a-Fetoprotein positively regulates cytochrome
c
-mediated caspase
activation and apoptosome complex formation
Lidia Semenkova
1,
*, Elena Dudich
1,
*, Igor Dudich
1
, Natalie Tokhtamisheva
1
, Edward Tatulov
2
,
Yury Okruzhnov
3
, Jesus Garcia-Foncillas
3
, Juan-Antonio Palop-Cubillo
4
and Timo Korpela
5
1
Institute of Immunological Engineering, Moscow, Russia;
2
Anticancer Drug Research Center, Moscow, Russia; Departments of
3
Oncology and
4
Organic Chemistry and Pharmacology, University of Navarra, Pamplona, Spain;

a-fetoprotein.
Apoptotic cell death is characterized by biochemical and
morphological changes, which are largely caused by caspase
activity. A class of cysteine proteases, known as caspases,
which are constitutively expressed in cells as inactive
proenzymes, require proteolytic cleavage to be activated.
In general, either receptor-induced or mitochondrion-
induced death signals stimulate activation of specific
adapterproteinsFADD/MORT1orApaf-1byformation
of the high-molecular-mass death-inducing complex or
apoptosome. The adapter proteins recruit initiator caspases
8 and 9 to activate them by autoprocessing. Once activated,
initiator caspases are ready to induce processing of down-
stream effector caspases 3 and 7 [1]. The mitochondrial
apoptosis pathway is mediated by cytochrome c (cyt-c)
release with the subsequent formation of the Apaf-1/cyt-c/
dATP/procaspase 9 apoptosome complex, leading to acti-
vation of caspase 9 and downstream effector caspases [2].
Chromatographic analysis of the apoptosome assembly
indicated that, in native cell lysates, Apaf-1 oligomerizes
into multimeric complexes of molecular mass  1.4 MDa
and  700 kDa, which in addition to processed caspase 9,
contain fully processed caspase 3 and 7 [3]. Caspases are
inhibited by a number of cellular inhibitor of apoptosis
proteins (cIAPs), which bind directly to procaspases 9 and 3
to prevent their cyt-c-mediated processing and activation
[4,5]. During apoptosis, a mitochondrial protein named
Smac/DIABLO [6] that directly binds to IAPs to remove
them from the apoptosome complex [4,7], cancels the
IAP-mediated caspase inhibition. Recently, another IAP-

AFP is specifically bound to the cell surface at 4 °Cand
internalized into the cytoplasm at 37 °C [15,16]. It has been
shown that AFP is internalized via coated pits and vesicles
before being delivered to endosomes [15,16]. Much evidence
of cell growth regulatory activity, including tumor suppres-
sion, has been reported for various species of the full-length
AFP molecule [17–22], its proteolytic fragments [23],
recombinant domains [24] and synthetic peptides [25–27].
It has been demonstrated that AFP realizes its tumor-
suppressive activity by triggering apoptosis, characterized
by typical morphological changes, growth arrest, cytotoxi-
city, and DNA fragmentation [20–22]. It was shown that
AFP induces apoptosis in malignant cells through activa-
tion of caspase 3, bypassing Fas/FasL and tumor necrosis
factor (TNF)/TNFR-dependent pathways and does not
require upstream activation of receptor-dependent initiatory
caspase 8 and caspase 1 [21]. Although these studies have
shown that a caspase cascade is initiated during AFP-
induced apoptosis, the mechanisms by which AFP triggers
caspase activation are unknown. Our previous experimental
data show that AFP does not require de novo protein
synthesis and RNA expression to trigger apoptosis, as it was
not blocked by actinomycin D or cycloheximide [20].
In this study, we aimed to determine how AFP activates
the caspase cascade. To understand the molecular mecha-
nisms of AFP-mediated apoptosis signaling, we established
a cell-free system, similar to that used for studies of cyt-c-
induced apoptosis [28,29]. We show here that AFP syner-
gistically enhances caspase activation and processing in the
presence of a low suboptimal dose of cyt-c and requires the

0.1 mg streptomycinÆmL
)1
in a humidified 5% (v/v)
atmosphere of CO
2
at 37 °C. For a passage, cells were
incubated in 0.25% (v/v) trypsin solution, then washed and
plated out.
Cytotoxicity assay
HepG2 cells were incubated with 5–7 l
M
AFP for deter-
mined time intervals of 2–14 h, and then assessed for their
viability by the trypan blue exclusion assay as described
previously [22]. Cells cultivated without additions were
taken as a control. The experimental data were expressed as
the percentage of dead cells relative to the total amount of
cells.
Preparation of cell-free extracts
Cell-free S-100 extracts were generated from human
hepatocarcinoma HepG2 as described [29,30]. Cells
(4 · 10
8
) were collected and washed (three times) in
50 mL NaCl/P
i
and once in 5 mL hypotonic cell extraction
buffer (containing 20 m
M
Hepes, pH 7.2, 10 m

stored in aliquots at )70 °C. Cyt c-free cytosolic extracts
were prepared in more mild conditions by the slightly
modified procedure described in [30].
In vitro
caspase activation
For in vitro caspase activation, 40 lg of the S-100 extract
(complete or after immunodepletion) was incubated for the
indicated times with bovine heart cyt-c (Sigma-Aldrich,
St Louis, MO, USA) and/or pure human AFP (5 l
M
)inthe
presence or absence of 1 m
M
dATP (Sigma) in 15 lLofa
reaction buffer (10 m
M
Hepes, pH 7.2, 25 m
M
NaCl, 2 m
M
MgCl
2
,5m
M
dithiothreitol, 5 m
M
EDTA, 0.1 m
M
phenyl-
methanesulfonyl fluoride) at 30 °C. To control specificity

M
dithiothreitol, 10% sucrose). Reactions
were terminated by dilution with 2.0 mL ice-cold 0.2 m
M
sodium phosphate buffer, pH 7.5, and fluorescence was
measured using a Perkin–Elmer MPF-44A fluorimeter
(k
exc
¼ 365 nm and k
em
¼ 440 nm for the AMC fluores-
cence or k
exc
¼ 400 nm and k
em
¼ 505 nm for the AFC
fluorescence). For each sample, caspase activity was
expressed in relative units, pmolÆmin
)1
Æmg
)1
, showing the
amount of cleaved substrate in pmol normalized for time of
reaction with substrates and cytosolic protein concentra-
tion, or in relative fluorescent units (FU) per fraction.
Immunoprecipitation and immunoblotting analysis
S-100 cytosolic extracts obtained from HepG2 cells were
immunodepleted from endogenous cyt-c, procaspase 9 or
procaspase 3 by immunoprecipitation with the corres-
ponding antibodies as described [31]. Briefly, 50 lLofthe

corresponding polyclonal antibody goat anti-(caspase 3) or
anti-(caspase 9). Bound antibodies were detected using
appropriate horseradish peroxidase-conjugated anti-rabbit
or anti-goat secondary IgGs (Santa Cruz) and developed by
enhanced chemiluminescence staining using ECL reagents
(Amersham Pharmacia Biotech). Gel calibration was per-
formed with the Low Molecular Weight Calibration Kit for
SDS Electrophoresis (Amersham Pharmacia Biotech).
Dot-blot analysis was performed as usual. Briefly, 1-lL
aliquots taken from the chromatographic fractions were
applied to the nitrocellulose membranes, then blocked by
defatted milk. The membranes were then probed with rabbit
polyclonal affinity-purified anti-(human AFP) IgG. Bound
antibodies were detected using appropriate peroxidase-
coupled secondary antibodies and developed as described
above.
Assay of cyt-c release
Cyt-c translocation from mitochondria to the cytoplasm
was assessed by direct immunochemical measurement of the
cyt-c in the cytosolic and mitochondrial fractions obtained
from HepG2 cells treated with AFP for various time
intervals. Briefly, cells (0.5 · 10
6
cells per well) in Dulbecco’s
modified Eagle’s medium with 10% fetal bovine serum were
plated on the flat-bottomed 24-well plates (Nunc) and
incubated for 24 h. Then 5 l
M
AFP was added to each well.
After various lengths of treatment (2–17 h), cells were

(mitochondrial fraction). Mitochondrial pellet was solubi-
lized by a 30-min incubation with 100 lL lysing buffer
(150 m
M
NaCl, 1% Nonidet P40, 0.5% deoxycholate, 0.1%
SDS, 50 m
M
Tris/HCl, pH 7.5, cocktail of protease inhi-
bitors). Thereafter, cellular debris was removed by a 10-min
centrifugation at 14 000 g at 4 °C. The supernatant com-
prising the membrane fraction was retained. Equal amounts
of cytosolic extracts and solubilized mitochondrial pellets
(50 lg protein) were fractionated by SDS/PAGE using 15%
polyacrylamide and then analysed by Western blot using the
cyt-c antibody 7H8.2C12, cyt-c oxidase subunit II antibody
(Molecular Probes), and b-actin antibody and ECL as
described above.
Direct protein–protein interaction assay
To determine possible interactions between AFP and
caspase 3, caspase 9 and cIAP-2, we used a direct copre-
cipitation assay with purified proteins. Before the experi-
ments, 25 lL Ni/Sepharose beads (Qiagen, Valencia, CA,
USA) were incubated for 1 h at 20 °C in a solution of assay
buffer (50 m
M
Tris/HCl, 100 m
M
KCl, 10% sucrose, 0.1%
Chaps, 0.5 m
M

cyt-c with or without 5.0 l
M
AFP. Before addition
to the S-100 extracts, AFP samples were dialyzed against the
elution buffer. Activated lysate proteins ( 1mg) were
applied (0.2 mLÆmin
)1
;4°C) to a 10/30 Superose-6 HR
column connected to an FPLC system (Amersham Phar-
macia Biotech). The column was eluted with elution buffer
(20 m
M
Hepes/KOH, 10 m
M
KCL, 1 m
M
EDTA, 1 m
M
EGTA, 1 m
M
dithiothreitol, 1.5 m
M
MgCl
2
,0.01m
M
phenylmethanesulfonyl fluoride, pH 7.2); 1-mL fractions
were collected. Aliquots of the fractions were taken for
measurement of caspase activity using the corresponding
fluorogenic substrates: DEVD-AMC for caspase 3 and Ac-

procaspase 9/cyt-c apoptosome cascade [28]. To determine
whether AFP is involved in this process, we established a
typical cell-free system using HepG2 cells and measured
caspase activation in this system with or without addition
of AFP. Two types of cell lysate were used for these
experiments: a typical S-100 cytosolic extract and a cyt-c-
free cytosolic extract, prepared by a mild procedure as
described previously [30]. Addition of AFP to the S-100
cytosolic extract triggered dATP-dependent induction of
caspase 3-specific DEVDase activity, which progressively
increased for at least 2 h (Fig. 2A). As a control, the
equivalent amount of human serum albumin was added to
the same cell-free system. No effect was observed at the level
of DEVDase activity. A low level of DEVDase activity was
also induced by dATP alone, evidently due to the presence
of a small amount of endogenous cyt-c in the preparations.
In the absence of dATP, AFP did not induce any caspase 3-
specific DEVDase activity at all.
To determine whether AFP can directly induce caspase
activation in cell-free cytosolic extract or requires the
presence of the basal level of cyt-c, we examined DEVDase
cleavage activity after addition of exogenous cyt-c and AFP
to the ÔsilentÕ cytosolic extracts with undetectable endo-
genous cyt-c. Figure 2B shows that no DEVDase activity
was detected in this type of cytosolic lysate stimulated with
dATP/AFP or with dATP and low suboptimal dose of cyt-c
even 1.5 h after treatment. A significant time-dependent
increase in DEVDase activity was observed in the same
reaction system only after addition of all three compounds:
AFP,dATPandcyt-c(Fig.2B).ThelowDEVDaseactivity

activation in a cell-free system, we examined S-100 extracts
for cleavage of procaspases 3 and 9 and corresponding
fluorogenic caspase substrates after addition of AFP/cyt-c/
dATP. Both procaspase 9 and procaspase 3 were processed
to their active forms, giving the corresponding fragments
p35/37 and p10 for caspase 9 and p17 and p12 for
caspase 3. However, when AFP was combined with cyt-c/
dATP, more complete cleavage of the procaspases was
observed (Fig. 3B,C). In addition, there was a dramatic
increase in caspase 3-like DEVDase activity and a notable
increase in caspase 9-like LEHDase activity on combined
treatment with AFP/cyt-c/dATP in comparison with cyt-c/
dATP (Fig. 3A). These data show that AFP positively
regulates both processing and activation of procaspases 9
and 3 in cell-free cytosolic extracts by amplification of the
low-dose cyt-c-mediated effects.
AFP induces caspase activation only in the presence
of the all components of the apoptosome complex
The above experiments demonstrated functional interfer-
ence of AFP with the cyt-c-mediated process of caspase
activation. We studied further the functional significance of
Fig. 2. AFP enhances cyt-c-mediated DEVDase activity in cell-free
cytosolic extracts. (A) AFP induces caspase 3 activation in cell-free
S-100 cytosolic extracts in the presence of dATP. Effect of endogenous
cyt-c. Aliquots of HepG2-derived cytosolic extract (25 lgprotein)
were treated for various times with AFP (5 l
M
) or as a control with the
same dose of human serum albumin in the presence of dATP (1 m
M

M
) and/or AFP (5 l
M
). (A) Proteolytic activities of caspase 9 and
3 in experimental lysates were assayed by monitoring the cleavage of
the corresponding fluorogenic substrates LEHD-AFC and Ac-DEVD-
AMC. The mean ± SD from four determinations is shown.
Processing of caspases was detected by immunoblotting with the cor-
responding antibodies that recognize the precursors and subunits of
active caspase 9 (B) and 3 (C).
4392 L. Semenkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003
AFP in regulation of activity of the apoptosome complex.
Cellular extracts were sequentially depleted of the main
active molecular compounds involved in the formation of
the apoptosome complex: endogenous cyt-c, procaspase 3,
or procaspase 9. Caspase activation was then induced by the
addition of cyt-c/dATP with or without AFP. AFP was
unable to induce caspase 3 activation in the absence of cyt-c
and/or dATP in the cyt-c-immunodepleted cytosolic extracts
(Fig. 4). However, addition of exogenous cyt-c together with
dATP produced DEVDase activity. Simultaneous addition
of all three compounds (AFP, cyt-c and dATP) resulted in
significant enhancement of total DEVDase activity com-
pared with that induced with cyt-c/dATP (Fig. 4).
We next determined whether AFP requires the presence
of procaspase 9 to induce caspase 3 activation mediated by
a suboptimal dose of cyt-c. HepG2 S-100 extracts were
depleted of procaspase 9 by immunoprecipitation with the
corresponding antibody and then treated with AFP/cyt-c or
cyt-c alone in the presence of dATP. Figure 5A,B shows

M
)inthe
presence of dATP (1 m
M
). Caspase 3 activity was measured by
monitoring cleavage of the fluorogenic substrate DEVD-AMC. The
mean ± SD from four determinations is shown.
Fig. 5. Procaspase 9 is required for AFP-mediated caspase 3 activation.
(A) S-100 cytosolic extract was immunodepleted of procaspase 9 by
immunoprecipitation with anti-(caspase 9). To confirm caspase 9
depletion, equal amounts (50 lg) of control untreated extract, cyt-c-
treated extract, extract treated with anti-RXR (control for possible
unspecific antibody-induced effects) and caspase 9-depleted extract
were analysed by immunoblotting with anti-(caspase 9). b-Actin was
used as a loading control. (B) Caspase 3 activation was induced in
different types of experimental extract: caspase 9-depleted extract,
complete extract, complete extract incubated with Ac-LEHD-CHO
and extract treated with anti-RXR. Extracts were activated by addition
(+) or in the absence (–) of appropriate doses of AFP (5 l
M
)and/or
cyt-c (0.2 l
M
) in the presence of dATP (1 m
M
). Caspase 3 activity was
measured by cleavage of the fluorogenic substrate DEVD-AMC. The
mean ± SD from four determinations is shown.
Ó FEBS 2003 AFP amplifies cytochrome c-mediated caspase activation (Eur. J. Biochem. 270) 4393
antibody. Depletion was controlled by immunoblotting

(fractions 8–10) and was detected only in fractions 14–15,
corresponding to the free form of the processed enzyme, as
described previously [31,33]. Thus, in the absence of
caspase 8 in the apoptosome complex, IETDase cleavage
activity in this region may represent effects induced by
active forms of caspase 9 [34]. The data obtained from
measurement of LEHDase cleavage activity showed signi-
ficantly lower fluorescent intensity and were difficult to
interpret (not shown). Our data demonstrate that AFP did
not induce any changes in IETDase activity in the position
of the active 700-kDa complex (fractions 8–10), but
DEVDase activity in this region was notably enhanced
compared with the effect of cyt-c alone (Fig. 7A,B). The
most significant AFP-mediated increase in DEVDase
cleavage activity was observed at  70–60 kDa (fractions
15–17), corresponding to the free active caspase 3
(Fig. 7B). Figure 7A shows that integral IETDase activity
at  90 kDa corresponding to free active caspases 9 and 8
(fractions 14–15) was also enhanced after AFP addition
(Fig. 7A).
The distribution of caspase 9 and caspase 3 precursors
and mature forms distinctly correlates with the corres-
ponding activity patterns (Fig. 7A,B). Caspase 9 was
processed under these conditions and showed two peaks
in the column for both experimental systems with and
without addition of AFP. The main peak of caspase 9-
specific material was located in fractions 9–10, whereas
the second peak was at fractions 13–15. It should be
mentioned that a smaller amount of the processed
caspase 9 was also detected in fractions 6–7, correspond-

(B) Caspase 9 activation in caspase 3-depleted extracts was induced by
addition of the appropriate doses of AFP (5 l
M
), cyt-c (0.2 l
M
), and
dATP (1 m
M
) and assessed by cleavage of the fluorogenic substrate
LEHD-AFC. The mean ± SD from four determinations is shown.
4394 L. Semenkova et al.(Eur. J. Biochem. 270) Ó FEBS 2003
were detected mainly in fractions 13–14 ( 160–
180 kDa), reflecting activity distribution (Fig. 7B). These
data indicate that, at low cyt-c, caspase 3, like caspase 9,
tends to form an intermediate  160–180-kDa complex
or migrate together with other protein aggregates in this
region. In the extracts stimulated with AFP/cyt-c/dATP,
we revealed the precursor form of caspase 3 in fractions
13–14, whereas processed caspase 3 was recovered
mainly in fractions 15–16, showing again that AFP
stimulates release of the free active caspase 3 from the
complex.
We have also monitored the distribution of AFP along
the chromatography pattern of the S-100 extracts after
addition of AFP/cyt-c/dATP and found this 70-kDa protein
in fractions corresponding to the high-molecular-mass
complexes (Fig. 7B, bottom). Pure protein migrates at the
position corresponding to its monomeric size compared
with the molecular mass standard. This indicates that AFP
may be involved in formation of the high-molecular-mass

distribution of Apaf-1 along the chromatographic pattern
of the apoptosome assembly, which was formed with and
without AFP (Fig. 7C). In cell extracts stimulated with low
cyt-c, Apaf-1 was recovered in two main peaks correspond-
ing to fractions 6–8 and 13–15, demonstrating that a low
suboptimal dose of cyt-c does not recruit all the available
Apaf-1 into the functional apoptosome and tends to form
the nonfunctional complex of molecular mass  1.4 MDa.
In the presence of AFP, Apaf-1 specificity was significantly
reduced in the biologically inactive  1.4-MDa complex
(fractions 6–7) [3,35], but notably increased in the region of
the  700-kDa apoptosome (fractions 8–10). These data
indicate that, at low cyt-c, AFP positively modulates
recruitment of Apaf-1 into the active  700-kDa apopto-
some complex.
Figure 7D shows that cIAP-2 distribution was not so
clearly affected by AFP addition as observed in the case of
Apaf-1. However, in the absence of AFP, full-length cIAP-2
was present in fractions 10–11, whereas fraction 9 mainly
contained fragmented IAP-2-specific material (Fig. 7D).
After the addition of AFP, the cIAP-2 specificity (including
full-length protein and its fragments) was distinctly reduced
in fractions 9–10 (Fig. 7D). The similar fragmentation
pattern for cIAP-1 and cIAP-2 has been described previ-
ously [36]. It was shown that fragmented cIAP-1 and cIAP-2
were more effective at protecting cells from apoptosis,
whereas full-length proteins lacked protective activity.
Removal of the RING domain by proteolysis restored the
antiapoptotic activity [36]. It was also shown that cIAP-1
was cleaved in vitro by pure caspase 3, producing similar

the cIAP-2 content, resulting in promotion of the release of
active caspases 3 and 9 from the complex.
Discussion
There is increasing evidence that AFP may selectively
induce activation of programmed cell death in tumor cells
[17–23], showing its potential for cancer treatment [10].
Various researchers have documented the tumor-selective
uptake of AFP by malignant cells [13–16], but the functional
significance of this phenomenon has not been clarified. The
exact molecular mechanisms of AFP-mediated apoptosis
also remain unclear. The present data explain some details
of the molecular interactions in this effect.
In this study we have investigated the ability of AFP to
directly activate the death program in a cell-free model of
apoptosis. Release of cyt-c into the cytoplasm of AFP-
treated cells suggests that a mitochondrion-dependent
mechanism of apoptosis signaling is involved. However,
these data do not exclude the possibility that another cyt-c-
independent pathway of AFP-mediated signaling of apop-
tosis is also involved in the sequential indirect induction of
cyt-c release with the onset of its activity. We found here
that AFP promotes low-dose cyt-c/dATP-mediated pro-
cessing and activation of procaspases 9 and 3 in a cell-free
system. These data show that AFP is directly involved in
regulating the mechanisms of caspase cascade activation
and suggest that it may be involved in regulating apopto-
some complex formation. We have demonstrated further
that AFP-mediated signaling of apoptosis requires the
presence of all the major members of the apoptosome
complex: cyt-c, dATP, caspases 9 and 3. To confirm that

tissues, such as developing immature immune cells, embry-
onic cells or tumor cells, certain natural control mechanisms
have to exist that select and direct developing cells toward
maturation and prevent their neoplastic transformation.
This study describes a naturally occurring protein, the
expression of which is restricted by developing immature
embryonic cells or cells undergoing malignant transforma-
tion [9–12]. Proteins with quite mundane functions in
healthy cells often behave very differently during cell suicide.
The selective proapoptotic activity of AFP, targeting only
neoplastic [17–23] and activated immune cells [9,10], indi-
cates that it is a natural effector in a fetoembryonic defense
system to prevent malignant transformation of developing
cells. Our data allow us to propose that AFP helps cells to
overcome their resistance to apoptosis by significant ampli-
fication of the apoptotic signals induced by other factors,
such as drugs and oxidative stress. AFP may help cells,
which are resistant to apoptotic stimuli for any reason, to
overcome their resistance, which is induced, for example, by
overexpression of heat shock proteins, cIAPs or any other
defects of apoptosome-dependent apoptotic pathways.
Tumor cells are characterized by defects in expression of
apoptosis-promoting proteins, such as Apaf-1 and p53, and
simultaneous overexpression of the antiapoptotic proteins
Hsp70, Bcl-2 and Bcl-x
L
, resulting in tumor-specific sup-
pression of apoptosis and enhancement of the malignance
and therapy resistance of tumors [39–43]. The existence of a
high background level of antiapoptotic factors in the cytosol

background level of cytosolic cyt-c has been shown in vivo in
the aging heart, with a significant decrease in the antiapop-
totic protein bcl-2 [49]. In certain types of cancer cell,
alterations in the regulation of apoptosis may contribute to
tumor malignancy and resistance to radiotherapy and
chemotherapy [50]. Sometimes dysfunctional apoptosome
activation in tumor cells is observed in the presence of the
required amount of cytosolic cyt-c, dATP, Apaf-1 and pro-
caspase 9, leading to significant enhancement of their
resistance to apoptotic stimuli including radiotherapy and
chemotherapy [51].
Our data indicate that AFP can be considered as a tumor-
specific regulator of cyt-c-mediated apoptotic signals.
In vivo, it may operate as a specific regulator of the
apoptosome dysfunction induced by the impaired release of
apoptogenic factors in the cytosol and/or the increased level
of cytosolic antiapoptotic proteins. It may operate to
amplify weak apoptotic signals induced by oxidative stress,
ionizing radiation or drugs to sensitize tumor cells to
chemotherapy. It seems to operate as a tumor-specific
regulator of apoptosis inhibitory proteins, but it remains to
be seen if it associates with and inhibits cIAPs other than
cIAP-2, and to determine the molecular mechanisms of
these interactions.
Acknowledgements
This work is supported in part by the International Science &
Technology Center, ISTC (grants Nos. 401-98 and 1878-01). We thank
Dr Alex Sazonov for the invaluable gift of recombinant caspase 3 and
caspase 9 and Dr Alex Chugunov for excellent assistance with FPLC
chromatography.

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