptophan synthase), no specific enthalpy change could be detected
upon mixing with apocytochrome c (data not shown). It thus
could be concluded that the apparent affinity of mAb5F8 for
apocytochrome c was between 8.7 × 105 and
3 × 106 M
1, i.e.
about 1000-3000-fold smaller than that (the mean of the ELISA and
microcalorimetric determinations) of mAb5F8 for native holocytochrome
c. To find out whether this "residual" affinity of the
antibody for apocytochrome c was due to a contamination of
the antigen preparation with 0.1% of holoprotein or to a weak binding
of the antibody to heme-free apocytochrome c, apocytochrome c (250 µM) was supplemented with 1.25 µM mAb5F8 (i.e. 2.5 µM binding sites), an amount sufficient to presaturate up to 1% of holoprotein that might be present in the apoprotein solution. This mixture was
placed in the syringe and injected in the cell containing mAb5F8 (10 µM). The affinity obtained by fitting, 8.7 × 105 M
1, was indistinguishable
from that obtained in the experiment of Fig. 3, thus ruling out that
the residual affinity of mAb5F8 for apocytochrome c might be
caused by an immunoreactive contaminant.

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Fig. 3.
Calorimetric titration of mAb5F8
(A) and mAB2B5 (B) with apocytochrome
c at 20 °C. A, the top
panel shows the heat signal (after substraction of base line) for
27 injections of 10-µl aliquots of buffer (50 mM sodium
phosphate, pH 7.5, with 5 mM DTT) with 250 µM
apocytochrome c into a 1.35-ml cell containing the same
buffer with 10 µM mAb5F8. The bottom panel
shows the integrated heat of each injection after correction for the
heat of dilution of apocytochrome c and normalization to the
amount of apocytochrome c injected (filled
squares). The curve through the points represents the
best fit to a model involving a single set of independent binding
sites. B, same as A (bottom panel)
when the reaction cell was filled with a 7 µM mAb2B5
solution. In B, the curve through the points
represents the best fit to a model involving two sets of identical
binding sites in interaction. In A and B, each
injection was made over a 25.2-s time interval with a 3.5-min pause
between injections, using a 250-µl syringe.
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Experiments similar to that described in Fig. 3A were
repeated with mAb2B5. The results obtained revealed a more complex
binding reaction, with two distinct binding modes (Fig. 3B).
Using as a model a unique set of independent binding sites provided a
rather satisfactory fit, with an affinity of about 2 × 106 M
1 and only one binding site
per antibody molecule, with the first, sharp titration mode, but failed
to describe the second part of the titration curve. A good fit of the
complete titration curve could be obtained using a model with two
identical interacting binding sites per antibody (Fig. 3B).
The affinities provided by this fit were 1.3 × 106 ± 0.4 × 106 M
1 for the first
site and 1.6 × 104 ± 0.8 × 104
M
1 for the second site, suggesting that the
first molecule of antigen bound to mAb2B5 hinders the binding of a
second molecule of antigen to the antibody.
To ascertain that the "biphasic" titration curve observed with
mAb2B5 was due to anticooperativity, the binding of native holocytochrome c to mAb2B5 was investigated. The data
obtained by titrating mAb2B5 (2.3 µM) with successively
five 8-µl and thirty-six 2-µl aliquots of holocytochrome
c (40 µM) could be well described assuming a
unique set of independent binding sites with an affinity of 2 × 108 ± 0.3 × 108
M
1 and 0.95 binding site per antibody
molecule (data not shown). That only about one site per mAb molecule
could be saturated with high affinity confirmed that the binding of the
antigen to mAb2B5 is anticooperative. Contrary to mAb2B5, mAb5F8 showed
no anticooperativity. This was demonstrated in a titration experiment
specially designed to ensure precise measurements of a high affinity
constant (Fig. 4). A mixture of mAb5F8
(1.64 µM) and holocytochrome c (2.8 µM) was introduced in the calorimeter cell in order to
start the titration just before the saturation transition, and
holocytochrome c (25.3 µM) was injected from
the syringe in small aliquots (3 µl) to ensure the collection of a
sufficient number of significant data points. The curvature of the
diagram in Fig. 4 indicated that the experiment should permit
resolution of the binding constant from the titration data. These data
could be well described according to a model with two identical
noninteracting binding sites per antibody molecule. The best fit was
obtained for 1.99 sites per mAb5F8 molecule, with an affinity of
1.8 × 109 ± 0.25 × 109
M
1. Thus, mAb5F8 showed neither
cooperativity nor anticooperativity for antigen binding, confirming the
results obtained with apocytochrome c.

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Fig. 4.
Calorimetric titration of mAb5F8 with
holocytochrome c at 20 °C. Top and
bottom panels are as described for Fig. 3, A and
B, respectively, with the following modifications: 18 injections of 3-µl aliquots of buffer (50 mM sodium
phosphate, pH 7.5) with 25.3 µM holocytochrome
c were made into the reaction cell containing the same
buffer with a mixture of mAb5F8 1.64 µM and
holocytochrome c 2.8 µM. A calibration
injection of 1 µl was performed prior to the injection series. Each
injection was made over a 5.93-s time interval with a 3.5-min pause
between injections, using a 100-µl syringe.
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Conformational Changes of Apocytochrome c upon mAb
Binding--
The possibility that a conformational change of
apocytochrome c might accompany its binding to the
monoclonal antibodies was examined by measuring the affinity of one of
the mAbs for the preformed complex between the apoprotein and the
second mAb. It was first verified that as expected from the positions
of the corresponding epitopes on native cytochrome c, the
two mAbs could bind simultaneously to the antigen. For that purpose,
holocytochrome c (4 µM) was first mixed with
mAb2B5 (5 µM) and incubated for 15 min at 20 °C. The
mixture was supplemented with mAb5F8 (5 µM), incubated
for 1 h at 20 °C, and submitted to analytical centrifugation. The rotor was accelerated to 55,000 rpm, and scans at 410 nm were made
at 15-min intervals. The absorbances in the cell after reaching full
speed was the same as that determined for the solution before centrifugation, indicating that no large aggregates had been formed. The sedimentation of the light absorbing material (i.e.
cytochrome c) showed a unique, symmetrical boundary, with an
observed sedimentation of 10.6 S. The value observed in a control
experiment with only holocytochrome c and mAb2B5 was 7.2 S. Comparing these two values shows that holocytochrome c was
bound to the two antibodies simultaneously. Binding of mAb5F8 to the
preformed complex between apocytochrome c and mAb2B5 was
therefore investigated by microcalorimetry. A mixture of apocytochrome
c (4 µM) with mAb2B5 (5 µM) was
titrated by injecting 8-µl aliquots of mAb5F8 (18.5 µM)
from the syringe. The results, represented in Fig.
5A (filled
symbols), showed a bimodal titration curve. A control experiment
in which apocytochrome c alone was titrated with mAb5F8
showed only the second binding mode (Fig. 5A, open symbols).
This demonstrated that the first binding mode observed in the presence
of mAb2B5 reflected the presence of a population of apocytochrome
c-mAb2B5 complex with high affinity for mA5F8. Attempts to
fit the data according to models in which the antigen was homogeneous
were unsuccessful, regardless of the assumptions made on the
affinities, number of binding sites per mAb5F8, and possible
interactions between sites. Therefore, the two binding modes observed
in Fig. 5A were further characterized experimentally, by
varying the concentration of the mAb5F8 solution injected. The first
binding mode (corresponding to the higher affinity) was characterized
using a low mAb5F8 concentration (2 µM). The results,
reported in Fig. 5B, were analyzed using a model with one
set of independent binding sites as described above, but with one
modification in the fitting procedure: whereas the concentration of
reactive antigen (i.e. the cytochrome c-mAb2B5 complex) was let free to vary, the stoichiometry of the binding reaction was maintained constant and equal to two antigen binding sites
per mAb5F8, the stoichiometry determined from the results in Fig.
3A. As shown in Fig. 5B, a meaningful fit of the
first transition was obtained for an antigen concentration of 0.6 µM (i.e. 15% of the total apocytochrome
c concentration), giving an association constant of 3.4 × 107 ± 0.4 × 107
M
1. The second binding mode (corresponding to
the lower affinity) was characterized using a high mAb5F8 concentration
(28 µM). The results presented in Fig. 5C
confirmed those of Fig. 5A. Fitting the second binding
transition with a model with one set of independent binding sites
provided values of 2.1 ± 0.07 for the number of binding sites per
mAb5F8 and 2.9 × 106 ± 0.6 × 106
M
1 for the affinity. These values were
indistinguishable from those obtained in the control without mAb2B5
(2.2 ± 0.1 and 3.0 × 106 ± 0.6 × 106 M
1, respectively). Thus, the
population of apocytochrome c-mAb2B5 complex molecules was
shown to contain about 15% of molecules recognized by mAb5F8 with a
10-15-fold higher affinity than the remaining 85% of the molecules,
which were recognized with the same affinity as free apocytochrome
c.

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Fig. 5.
Calorimetric binding isotherms of mA5F8 to
apocytochrome c and to the mAb2B5-apocytochrome
c complex at 20 °C. The integrated heat is
shown for each injection of a series of 8-µl injections of buffer (50 mM sodium phosphate, pH 7.5, with 5 mM DTT)
with 18.5 µM (A), 2 µM
(B), and 28 µM (C) mAb5F8 into the
reaction cell containing the same buffer with either 4 µM
apocytochrome c (open squares) or a preincubated
mixture of apocytochrome c (4 µM) and mAB2B5
(5 µM) (filled squares). Heats of association
are given after correction for the heat of dilution of mAB5F8 and
normalization to the amount of mAB5F8 injected. Each injection was made
over a 20.16-s time interval with a 3.5-min pause between injections,
using a 250-µl syringe. In B and C, the
curve through the points represents the best fit to a model
involving a single set of independent binding sites. In B,
the fit was obtained by leaving the concentration of antigen
(i.e. the cytochrome c-mAb2B5 complex) free to
vary in the fitting procedure while maintaining a stoichiometry of two
antigen binding sites per mAb5F8 molecules. In C, the normal
fitting procedure was used. The two sets of experimental data
(open and closed squares) gave the same
theoretical curve.
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The number of binding sites per mAb and affinities obtained in all the
experiments described above are summarized in Table II, together with
the free energy (calculated from the equilibrium constant), enthalpy
(obtained from the microcalorimetry measurements), and entropy
(calculated from the free energy and the enthalpy) of binding.
 |
DISCUSSION |
As compared with previous reports, the present work has
brought new results on the purification, immunoreactivity, and
conformation of apocytochrome c and its noncovalent complex
with heme. These various aspects will be discussed successively.
By introducing a new method (differential ammonium sulfate
precipitation) to separate the apoprotein from residual heme-containing molecules, a protocol has been set up for the large scale preparation of apocytochrome c. Hundreds of milligrams of pure
apocytochrome c can thus easily be obtained in one run. It
has been demonstrated that this procedure does not affect the integrity
of the polypeptide chain. In particular, our mass spectrometry
measurements demonstrate that the methionines are not oxidized, which
is of particular importance because, in native cytochrome c,
the sulfur of methionine 80 is engaged in a coordination bond with the
iron of the heme (26).
All of the spectral and hydrodynamic properties that we observed with
our preparation of apocytochrome c fully confirmed those previously reported for the free apoprotein (10, 11) and the noncovalent complex (13) obtained with previous purification procedures. We also showed that despite the similarities of the absorption spectra of the heme in holocytochrome c and in
the noncovalent complex (13), the heme environment appears drastically different in these two species, because their CD spectra in the visible
range are widely different. Additionally, we brought direct evidence
indicating that the noncovalent complex is monomeric but shows a strong
tendency to slowly form high molecular weight aggregates even in dilute
(6 µM) solution. Surprisingly in view of its very low
solubility in the presence of ammonium sulfate, apocytochrome
c is much less prone to aggregation, even though we observed
during microcalorimetry measurements that it also forms some aggregates
upon standing for several hours in concentrated (250 µM) solutions.
The results obtained from antibody binding studies will now be
discussed. From a methodological point of view, it is worth emphasizing
the complementarity of the ELISA- and ITC-based methods for measuring
affinities. Indeed, low affinities such as those of the two antibodies
for apocytochrome c could well be measured by ITC but not by
ELISA. Conversely, very high affinities, such as those for native
holocytochrome c, were easily determined by ELISA but
required a modification of the standard ITC procedure. One should,
however, point out that in contrast to the limitations previously
encountered (31), the increased sensitivity of presently available
microcalorimeters provided reasonable titration calorimetry estimates
of the affinities of mAbs 2B5 and 5F8 for holocytochrome c.
Despite the fact that due to these high binding constants, the ITC
affinity measurements were made close to the limit of sensitivity of
the method, the values obtained by ELISA and ITC were in reasonably
good agreement. The agreement was particularly good for the
mAb5F8/holocytochrome c complex, although its dissociation constant is as low as 3 × 10
10 M, when
the titration was "sensitized" by premixing the antigen with the
appropriate amount of antibody (see under "Results"). The values we
obtained were, however, significantly smaller than those previously
reported for mAb5F8 (32) and mAb2B5 (30). The latter were measured at
25 °C, a temperature 5 °C above that of the present experiments
(20 °C, Table II). Prior to comparison, values of the affinity
constants must therefore be corrected for the temperature difference.
Based on the changes in heat capacities (
Cp) accompanying
the two binding reactions measured by titration calorimetry by Raman
et al. (30), corrected values of the affinity constants at
20 °C were of 2.3 × 1010 M
1
and 2.6 × 109 M
1 for mAb5F8
and mAb2B5, respectively. Hence, the affinity constants of the two mAbs
determined in this study (Table II) were 13-fold smaller than those
derived at the same temperature from the studies of Raman et
al. (30, 31). The reason why the affinity of mAb5F8 for cytochrome
c had been overestimated in previous studies has already
been discussed (32). The discrepancy between our values and that
reported previously for mAb2B5 is more difficult to explain. It may
originate from the fact, demonstrated in our microcalorimetry measurements, that this antibody binds efficiently only one cytochrome c molecule per IgG, a feature that the authors did not take
into account in their fitting model (30).
The microcalorimetry experiments that we report provided dissociation
constants in the micromolar range (Table II) for the complexes between
the two antibodies and apocytochrome c, whereas this method
failed to detect any interaction between this antigen and a control
antibody (mAb164) directed against a foreign protein. Moreover,
premixing apocytochrome c in a 100:1 molar ratio with mAb5F8
ruled out the possibility that the enthalpy of binding detected by
microcalorimetry might be due to a small contamination of the
apoprotein preparation by residual holocytochrome c. Thus, contrarily to earlier conclusions, antibodies 2B5 and 5F8 recognize the
apoprotein. Although their affinities for this antigen are much smaller
than for holocytochrome c (by factors 270 and 2700 for 2B5
and 5F8, respectively), these weak but significant interactions should
be taken into account whenever these antibodies are used at high
concentrations. Thus, in experiments aimed at monitoring the regain of
native-like epitopes during the refolding of holocytochrome c, antibody binding sites concentrations ranging between
13.3 and 48 µM were used (28). These concentrations were
at least 10-fold higher than the dissociation constants that we
determined for the corresponding antibodies. Thus, under these
conditions, the antibodies could practically saturate apocytochrome
c, i.e. the unfolded protein. This indicates that
the antibodies might bind to the polypeptide chain even before it folds
into a native-like conformation. This casts some doubts on the
conclusion that the early holocytochrome c folding
intermediates formed within less than 100 ms contain native-like
epitopes (28).
The large difference in the affinities of mAbs 2B5 and 5F8 for apo- and
holocytochrome c confirm that these antibodies can indeed be
used to probe the conformation of the polypeptide chain. Hence, because
both antibodies recognize the noncovalent complex much better than
unfolded apocytochrome c and nearly as well as native
holocytochrome c, one can conclude that the noncovalent heme-apocytochrome c complex carries pseudo-native epitopes.
What does "pseudo-native" mean? Two extreme points of view could be adopted. One is that in the absence of antibody, each individual molecule of the noncovalent complex would carry a slightly distorted epitope that can be recognized by the antibody. The ratio of about 10 between the affinities of the native and the distorted epitope could
then result from the loss of as little as one elementary interaction
with a free energy of about 1.5 kcal/mol, like a hydrogen bond or one
hydrophobic contact. Alternatively, it could reflect an "induced
fit" of the epitope upon binding to the antibody, which would cost
about 1.5 kcal/mol and therefore decrease the affinity by a factor 10. Another point of view, based on the two-state hypothesis for protein
folding and adopted by Furie et al. (33) in their
"Kconf" model, would be that the antibody
recognizes only the native antigen and that the affinity exhibited for
a distorted antigen reflects a pre-existing equilibrium between the
unfolded, nonimmunoreactive conformation of the protein and its native,
fully immunoreactive conformation. Accordingly, the noncovalent complex
would exist in a dynamic equilibrium between the unfolded and native
conformations of cytochrome c. The ratio of the apparent
affinity of a monoclonal antibody for the noncovalent complex
(Knc) to the real affinity of that antibody for
holocytochrome c (Kh) would then be
related to the equilibrium constant (Kconf) between the nonreactive and the native conformations of the protein in
the population of noncovalent complex molecules by the equation Kconf = Knc/(Kh
Knc) (33). Because the folding of a small protein such as cytochrome c is highly cooperative, a strong
prediction of this model is that the value of
Kconf should be independent of the antibody
used, because it would reflect the intrinsic conformational equilibrium
of the antigen. Within the precision of the experimental measurements,
this prediction was reasonably well verified for the noncovalent
complex. Indeed, the value of Kconf was
estimated (from the average of the ELISA and microcalorimetric
determinations of Kh) to be 0.25 for mAb2B5 and
0.14 for mAb5F8. Thus, the immunoreactivity of the noncovalent complex
seemed at first sight compatible with the existence of a cooperative
conformational equilibrium between the unfolded and the native
conformations of the polypeptide chain.
The Kconf model assumes that the antibody has no
affinity at all for the unfolded antigen. Yet the two antibodies show a
small, but certainly not negligible, affinity for apocytochrome
c. Again, one might assume that these small affinities in
fact reflect the existence of a spontaneous equilibrium between a small
fraction of native-like molecules and a majority of unfolded,
nonimmunoreactive molecules in the apocytochrome c.
Accordingly, comparing the affinities of both antibodies for apo- and
holocytochrome c indicates that the
Kconf would be about 4 × 10
3
for mAb2B5 and 3.5 × 10
4 for mAb5F8. Then, about
0.1% only of the molecules would be native, a fraction that by no
means could be detected by the spectroscopic or hydrodynamic methods
used to characterize apocytochrome c. That the values of
Kconf of apocytochrome c estimated
for the two antibodies differ by a factor about 10 suggests that a
concerted conformational equilibrium between unfolded and native
apocytochrome c cannot account for the small
immunoreactivity detected in the apoprotein with the two antibodies.
This conclusion is supported by the comparison of the enthalpies of
binding of both antibodies to apo- and holocytochrome c.
Indeed, the Kconf model predicts that the
difference between the enthalpies of binding of the "distorted" antigen and of the native antigen to the antibody should be equal to
the enthalpy of the transition from the unfolded to the native conformation. Hence, the enthalpy differences determined for both antibodies should be similar. The same should hold for the entropy differences. Whereas the precision of the free energies (and hence of
the entropies) of binding is not very good because they rely on the
determination of the equilibrium constants, the values of the
enthalpies of binding can be determined much more precisely from the
total heat exchange during the transition. The values of 
H
(i.e.
H°apo
H°holo)
calculated for mAbs 2B5 and 5F8 from the enthalpies of binding reported
in Table II are 4.1 and 7.2 kcal/mol, respectively. The large
difference between these two values is clearly not compatible with the
prediction made on the basis of a concerted conformational equilibrium
between fully unfolded and native apocytochrome c molecules.
The basic assumption of the Kconf model is
therefore definitely not acceptable in the case of apocytochrome
c. Rather, it seems likely that the two epitopes undergo
nonconcerted equilibria or induced fits.
Yet the conformations of the two epitopes cannot be considered as
entirely independent within the apocytochrome c polypeptide chain. Indeed, the binding of mAb2B5 to its epitope on apocytochrome c results in an increased affinity of mAb5F8 for a fraction
(about 15%) of the antigen molecules. We shall first discuss the
properties of the 2B5/apocytochrome c molecules that bind
mAb5F8 with a high affinity. This affinity is about 10-fold higher than
that for apocytochrome c alone, but still 100-fold lower
than that for holocytochrome c. Thus, the conformation of
the polypeptide chain is certainly not entirely native. Yet the binding
of mAb2B5 appears to have either shifted a preexisting conformational
equilibrium, or induced a conformational change, that brings the
polypeptide chain somehow closer to its native conformation and creates
a better fit between mAb5F8 and its cognate epitope. The amplitude of
the enthalpy of binding of mAb5F8 to these molecules is unusually large
(
118.2 kcal/mol) when compared with the binding enthalpies of
monoclonal antibodies to protein antigens. In particular, it is about
5-fold larger than its binding enthalpy to native holocytochrome c. At the same time, the amplitude of the binding entropy is
also exceptionally high (
368 cal/mol/K) and corresponds to about
11-fold the entropy of binding of mAb5F8 to native holocytochrome
c. This strongly suggests that the binding of mAb5F8 to the
"high affinity" mAb2B5/apocytochrome c molecules is
accompanied by an important conformational change of the polypeptide
chain. Considering that the measured enthalpy (or measured entropy)
corresponds to the sum of the enthalpy (respectively entropy) of this
conformational change and of the enthalpy (respectively entropy) of
binding of mAb5F8 to the reorganized molecule, one can estimate to
about 96 kcal/mol and 335 cal/mol/K the enthalpy and entropy,
respectively, associated with the change in conformation undergone by
the mAb2B5-bound apocytochrome c when it binds to mAb5F8.
This suggests that within the 2B5/apocytochrome/5F8 ternary complex,
apocytochrome c becomes highly ordered, as shown in
particular by the strongly negative value of the entropy. Although no
direct structural evidence exists that supports this hypothesis, one
may speculate that this ordered structure may be close to the native
conformation of holocytochrome c. Regardless of the exact
nature of this conformation, these results indicate that like heme
binding, the simultaneous binding of the two monoclonal antibodies to
apocytochrome c represents a case where ligand binding is
the principal determinant of a protein conformation, as nicely
illustrated in the case of the p21H-ras protein
(34) and extensively discussed for a variety of proteins (35).
Finally, the fact that only a fraction of the mAb2B5-bound
apocytochrome c molecules have a high affinity for mAb5F8
suggests that the binding of mAb2B5 to its epitope hinders some
intramolecular movements within the apocytochrome c
polypeptide chain, thus preventing the interconversion of two
subpopulations of mAb2B5/apocytochrome c complexes, one with
high affinity and one with low affinity for the second antibody mAb5F8.
As discussed above, the binding of mAb2B5 may represent a key
interaction in the antibody induced folding of apocytochrome
c. One may therefore draw an analogy between the low
affinity subpopulation of mAb2B5/apocytochrome molecules and
kinetically trapped folding intermediates that need to overcome the
activation barrier surrounding a metastable state before they can
proceed to the native conformation.
In conclusion, we wish to point out the wealth of information that
conformation-specific monoclonal antibodies can provide in a case where
powerful structural approaches such as NMR or x-ray crystallography are
inapplicable to investigate protein conformation and dynamics. Not only
did they provide valuable information on the noncovalent
heme-apocytochrome c complex, but they also shed some light
over the folding properties of the cytochrome c polypeptide chain.