THE
ISOLATION
AND
PROPERTIES
OF
SOME
SOLUBLE
PROTEINS
FROM
WOOL
II.
THE
PREFERENTIAL
EXTRACTION
OF
HIGH-SULPHUR
PROTEINS
By
J.
M.
GILLESPIE*
[Manuscript
received September 15, 1961]
Suiwmary
A
study
has
been
made
of
the
preferential
extraction
of
high·
sulphur
protein
components
from
wool
by
alkaline
solutions
of
potassium
thioglycollate.
It
was
found
that
in
general
the
high.
sulphur
proteins
were
extracted
in
quantity
at
lower
values
of
pH,
temperature,
and
time
than
the
low·
sulphur
proteins.
The
extraction
of
low·
sulphur
proteins
was
repressed
by
small
increases
in
salt
concentration
and
by
the
addition
of
divalent
cations
such
as
zinc
and
calcium,
whereas
the
solubility
of
the
high.
sulphur
proteins
was
much
less
affected
by
these
additions.
When
wool
was
extracted
with
O·
8M
potassium
thioglycollate
at
pH
values
between
10
and
IO· 5,
the
extraction
of
low·
sulphur
proteins
was
almost
completely
suppressed
and
a
high
yield
of
high-sulphur
protein
was
obtained.
1.
INTRODUCTION
During
previous studies (Gillespie 1958, 1960a), proteins
extracted
from wool
with
alkaline solutions
of
potassium
thioglycollate
and
then
alkylated
and
fractionated
into
low-
and
high-sulphur components (containing
2-2·5
and
5·8%
S respectively)
were observed
to
yield less
of
the
high-sulphur
protein
than
would
have
been expected
from
the
amount
of
y-keratose (i.e. oxidized high-sulphur protein)
obtained
from
oxidized wool (Alexander
and
Hudson
1954; Corfield, Robson,
and
Skinner 1958).
As
little
was
known
about
the
best
conditions for isolating these high-sulphur proteins
from reduced wool, a systematic
study
has
now been
made
of
variables such
as
pH,
temperature,
and
concentration
of
extractant
likely
to
influence
this
procedure.
Methods were also
sought
for preferential
extraction
of
these
proteins similar
to
those available for
the
preferential
extraction
of
y-keratose from oxidized wool
(Corfield, Robson,
and
Skinner 1958;
Burley
and
Horden
1959; Rogers 1959).
The
availability
of
high-sulphur
protein
in
the
-SH
form,
substantially
free from low-
sulphur
protein, could open
the
way
for
the
production
of
alkylated
derivatives
with
positive charges
on
the
sulphur
side-chains (by alkylating
with
,B-bromoethylamine
(Lindley 1956)) or
with
no
charge
by
alkylating, for example,
with
iodoethanol,
methyl
iodide, or iodoacetamide
and
for
the
re-formation
of
disulphide-containing
proteins.
There was some previous evidence
that
high-sulphur proteins could be pre-
ferentially
extracted
from reduced wool
in
Simmonds
and
Stell's (1956) analysis
of
"extract
A"
of
Gillespie
and
Lennox (1955).
Protein,
prepared
by
extracting
wool
* DiviRion
of
Protein
ChemiAtry, C.H.I.R.O.
Wool
ReAearch Laborat,ories,
Parkville,
Vic.
ISOLATION
OF
SOLUBLE
PROTEINS
FROM
WOOL.
II
263
at
50°C
with
0 ·IM thioglycollate
at
pH
9·9
for 20 min, was shown
to
contain more
sulphur
than
wool
and
also
to
be richer
in
serine
and
threonine residues.
As previous work has shown
that
these
high-sulphur proteins
are
electro-
phoretically
very
heterogeneous (Gillespie 1959, 1960b) some
attention
has
also been
paid
to
the
preferential isolation
of
individual components.
Conditions
of
extraction
were chosen such
that
less
than
50%
of
wool substance
was
extracted,
for,
although
it
was possible
to
extract
larger
amounts
of
protein
by
using more alkaline solutions
of
thioglycollate,
it
was
thought
that
this
might
cause
side reactions such as racemization
and
amide hydrolysis.
When
required,
the
more difficultly
extractable
protein
components
of
wool were
obtained
by
incorpor-
ating
urea
in
the
alkaline thioglycollate
extractant.
II.
MATERIALS
AND
METHODS
(a)
Wool
The
slubbing used was from dry-combed
top
prepared
from degreased Merino
64's wool (MW 127). This was washed
in
three
changes
of
petroleum
ether,
in
ethanol,
water,
again
in
ethanol,
and
was finally allowed
to
dry
and
equilibrate
at
68°F
and
70%
R.H.
Aliquots
of
wool were weighed
in
a
constant-temperature
humidity
room
and
moisture. determinations were
made
on
the
wool used
in
each
experiment.
(b)
Reagents
A.R. grade thioglycollic acid was distilled
under
vacuum
(20 mm)
and
the
fraction boiling
at
107°C collected
and
stored
at
-20°C.
Solutions were
made
in
freshly boiled
and
cooled distilled
water
and
adjusted
to
the
required
pH
by
adding
potassium hydroxide. Ammonia
(0
.
175M)
was
added
to
the
solutions
to
react
with
any
residual thioesters.
Lithium
bromide solutions were freed
of
a
purple
impurity
by
adding
a few
crystals
of
sodium thiosulphate,
the
slight excess
of
the
thiosulphate
also serving
to
remove bromine produced during heating.
Colourless iodoacetic acid (Light & Co.
Ltd.),
was
stored
at
-20°0
and
dissolved
immediately before use.
(c)
Optical Density Measurements
These were
made
in
a Beckman
DU
spectrophotometer.
The
extinction
coefficients
of
representative high-
and
low-sulphur fractions were measured, using
solutionR
of
concentration known from
dry
weight
determination
(Armstrong
et
al.
HI47).
The
principal value
of
the
optical
density
method
of
protein
determination
has
been
its
convenience
and
rapidity
in making comparative studies
of
various methods
of
ext,racting'
the
proteins.
(d)
Extraction
of
Wool Proteins
Unless otherwise
stated
I-g samples
of
wool were immersed
in
30
ml
of
extractant
in
glaRs-stoppered
test
tubes. To
wet
the
wool thoroughly
the
tubeR were
evacuated
264
J.
M.
GILLESPIE
with
a
water-pump,
then
thoroughly
shaken
and
allowed
to
stand
at
the
required
temperature
for
the
appropriate
time
with
occasional swirling.
The
final
pH
was
measured
and
undissolved wool
removed
by
filtration
on
a
Buchner
funnel
under
vacuum.
It
is
this
final
pH
value
which is referred
to
in
subsequent
sections.
If
the
weight
of
residual
wool was needed,
the
residue was
washed
with
five 20-ml
aliquots
of
distilled
water,
and
dried
to
constant
weight
at
105
D
C.
(e)
Conversion
to
S-Carboxymethyl Kerateines
(SCMK)
To
20
ml
of
the
extract
was
added
10
ml
of
a solution
containing
0·95
g iodo-
acetic
acid
and
2·0
g
trishydroxymethylaminomethane,
the
final
pH
being
between
8
and
9·0.
When
the
nitroprusside
test
(Feigl 1947)
became
negative
the
excess
iodoacetate
was allowed
to
react
with
a slight excess
of
potassium
thioglycollate.
The
alkylated
proteins
were dialysed
in
"Cellophane"
tubing
(Visking 18/32) for
18
hr
against
running
tap
water,
the
volume
was measured,
and,
after
dilution
of
an
aliquot
with
an
equal
volume
of
50%
v/v acetic
acid
to
give optically clear
solutions,
the
optical
density
at
277 mIL was
determined
against
an
appropriate
blank.
(f) Separation into Fractions
of
High- and Low-sulphur Content
The
low-sulphur
protein
fraction
was
precipitated
by
dialysing
the
protein
solution for 18
hr
against
acetate
buffer (Gillespie 1960a).
In
the
initial
studies
buffer
at
pH
4·1
and
of
ionic
strength
0·1
was
employed
but
subsequently
the
pH
was increased
to
4·4
and
the
ionic
strength
to
0·5.
The
precipitate
of
low-sulphur
protein
was recovered from
the
residual solution
of
high-sulphur
protein
by
filtration
or
centrifugation.
The
protein
content
of
the
supernatant
fraction was
determined
from
an
optical
density
measurement
at
277 mIL.
(g)
Calculations
The
difference
between
the
optical
densities
of
the
unfractionated
proteins
in
Section
II(e)
and
the
supernatant
derived
from
it
gave
the
optical
density
equivalent
to
the
low-sulphur component.
This
was
in
excellent
agreement
with
the
value
determined
directly
on
a
solution
of
the
precipitated
protein.
By
using extinction
coefficients
(Ei~m'
277 mIL)
of
8·6
and
5·4
for
the
low-
and
high-sulphur
proteins,
respectively,
and
making
suitable
corrections for
dilution
during
dialysis,
the
con-
centration
of
each
in
the
extraction
fluid was
obtained.
Their
sum
gave
the
total
amount
of
protein
extracted,
in
good
agreement
with
the
value
calculated
from
the
weight
of
residual wool.
In
some
experiments
the
yields
of
extracted
protein
were
also
measured
by
a semi-microKjeldahl procedure.
Throughout
this
paper
amounts
of
protein
extracted
are
expressed as a
percentage
of
the
weight
of
dry
wool.
(h)
Preparation
of
High-sulphur Protein
For
use
in
experiments
on
the
separation
of
low-
and
high-sulphur
proteins
from
mixtures,
a
preparation
of
high-sulphur
protein
was
made
by
extracting
wool
for 2
hr
at
40
D
C
with
O·
8M
potassium
thioglycollate
at
pH
10·2.
The
extracted
protein,
consisting
mostly
of
high-sulphur
protein,
was dialysed
at
1
DC
against
a large
volume
of
o ·IM
potassium
thioglycollate
at
pH
9,
then
alkylated
and
fractionated
as described
in
Sections II(e)
and
II(f),
then
dialysed
and
freeze-dried.
ISOLATION
OF
SOLUBLE
PROTEINS
FROM
WOOL.
II
265
(i) Electrophoresis
Moving-boundary electrophoresis was carried
out
as described previously
(Gillespie
1960a) using
an
acetic acid-sodium
acetate
buffer
of
pH
4·5
and
ionic
strength
0·1,
or a glycine
or
,B-alanine-NaOH buffer
of
pH
11·0
and
ionic
strength
0·1.
III.
RESULTS
(a)
Relation
between
pH
and Solubility at 40°0
Figure 1 shows
the
relationship between
the
pH
of
the
0 ·IM potassium thiogly-
collate
extractant
and
the
amounts
of
high-
and
low-sulphur protein
extracted
from
wool. Whereas
the
solubility
of
the
low-sulphur proteins continued
to
increase
with
increasing
pH,
that
of
the
high-sulphur proteins
apparently
reached a
maximum
at
-
40
o
"'
~
30
o
Ul
Ul
o
.J
o
~
20
"'
"
«
...
z
"'
U
II
10
"'
..
(a)
LOW-SULPHUR
FRACTION
01
u--
ii'
8 9
10
11
pH
40
30
20
10
(b)
HIGH-SULPHUR
FRACTION
'"
0,1 I
8 9
10
11
Fig.
I.-Curves
relating
extraction
of
proteins
from
wool
by
O·
1M
potassium
thioglycollate
at
40°C for 160
min
and
pH:
(a)
low-sulphur
fra<;tionj
(b)
high-sulphur
fraction
prepared
by
precipitation
at
pH
4·1
and
ionic
strength
O·
1
(6.),
and
at
pH
4·4
and
ionic
strength
O·
5
CAl.
pH
10·1
when these were measured
by
precipitation
by
dialysis against
pH
4·1
buffer,
and
further
increases
in
pH
caused a decrease
in
the
amount
of
these proteins
extracted.
Similar results were observed
at
lower
temperatures
but
the
curves were
displaced so
that,
for extraction
of
equal amounts, a higher
pH
value
or
a longer
time
of
extraction
was needed.
At
both
high
and
low
temperatures
the
position
of
the
maximum
in
the
pH
extraction
curve for high-sulphur protein closely corres-
ponded
with
the
beginning
of
the
steep
portion
of
the
corresponding curve for low-
sulphur protein, suggesting
that
the
presence
of
increasing
amounts
of
low-sulphur
protein decreased
the
recovery
of
high-sulphur protein. This is
due
to
binding
of
the
high-sulphur
protein
by
the
low-sulphur protein
and
their
subsequent precipi-
tation
together, when
the
precipitation
takes
place
at
a
pH
between
their
isoelectric points (Gillespie, O'Donnell,
and
Thompson 1962).
It
can be avoided
266
J.
M.
GILLESPIE
by
precipitation
at
pH
4·4
in
the
presence
of
a high concentration
of
salt
(Fig. l(b)),
or glycine or
by
precipitation
outside
the
inter-isoelectric region
with
zinc acetate.
In
the
experiments which follow
the
two
types
of
proteins
have
been
separated
by
precipitation
at
pH
4·4
with
sodium
acetate-acetic
acid buffer
of
ionic
strength
0·5.
(b)
Relation between
pH
and Extraction at Various Times and Temperatures
The effect
of
temperature
and
time
on
the
extractability
of
high-sulphur
protein
by
0
·IM
potassium
thioglycollate is shown
in
Figure
2(a).
It
can
be seen
that
at
40°C,
as
the
pH
increased above 9,
the
yield
of
protein
increased,
the
increase being
very
marked
at
pH
values
near
10.
At
lower
temperatures
the
corresponding curves were
displaced
toward
higher
pH
values. Curves showing
the
relation
between
pH
and
the
simultaneous
extraction
of
low-sulphur proteins,
not
recorded here, were similar
in
shape
to
the
one
prepared
at
40°C (Fig.
1)
but
were displaced
to
higher
pH
values.
~
w
>
30
.J
o
Ul
Ul
i5
.J
20
o
o
3:
~
10
«
...
z
w
U
0:
0
W 8
..
(a)
3HR.400C
9
3HR,
20°C
~-
I .
Iv
11
pH
30
(bl
20~~500C
40°C
°
30°C
"r'ooc
o
50
100
3'0
TIME
(MIN)
IONIC
STRENGTH
Fig.
2.-0urves
showing
the
extent
of
extraction
of
proteins
from
wool
by
O·
1M
potassium
thioglycollate:
(a) effect
of
pH
on
the
extraction
of
high-sulphur
protein;
(b)
rate
of
extraction
of
high-sulphur
protein
at
pH
10·5;
(0) effect
of
ionic
strength-extraction
period
60
min
at
pH
10·
5
and
40°0.
The
rate
of
extraction
of
high-sulphur
protein
by
0 ·IM
potassium
thioglycollate
was measured
at
four
temperatures. The results (Fig. 2(b)) indicate
that
up
to
80
min
equilibrium was
not
reached
and
that
temperature,
pH,
and
time
are
mutually
linked
variables.
From
these
curves,
the
initial
rate
of
reaction
and
the
activation
energy
of
the
process were calculated, a value
of
about
3800 kcal/mole for
the
latter
parameter
being obtained.
(c)
Effect
of
Ionic Strength
High-
and
low-sulphur proteins were
extracted
from wool
at
pH
10·5
by
O'IM
potassium thioglycollate
at
40°C
in
the
presence
of
varying
amounts
of
potassium
carbonate. The curves
in
Figure
2(c)
show
that
each
increase
in
ionic
strength
caused
a corresponding decrease
in
the
extractability
of
the
low-sulphur proteins,
but
not
of
the
high-sulphur proteins
until
the
ionic
strength
exceeded
1·0.
Furthermore
the
curves show
that
by
selecting a suitable ionic
strength,
preferential
extraction
of
high-sulphur
protein
could be obtained,
although
at
reduced yield. As
might
be
expected
salts
other
than
sodium carbonate showed
varying
effectiveness
in
this
regard.