The Mechanism of 2,2,2-Trichloroacetic Acid-Induced Protein Precipitation

Abstract

The mechanism of 2,2,2-trichloroacetic acid (TCA)-induced precipitation of proteins is studied. The TCA-induced protein precipitation curves are observed to be U-shaped. It is bound that the protein-precipitate-inducing effects of TCA are due to the three chloro groups in the molecule. Using cardiotoxin III (CTX III) isolated from the Taiwan cobra (Naja naja atra), as a model protein, we attempt to understand the molecular basis for the TCA-induced effects. Employing circular dichroism, proton-deuterium exchange in conjunction with conventional 2D NMR techniques, and 1-anilino naphthalene-8-sulfonate-binding experiments, we demonstrate that CTX III is in a partially structured state similar to the 'A state' in 3% w/v TCA. It is postulated that the formation of this 'sticky' partial structured 'A state' in the TCA-induced unfolding pathway is responsible for the acid-induced protein precipitation.

Full text

Journal
of
Protein Chemistry, Vol.
16, No. 4,
1997
The
Mechanism
of
2,2,2-Trichloroacetic
Acid-Induced
Protein
Precipitation
T.
Sivaraman,,
1
T. K. S.
Kumar,
1
G.
Jayaraman,
1
and C.
Yu
1
-
2
Received
December
11,
1996
The
mechanism
of
2,2,2-trichloroacetic
acid
(TCA)-induced
precipitation
of
proteins
is
studied.
The
TCA-induced protein
precipitation
curves
are
observed
to be
U-shaped.
It is
bound
that
the
protein-precipitate-inducing
effects
of TCA are due to the
three
chloro
groups
in the
molecule. Using cardiotoxin
III
(CTX III) isolated
from
the
Taiwan cobra
(Naja
naja
atra),
as a
model protein,
we
attempt
to
understand
the
molecular
basis
for the
TCA-induced
effects.
Employing
circular
dichroism,
proton-deuterium
exchange
in
conjunction
with
conventional
2D NMR
techniques,
and
1-anilino
naphthalene-8-
sulfonate-binding
experiments,
we
demonstrate that
CTX III is in a
partially
structured
state
similar
to the 'A
state'
in 3% w/v
TCA.
It is
postulated
that
the
formation
of
this
'sticky'
partial
structured
'A
state'
in the
TCA-induced
unfolding
pathway
is
responsible
for
the
acid-induced
protein
precipitation.
KEY
WORDS: A-state;
CTX
III; protein precipitation; TCA.
1.
INTRODUCTION
One of the
most challenging problems
in
biology
today
is to
unravel
the
mechanisms
of
protein
folding
(Baldwin,
1995).
The
high cooperativity
of
the
process
and its
rapidity
are the two
major
impediments
to
solving
the
protein folding problem
(Hughson
et al,
1990;
Dobson,
1994).
Protein aggregation
is a
widespread phenome-
non
that occurs during protein folidng
in
vivo
and
in
vitro (Goldberg
et
al.,
1991).
The
formation
of
aggregates
is
often considered
to be a
nonspecific
association
of
partially folded polypeptide chains
through hydrophobic interactions
to
form
a
precipitate
(De
Young
et al,
1993).
Understanding
the
mechanism
of
protein association/aggregation
is
important
to
solving
the
problem
of
formation
of
inclusion bodies during overexpression
of
recom-
binant proteins
in
foreign hosts (Kumar
et
al.,
1996a;
Mitraki
and
King, 1989)
and
also
in the
prevention/cure
of
various human diseases which
are
believed
to be
caused
due to
protein
aggregation/association (Thomas
et
al., 1995;
Wetzel, 1994)
in the
cell.
2,2,2-Trichloroacetic acid (TCA)
is a
well-
known
protein-precipitating agent. Understanding
the
mechanism
by
which
TCA
induces precipitation
of
proteins could
offer
useful
clues
to the
solubility
problems associated with inclusion bodies formed
in
vivo.
In
this context,
in the
present study,
we
embark
on
investigating
the
molecular mechanism
underlying
the
TCA-induced precipitation
of
proteins.
2.
MATERIALS
AND
METHODS
Hen
egg-white lysozyme, TCA, acetic acid,
monochloroacetic acid, dichloroacetic acid, tri-
fluoroacetic
acid,
and
tribromoacetic acid were
purchased
from
E.
Merk, Germany. Bovine serum
albumin
(BSA)
and
1-anilino naphthalene-8-
sulfonic
acid (Mg
2+
salt) (ANS) were
from
Sigma
Chemical Co., USA. Cardiotoxin analgoue
III
(CTX III)
was
purified
from
Taiwan cobra venom
Department
of
Chemistry, National Tsing
Hua
University,
Hsinchu, Taiwan.
2 To
whom correspondence should
be
addressed; e-mail:
cyu@chem.nthu.edu.tw.
291
0277-8033/97/0500-0291$12.50/0© 1997
Plenum
Publishing
Corporation

292
Sivaraman,
Kumar, Jayaraman,
and Yu
(Naja
naja
atra)
as per the
procedure
of
Yang
et al.
(1981).
All
other chemicals used were
of
high-quality
analytical
grade.
Protein solutions were
made
in
deionized water.
All
experiments, unless
otherwise mentioned, were carried
out at 25 ±
2°C.
The
concentrations
of the
various acids used
are
reported
as
percentage weight
to
volume (w/v).
2.1. Treatment with Acids
The
proteins, lysozyme, BSA,
and CTX III
were
treated
with
TCA and
other
acids
by the
method
of
Sagar
and
Pandit
(1983).
Protein
solutions
containing appropriate concentrations
of
the
respective acids were prepared
by the
addition
of
requisite amounts
of the
stock solutions
of the
individual acids
to
aqueous
solutions
of
proteins.
The
acid(s)-treated protein solutions were incub-
ated
at
25°C
for
2hr.
The
precipitated protein
samples were pelleted down quickly
by
centrifuga-
tion
at
3000
rpm for 20
min. Protein concentrations
in
the
suspernatant were determined using
the
respective molar extinction
coefficients
of the
proteins
at 280 nm. All UV
spectrophotometric
measurements were carried
out on a
Hitachi Model
U-3300
spectrophotometer.
Irrespective
of the
protein concentration used,
it was
found
possible
to
redissolve
the
precipitated protein. Degradation
of
the
redissolved
protein
was
assessed
by
recording
a
one-dimensional
NMR
spectrum
and
comparing
it
with
that
of the
untreated
CTX III
(native protein)
sample.
The
redissolved protein
was
also checked
for
covalent damage
on a
reverse-phase
HPLC
(C
18
) column.
2.2.
Circular
Dichrosim
All
CD
spectra were recorded
on a
Jasco J-720
spectropolarimeter
using 0.02-
and
0.2-cm-
pathlength cells.
The
results
are
expressed
as
mean
residue ellipiticity [6], which
is
defined
as
[9]
=
0
obs
/60/c,
where
0
obs
is the
observed ellipticity
in
degrees,
c is the
concentration
of the
protein
in
dmol/L,
/ is the
length
of the
light path
in cm, and
the
constant
60 is the
number
of
residues
in the
protein (CTX III).
2.3.
Proton-Deuterium
Exchange
and
Two-
Dimensional
1
H-NMR Spectroscopy
The
proton-deuterium exchange experiments
on the CTX III
sample were performed
by
dissolving
appropriate amounts
of the
protein
(CTX III)
in D
2
O
containing
3%
TCA.
The
proton-deuterium
exchange
of the
unprotected
backbone
amide protons
was
allowed
to
take place
at 5°C for
^hr.
The
exchange process
was
then
stopped
by
incubating
the
sample
at
0°C.
TCA was
then
removed
by
centrifugation
at
3000
rpm
using
Pharmacia
high
pore spin column
for 3
min.
The
pH of the
solution
was
then adjusted
to 3.0 to
decrease
the
rate
of
proton-deuterium exchange.
TOCSY
(Bax
and
Davies, 1985) spectra
of
this
sample were acquired
on a
Bruker DMX-600
spectrometer
at
20°C. Assignments
of the
protected
amide
protons were obtained
by
comparison
of the
TOCSY
spectrum
of the
native
CTX III
dissolved
in
H
2
O
solution (Bhaskaran
et
al., 1994a).
2.4.
ANS
Binding
All
fluorescence
measurements were carried
out
on a
Hitachi
F-3010
spectrofluorimeter.
Appropriate volumes containing 400,M
ANS and
20
/j,
M CTX III
with
the
requisite concentrations
of
TCA
were mixed
and the ANS
emission spectra
were
recorded between
370 and 600 nm
using
an
excitation wavelength
of 350 nm.
Appropriate
background
corrections were made
in all the
spectra.
3.
RESULTS
AND
DISCUSSION
In
the
present study,
we
investigate
the
chemical
and
physical basis
of the
TCA-induced
protein precipitation. Trichloroacetic acid
has
been
routinely
used
as a
protein-precipitating agent.
We
studied
the
action
of TCA
over
a
wide
range
of
concentrations
(0-70%
w/v)
on
three proteins;
namely,
BSA, lysozyme,
CTX
III.
BSA is an
acidic
protein
(pi =
5.60),
while
CTX III and
lysozyme
are
basic
proteins
with
pI's
of
9.38
and
9.12,
respectively.
As the
shapes
of the
protein
precipitatin
curves (Fig.
1) of all the
three proteins
are
similar (U-shaped),
it
appears that
the
protein-precipitating
action
of TCA is
mostly
independent
of the
nature
of the
protein(s) used.
In
general,
the
action
of TCA on all the
proteins used
can
be
classified
to
occur
in
three stages
as
shown
in
Fig.
1. In the first
stage, occurring below
5%,
most
of
the
protein
is
found
to
remain
in
solution.
However,
in
stage
2,
occurring between
5 and 40%
TCA
concentrations, most
of the
protein precipit-
ates
(in CTX III
complete protein precipitation

TCA-Induced Protein Precipitation
293
Fig,
1.
Extent
of
precipitation
of
BSA, lysozyme,
and CTX III
at
various concentrations
of
TCA.
occurs over
a
narrower acid concentration range
of
15-35%).
When
the
acid concentrations
are
raised
above
50%
(stage
3), all the
proteins
is
found
to
redissolve back into
the
solution (Fig.
1). The
differential
action
of TCA in
terms
of
protein
precipitation
at
various concentrations indicates
that acid-induced structural transitions occur
in the
proteins.
At
this juncture,
two
fundamental
questions arise. First, what physical-chemical
properties
of TCA are
responsible
for its
(TCA)
protein-precipitating action?
And
second, what
structural
transition
does
TCA
induce
in the
protein which renders
the
protein precipitation-
competent?
In the
present study,
we
address
both
these
questions
and we
believe that
the
answers
should
help
in the
understanding
the
molecular
basis
for the
TCA-induced protein precipitation.
Figure
2
shows
the
precipitation
profiles
of
Fig.
2.
Extent
of
precipitation
of CTX III in
HC1, acetic acid,
monochloroacetic acid, dichloroacetic
acid,
and
trifluoroacetic
acid.
CTX III in
various other acids. Acetic acid
and
monochloroacetic acid
are
mostly unable
to
induce
precipitation
of the
protein. However, dichloro
acetic acid induces
CTX III
precipitation only
to a
small
extent.
The
results
of
these experiments show
that
the
presence
of
three chloro groups
(on the
alpha-carbon atom)
in the
acetic acid molecule
is
important
for the
protein-precipitating action.
It
may
be
argued that
the
protein precipitation could
be a
/?H-dependent
effect,
because
the
increase
in
the
number
of
electronegative chloro groups
effectively
increases
the
acidity
of the
acid.
However,
HC1 (a
stronger acid than TCA)
is
unable
to
induce
the
protein (CTX III) precipita-
tion even
up to a
concentration
of 70%
(Fig.
2). It
is
interesting
to
note
(Fig.
2)
that trifluoroacetic
acid
(TFA),
which
is a
stronger acid than
TCA and
possesses
three
fluoro
groups instead
of
chloro
groups
as in
TCA,
is not a
potent
protein-
precipitation-inducing
agent.
The
maximal protein
precipitation
noticed
with
TFA was
only
20%
(Fig.
2).
These
results demonstrate that
the pH of the
solution
is not the
dictating
force
in
inducing
protein precipitation.
It
also implies that
the
acid-induced protein precipitation
is
unique
to TCA
and
appears
to be
strongly dependent
on the
three
chloro groups present
in the
molecule.
The
molecular basis
of the
TCA-induced
protein precipitation could
be
best understood
by
studying
the
structural
change(s)
occurring
in the
protein upon addition
of
TCA.
Hence,
we
decided
to
investigate
the
TCA-induced structural transi-
tions
in CTX
III.
CTX III is a
well-characterized, all-/3-sheet,
low-molecular-weight
(6.7 kDa) protein (Bhaskaran
et
al,
1994a;
Yu et al,
1994; Kumar
et al,
1995,
1996a).
The
protein (CTX III)
is
three-finger-
shaped,
with
three loops emerging
from
the
globular head (Kumar
et al,
1996b>;
Bhaskaran
et
al., 19940).
The
three loops
in the
molecule
are
held
together
by
four
disulfide
bridges.
The
secondary structure
of CTX III
consists
of a
double-
and a
triple-stranded antiparallel j3-sheet
(Bhaskaran
et
al., 1994a).
The
mechanism
of
TCA-induced protein
precipitation reaction could
be
unraveled
by
examining
the
structural
transitions
that
occur
at
different
(selected) points along
the
U-shaped
precipitation
profile(s) (Fig.
1) of CTX
III. Below
3%
TCA
concentration,
the
solution containing
CTX III
remains clear.
The CD
spectra
(in the
far-
and
near-UV regions)
of CTX III in 1% TCA

294
Sivaraman,
Kumar,
Jayaraman,
and Yu
showed that
no
significant
structural change(s)
occurred
in the
protein
(data
not
shown). Similarly,
CTX III
solution also remained clear upon addition
of
TCA at
concentrations greater than 35%.
The
CD
spectra
of CTX III in 45% TCA in the
near-UV
region show that
the
positive
CD
band
at 270 nm
(indicative
of the
tertiary structural interactions
in
the
native protein)
is
completely lost
in 45%
TCA,
signifying
that
the
protein
is
completely unfolded
(Fig.
3).
However,
the
far-UV
CD
spectra
of the
45%
TCA-treated
CTX III
could
not be
recorded
because
of
optical interference
by TCA at
high
concentrations.
It is
clear that
at
concentrations
of
TCA
below
3%, CTX III
maintains
its
native
structure
and it
appears
to be
disordered
at
concentrations
of TCA
greater than 40%.
CTX III
tends
to
precipitate
at TCA
concentrations between
3%
and 4%. In
order
to
determine accurately
the
concentration
at
which
CTX III
begins
to
precipitate,
we
monitored (Fig.
4) the
TCA-induced
protein precipitation over
a
narrow range
of TCA
concentrations
(3-4%).
It can be
observed that
CTX III
begins
to
precipitate
at a TCA
concentration
of
3.24% (Fig.
4). We
strongly
believe that understanding
the
structural status
of
Fig.
4.
TCA-induced
precipitation
of CTX III
monotired
in the
acid
concentration range
of 3% w/v to 4%
w/v.
CTX III at
concentrations
just
below
the
precipitation
point
can
yield important clue(s)
to
the
molecular mechanism underlying
the
precipitation
of
proteins
by
TCA.
Hence,
we
studied
the
structural characteristics
of CTX III
using various
spectroscopic
techniques
in 3%
TCA.
It is
pertinent
to
mention that
the
results
of our CD
and
fluorescence
experiments
with
CTX III
showed
no or
very little difference(s)
in the
spectral
Fig.
3. (A) The
far-
and (B)
near-
UV CD
spectra
of CTX
III.
(a)
native
CTX
III;
(b) 3%
TCA-treated
CTX
III;
(c) 45%
TCA-treated
CTX
III.

TCA-Induced Protein Precipitation
295
characteristics
of the
protein obtained
at TCA
concentrations
of 3% and
3.2% (data
not
shown).
The
protein (CTX III) solution treated
with
3%
TCA
turned turbid upon standing
for
more than
7
days.
The CD
spectrum
of
native
CTX III in
water
in
the
near-UV region (which
is
indicative
of the
tertiary structure interactions
in the
protein) shows
a
broad band centered around
270 nm
(Fig.
3). A
negative ellipticity band
with
a
maxima
at 214 nm
can be
visualized
in the
far-UV
CD
spectrum
of
the
protein (CTX III)
and is
indicative
of the
J3
-sheet conformation
in the
backbone
of the
protein (Fig.
3). The
near-UV
CD
spectrum
of
CTX III
treated with
3% TCA
shows
a
drastic
decrease
in the CD
signal centered
at 270 nm,
implying
a
significant
loss
in the
teritary structural
interactions
in the
protein (Fig.
3).
However,
the
far-UV
CD
spectrum
of the 3%
TCA-treated
CTX
III
shows very little change
in the
spectrum
as
compared
to the one
obtained
for the
native
protein (Fig.
3). The
214-nm
CD
signal
signifying
|8-sheet
conformation
in the
protein remains intact
in
3%
TCA. Thus,
the
protein (CTX III)
in 3%
TCA
presents structural characteristics
of a
'molten
glubule'
or 'A
state'
wherein
a
protein
is
believed
to be
compact with pronounced secondary structure
but no
rigid tertiary structure. Acid-induced
'molten globule'
or 'A
state(s)'
have been realized
in
the
unfolding/folding pathways
of
many proteins
(Ptitsyn,
1992). Interestingly, several proteins
in the
'A
state'
have been shown
to be
'sticky'
and
prone
to
aggregation
(Goto
and
Fink, 1990;
Goto
et
al.,
1990;
Goto,
1991).
In the
context,
it
appears that
the
TCA-induced protein precipitation
is
mediated
by
the
formation
of
'sticky' aggregation/association
sensitive
'A
state'
as
observed
in CTX III in 3%
TCA in the
present study.
It is
important
to
understand whether
the
process
of
protein pre-
cipitation
induced
by TCA is due to
irreversible
aggregation
or
reversible association
of the 'A
state.'
We
dissolved
the
precipitate
of CTX III
obtained
at 15% TCA in
water
and
measured
the
CD and 1D
H-NMR spectra
of the
redissolved
protein.
The CD and 1D
'H-NMR spectra
of the
redissolved protein (CTX III) were
very
similar
(data
not
shown)
to
those observed
for the
native
protein, indicating that TCA-mediated protein
precipitation
is
completely reversible
and
that
the
precipitation
of the
protein
is due to
reversible
association
and not to
aggregation
of the
protein
in
the 'A
state.'
CTX III is an
all-/3-sheet protein
made
from
five
/3-strands extending between
residues, 2-5,
10-15,
20-25,
35-39,
and
51-55
(Bhaskaran
et
al., 1994a).
We
probed
the
intactness
of
these secondary structural elements
in the 3%
TCA-treated
CTX III
sample
by 2D NMR
experiments using
the
proton-deuterium
exchange
technique. This technqiue
has
been
successfully
used
in the
characterization
of the
backbone
folding
in
stable, equilibrium intermediates
in
several
proteins (Baldwin, 1991; Harding
et
al., 1991;
Fan et
al., 1993).
The
logic involved
in the
proton-
deuterium
exchange technique
is
simple. When
a
protein
is
dissolved
in
D2O,
the
exposed hydrogen
atoms
of all
those amide protons
in the
backbone
which
are not
involved
in the
secondary structure
(not
forming
hydrogen bonding
with
the
carbonyl
groups
of the
backbone)
are
substituted rapidly
by
the
deuterium atom. However,
the
protons
involved
in the
hydrogen bonding resulting
in the
secondary structure formation
are
protected
from
the
deuterium exchange.
The
amide proton
exchange
can be
estimated
by
monitoring
the
(NH—CaH) cross peaks
in the fingerprint
region
of
the
TOCSY
spectrum
of the
protein.
The
TOCSY
spectrum (Fig.
5) of the 3% TCA
treated
in
D
2
O
showed that
17 out of the 23
amide protons
involved
in the
secondary structure hydrogen
Fig.
5.
Two-dimensional
total
correlation spectroscopy
(TOCSY)
of the 3% TCA
(w/v)-treated
CTX III in D
2
O
(pH3.0,
20°C).
The
spectrum
shows
the
NH-CaH cross-peaks
of
residues protected
from
exchange.
The
TOCSY
spectrum
was
recorded
on a
DMX-600 spectrometer.

296
Sivaraman,
Kumar,
Jayaraman,
and Yu
bonding
[in the
native protein (Bhaskaran
et al,
1994a)]
are
protected
from
exchange.
The
remaining
six
amide protons which
are
exchanged
out
are
those
of
residues
located
at the
edge
of the
j8-strands.
The
core region (triple-stranded
0-sheet
segment)
of the CTX III
molecule appears
to be
unperturbed
in 3%
TCA.
It
should
be
mentioned
that
the
amide proton
of
some residues
not
involved
in the
secondary structure formation
in the
native
CTX III are
also protected
in 3%
TCA.
For
example, Met26, which
is
believed
to be
buried
in
the
native
state
and
strongly involved
in
biological
activity
of CTX
III,
is
protected
from
exchange
in
3%
TCA.
On the
whole, proton-deuterium
exchange experiments corroborate
the CD
results
indicating
that
CTX III in 3% TCA
exists
in a
partially structured
state
similar
to the
'A-state.'
ANS,
a fluorescence
probe,
is
known
to
bind
to
proteins
in
their
'A-state'
more strongly than
in the
native
or
completely
unfolded
states
(Ptitsyn,
1992).
Thus,
if CTX III
treated
with
3% TCA
exists
in the
'A
state'
and if it
possess exposed
and
sticky
hydrophobic patches responsible
for
protein pre-
cipitation, then
the
emission intensity
is
expected
to
be
high
in the 3%
TCA-treated
CTX III
sample
as
compared
to the
native
or
completely unfolded
state(s). Figure
6
reveals that
the
emission intensity
of
ANS is
maximum (Imax
=
353) when bound
to
the 3%
TCA-treated
CTX
III.
The ANS
emission
intensity
(Imax
= 95) is
least when bound
to the
completely
unfolded state
of CTX III in 45%
TCA.
Interestingly,
the
A
max
of ANS
emission when
bound
to the
native
CTX III is 498 nm and the
A
mux
of ANS
emission blue
shifts
to 484 nm
when
bound
to the 3%
TCA-treated
CTX III
sample,
indicative
of
greater exposure
of
hydrophobic sites
conducive
for ANS
binding
in the 3%
TCA-treated
CTX III
sample (Fig.
6).
Thus,
the
results
of the
ANS
binding experiments clearly demonstrate that
CTX
III in 3% TCA
exists
in the 'A
state'
with
greater exposure
of
hydrophobic sites
as
compared
to the
native protein.
It is
envisaged that increased
exposure
of
hydrophobic patches
in the 'A
state'
could render
the
protein association-sensitive.
Though
in
this
paper
a
systematic study
was
only
carried
out on CTX
III,
we
believe
that
the
phenomenon reported here also operates
in the
case
of
TCA-induced precipitation
of
other
proteins.
As the 3%
TCA-treated protein remains
in
solution,
it is
pertinent
to
address
the
question
as to
whether
the 'A
state' described
in
this
paper
precedes
the
actual intermediate
state
responsible
for
protein precipitation
or
whether
it is a
stage
preceding
the
'sticky' intermediate state.
We
found
that
the 3%
TCA-treated
CTX III
samples when
Fig.
6.
Emission spectra
of ANS
bound
to CTX III in (a)
water,
(b) CTX III in 3% w/v
TCA,
and
(c) CTX III in 45% w/v
TCA.

TCA-Induced Protein Precipitation
297
Fig.
7.
Mean residue
ellipticity
plots
of CTX III as a
function
of HC1
concentration
at
270 nm
(open circles)
and
215nm
(filled
circles).
left
for
several days turn turbid
and
eventually
precipitate. Thus,
we
believe that
the 'A
state'
in
3% TCA
should
possess
structural
features
very
similar
to the
intermediate which
is
actually
involved
in the
instantaneous precipitation.
It is
relevant
to
mention that TCA-precipitated RNase
A
when redissolved
in
appropriate
buffers
also
exists
in the 'A
state.' Thus,
it
appears that
TCA
unfolds
proteins
and
induces
a
'sticky'
A
state
responsible
for
association,
leading
to
precipitation.
However, proteins
fail
to
precipitate
at
higher
concentrations
of TCA
greater than
50%
because
the
protein molecules bypass
the
'sticky'
'A
state'
and
directly melt
to
yield
an
association-insensitive
unfolded
state.
It is
worth mentioning that
unfolding
of CTX III in HC1 is a
two-state process
(Fig.
7)
with
no
stable
intermediate(s).
This
may be
the
plausible reason
as to why
protein precipitation
does
not
occur
in
other acids, such
as
HC1.
In
this paper,
we
demonstrate that TCA-
induced protein precipitation
is
specific
and
strongly
depends
on the
three chloro groups
in the
molecule. Using
CTX
III,
we
demonstrate that
TCA
induces
a
'sticky'
association
sensitive
'A
state' which
is
responsible
for the
observed
precipitation
of
proteins
in
TCA.
ACKNOWLEDGMENTS
This work
was
supported
by
Taiwan National
Science Council grants
NSC
85-2113-M007-006
and
NSC
85-2311-B007-017.
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