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Small polypeptide components of the Bacillus thuringiensis parasporal crystalline inclusion
Vol. 41,No.
$1970
BIOCHEMICAL
SMALL POLYPEPTIDE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
COMPONENTS OF THE BACILLUS THURINGIENSIS
PARASPORAL CRYSTALLINE Vaughn
B. Sayles,
Jr.,
INCLUSION*
John N. Aronson+,
and Arthur
Rosenthal
Department of Chemistry University of New York at Albany Albany, New York 12203
State
October 10, 1970
Received
SUMMARY The bipyramidal protein crystals isolated from sporulated Bacillus thuringiensis cells can be solubilized in SM urea-0.5% dithiothreitol to yield The principal fraction of the solubicomponents of very low molecular weight. lized crystal protein elutes from Sephadex G-15 after bacitracin (mol. wt. 1.4 x 103). Sedimentation equilibrium experiments indicate a molecular weight of approximately 1.4 x 103. Electrophoresis of the solubilized crystal protein on 7% polyacrylamide gels containing 0.1% sodium dodecylsulfate reveals two major bands, one which migrates slightly behind bromophenol blue and a secondary band which moves slightly ahead of the dye front. The parasporal is
a bipyramidal
inclusion
crystalline
protein The crystal
Lepidoptera
larvae
(1,2).
composition
(1,3),
is
crystal
below
The effects cysteine
agent,
surprisingly
of alkaline (6),
solutions
and urea with
(4)
or denaturing
agent.
(a),
protein.
molecular
Reported
have
which
in
insoluble.
Protein
agent
or strong
studied.
(4),
weights
is
toxic
is
for
released
denaturing
agent
(4),
of a reducing
has been in the agent
effective protein
should
be addressed. 1126
the
is present. (5,6),
found
to increase of the
and a denaturing and guanidine-
in solubilizing
the crystal
widely.
*This work is taken in part from the thesis submitted by V. B. Sayles, Graduate School of the State University of New York at Albany in partial ment of the requirements for the M.S. degree. t To whom inquiries
from
concentration
(7),
vary
by
acid
thioglycollate
urea-S-mercaptoethanol
solubilized
amino
only
Solubility
to be quite
thuringiensis
when ingested
a normal
the pH or an increase
Combinations
proven
of Bacillus
despite
of dithiothreitol
such as urea-dithiothreitol
S-mercaptoethanol
sporulation
protein,
have been
an increase
during
substance
pH 11.8 when a reducing
proportionally reducing
formed
A study
Jr.
to the fulfill-
Vol. 41,No.
(9)
determined
crystal
uncorrected
protein
(6) with
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5, 1970
sedimentation
of 4.1s
(Svedberg
collate
G-25
column
obtained
evidence
for
corresponding from her
laboratory
dialyzed,
reducing
and then
(8) material
Varying
results
Somerville
phoresis.
(10)
reported
a single
spores
and crystals.
of 40,000-to-200,000 techniques
of both
evident
obtained
of urea
at 20°C,
In a later
study and
by a factor
using
and found
in some preparations.
have been
co-
of two,
Schlieren
pattern
+ 9,000.
et al.
concentration
M thiogly-
gave a homogeneous
been
containing
as many as twelve
equilibrium
on
S-mercaptoethanol-guanidine
also
extract
a high
in
10,000.
have
&mercaptoethanol
fractionated
of 0.83s
of 8-mercaptoethanol
of 80,000
Lecadet
sedimentation
coefficient
solubilized
by sedimentation weight
protein
of different
of approximately
the concentration
analyzed
and a molecular
constituents
weight
solubilised
0.4S-to-0.8s.
of crystal
gave a sedimentation
to a molecular
alkali
low3 M p-mercuribensoate-0.1
with
three
for
and approximately solution
equilibrated
Her fraction
efficients.
obtained
from
gel
polyacrylamide fast
moving
electro-
band from
Cooksey
a variable
gel
(11)
number
a urea-
used gels of bands
Molecular
weight
exclusion
chromatographic
with
approximations
(2). Experiments
phoresis,
and gel of g.
using
l-l.5
sedimentation,
chromatography
thuringiensis
crystal consisted
are
protein of more
polyacrylamide
reported
in this
solubilized than
gel
electro-
paper.
The major
in urea-mercaptoethanol
one small
polypeptide
of molecular
x 103. Sporulated
cultures
grown
in the
trace
metals,
7,000
x g in a Sorvall
times
by centrifugation
and the spores by Gingrich
equilibrium
exclusion
or urea-dithiothreitol weight
units)
a thioglycollate-solubilised
a Sephadex
portion
coefficients
citrate-salts
of Bacillus
medium
0.1% w/v glucose,
were (13).
of Vogel
removed
and Bonner
and 0.04% w/v
RCZ-B centrifuge through
thuringiensis
distilled
after
(12)
The pellet
water,
suspended
procedure
the spores
1127
were
thuringiensis
supplemented
acids
at 2'C.
by a floatation
The residue
casamino
var.
were
in distilled
siphoned
with
harvested
was washed
similar
(1)
to that
at
three water, described
off was pelleted
Vol. 41, No. 5, 1970
and washed
by centrifugation
was suspended vegetative
debris
crystals
x g.
washed
The washed
bv phase
contrast
at room temperature particulate
times
pellet
pH 7.2,
was removed
system (14).
through than
The crystal described
95% free
by centrifugation
crystals, were
speed
of CC14
water
each experiment at full
and
The sedimented
distilled
preparations
for
The pellet
spores
and Morrison
of greater
microscopy.
x g.
in a biphasic
by centrifugation
consisted
at 7,000
and remaining
by Pendleton
in the solutions
matter
water
by distribution
as described three
distilled
buffer,
removed
1% Na2S04
were
judged
were
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
through
in 0.2 M phosphate
and aqueous
2,500
BIOCHEMICAL
at as
incubated and residual
on an International
HN centrifuge. Preparations a Sephadex gave in
G-15
solubilized column
the same basic
the first
weight
Figure
12,400)
1:
dithiothreitol at 4'C with dithiothreitol
peak
with
with
0.2 M NaCl containing
pattern,
although
following
the void
and bacitracin
Fractionation
in
of crystal
0.2 M NaCl-0.01% at a flow
G-15
the
volume
(molecular
on a Sephadex
(w
8 M urea-0.5%
dithiothreitol rate
latter (Fig.
protein
1). about
(-o->,
protein
Cytochrome 1,400)
from
were
was present
C (molecular used as markers.
in 8 M urea-0.5%
x 45 cm).
of 6 ml/hour.
1128
case less
solubilized (1.3
and eluted
0.01% or 0.1% dithiothreitol
weight
column
dithiothreitol
or with
Samples
were
eluted
0.2 M NaCl-0.1%
BIOCHEMICAL
Vol. 41, No. 5, 1970
Use of other
gel
elution
0.2 M NaCl containing
with
columns
two peaks,
results: cytochrome
sometimes
C and the
Upon further
(Sephadex
in
being
solubilized
material
longer
as shown in Fig. The lower equilibrium with
in
shoulders,
experiments
bring
about
set
experiments
(or
further
after
as before
between
bacitracin). from
with
the first
the first
indicated
that
to minimum
higher
and
gave similar
collected
disaggregated
sub-units,
mercaptan
but
concentrations,
disaggregation.
material
a Spinco
scanner
(eluted
obtained
solution
weight
with
P-2)
in the region
of the material
completely
molecular
or Bio-Gel
appeared
These
the mercaptan
1) would
G-25,
peak of mercaptan
in size.
was not
a photoelectric
with
two peaks were
diminished
incubations
Sephadex
0.1% or 1.0% 8-mercaptoethanol
the solvent
peak and rechromatography, peak eluted
G-50,
concentrated
incubation
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Model
was studied
in sedimentation
E analytical
ultracentrifuge
at 280 nm with
the monochromator
equipped
slit
width
... . .
0.90
. .
1 .5
i
"
I
. .
1.00 t
. .
49.5
50.0
50.5
51.0
r2 (cm2)
Figure mentation
2:
Plot
of In c versus
equilibrium
in 8 M urea-0.01%
pattern.
was from
sample
was spun at 60,000rpm The cell
peak near
length
no.
obtained
from
The sample
dithiothreitol
sample
10.9Oc.
r2
was crystal
and fractionated 28 of the
cm.
1129
model
trace
protein
as shown
.Ol% dithiothreitol
on a Spinco
was 1.6
the scanner
E analytical
of sedi-
solubilized
in Fig.
1.
elution. ultracentrifuge
The The at
BIOCHEMICAL
Vol. 41, No. 5, 1970
lmm and the centered
about
(as shown radial
scanner
used
width
fraction
in Fig.
distance
The plot
slit
no.
1) is were
portrayed
in Fig. from
eluted
2.
radius
average
the formula
of analysis with
Values
the scanner
versus
the weight
into
Results
mm.
28 of the sample
of In concentration
substitution
0.06
determined
to approximate
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.01% dithiothreitol
of concentration
trace
squared,
molecular
of a portion
obtained
as shown weight
and
at equilibrium. in Fig.
2, was
of the protein
by
(15):
M
2RT
=
(1 - ;p)w2 where
M is
the weight
T = temperature protein,
in degrees
In c versus p = 1.0
concentrations
reveals
concentration with
0.75
q
gel
further
of crystal
analyzed
electrophoresis.
gels
containing
similar
Disc
to that
described
ammonium persulfate
two drops was run in
the upper
dodecyl
results,
column
by Davis
were
at 3 milliamperes/gel
the amidoschwartz-ethanol-acetic
2 at lower
the dithiothreitol
M = 1 x lo3 were
8 M urea-l%
obtained
by polyacrylamide
was performed
on 7% polyacrylamide
(SDS).
(16).
Gels
The procedure
polymerized
riboflavin
were
used
layered
with
in
above
at 4'C for acid
large
pore
2 to 3 hours. system
(17).
1130
described
either
pH 8.3,
the urea-mercaptan the
was 0.07%
and the 0.0124
buffer,
Samples
&mercaptoethanol
fractionation
sulfate
or 4.4 x lo-4%
of 50% sucrose
in
by plotting
of Fig.
had altered
in
electrophoresis
reservoir.
velocity
obtained
the plot
(2-amino-Z-(hydroxymethyl)-l,3-propanediol)-glycine 0.1% SDS in
of the
of 1.4 x lo3 was calculated in
solubilized
and after
0.1% sodium
deg.,
as well.
protein
before
weight
Similar
preparations
volume
of the line
preparation
slightly.
erg/mole
wL = the angular
The break
column
x 10'
specific
solution, the slope
used.
the
of the sample
Samples were
is
R = 8.3114
partial
molecular
were
that
S-mercaptoethanol
; = the
c)/dr'
An approximate
and v
weight,
of the protein
and d(ln r2.
molecular
Kelvin,
p = the density
radians2/second2,
when
average
gel.
The gels
M TRIS
was also
containing Electrophoresis were
by Kaplan
stained
and Criddle
BIOCHEMICAL
vol. 41, No. 5, 1970
Figure
3:
Band patterns
on 7% polyacrylamide
4.4 x lQe4% riboflavin buffer run
at 3 milliamps/gel
were: crystal G-50.
(1)
blue
crystals
protein Migration
Typical bilized major front.
and containing
at pH 8.3 was also
of bromophenol
bands
indicated
recovered
from
the lowest
results
Analyzer.
normally
as found
the same as literature
values
about
1.4 x lo3
must
contain
3).
at most
on the above gels
eluted
(2) from
solubilized Sephadex
moving
and analyzed
the normal
Since
amino of intact
a single
13 different
1131
a non-fractionated bands,
amino
solubut
by the bromophenol
in hydrolysates (1,
peak
slower
occupied
revealed
in the same proportion
wt.
3 in which
was hydrolyzed
Analysis
position
the anode.
to have several
the position sample
Samples
(usual
were
B-mercaptoethanol;
mol.
are shown in Fig.
was seen
near
towards
Samples
dye was used
in 8 M urea-l%
with
M TRIS-glycine
reservoir.
by the arrows).
was downward
A fractionated
Amino Acid
is
photopolymerized The 0.0124
0.1% SDS.
and no marker
at 4"C,
RESEARCH COMMUNICATIONS
gels
0.1% SDS in the upper
solubilized
preparation
AND BlOPHYSlCAl
blue
on a Spinco acids
(Table
crystals,
polypeptide acids,
also dye 120C 1)
essentially of mol.
it
two
must be
wt.
BIOCHEMICAL
Vol.41,No.5,1970
concluded
that
crystal
the low-molecular
proteins
is
the
allowed
crystal
The aggregation reported
in
solubilized
exhibited
protein
to stand
weight
polypeptide
fraction
from
solubilized
heterogeneous.
The aggregation from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
some time
by the
readily
low molecular
reversible,
and of samples
phenomenon
could
A small
peptide
crystal
appears
to retain
polypeptides
by analysis
further
the diversity
the literature. protein
as judged
incubated
explain
weight
in
the proper
of molecular
fraction
from
toxicity
of samples
chmotrypsin
solution.
weight
digests
(18).
TABLE I Amino
acid
composition
from
crystal
of low molecular protein
weight
fractionated
Sephadex
material
on
G-50
per cent by weight
per cent by weight
amino
lysine
4.0
glycine
4.0
histidine
3.0
alanine
4.8
arginine
10.2
half
12.7
valine
6.1
amino
acid
aspartic
acid
acid
cystine
0.9
threonine
5.5
methionine
0.0
serine
3.3
isoleucine
6.0
14.5
leucine
8.9
3.0
tyrosine
5.2
phenylalanine
5.4
glutamic
acid
proline
ACKNOWLEDGEMENT This
work
was supported
in part
by National
GB-7997. 1132
Science
Foundation
values
grant
of
Vol.41,No.5,
BIOCHEMICAL
1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES A. M., Ann. --
Rev. -y-) of Entomol
12 -3
287-321
1.
Heimpel,
2.
Rogoff,
3.
Spencer,
E. Y. , 2.
4.
Lecadet,
M. M., 2. g. Acad. -
5.
Young, I. E. and Fitz-James, 483-498 (1959).
6.
Lecadet,
7.
Delafield, F. P., Sommerville, 96, 713-720 (1968).
8.
Glatron, M. F., Lecadet, M. M., and Dedonder, Paris , 269, 1338-1341 (1969), D.
9.
Holmes,
M. H. and Yousten,
A. A.,
Invertebrate
Somerville, 96, 721-726
H. J., (1968).
11.
Cooksey,
12.
Vogel,
13.
Gingrich,
14.
Pendleton,
15.
Svedberg, T. and Pedersen, Press (1940).
16.
Davis,
17.
Kaplan,
18.
Fast,
K. E.,
R. E., I.
B. J.,
&.
106,
-Ann. -N. -Y. -Acad.
P. G. and Angus,
Biol.
R. S., T. A.,
195-198
14,
Invert.
1133
D.
C. g. -Acad.
572-581
SC.
(1965).
S. C., -J. w.,
218,
180-184
97-106
404-427
Pathol.,
(1956).
(1968).
212,
Biochim.Biophys. J.
5,
(1968).
Nature,
-121,
Cytol.,
S. C., -J. s.,
728-729
The Ultracentrifuge,
z.,
(1969).
D.
(1966),
M. R.,
Chem.,
lo,
R. B.,
K. O.,
Biochem.
and Rittenberg,
-Pathol.,
R. and Morrison,
(1967),
and Rittenberg,
445-454
D. M., J.
2. ~Invert.
D. and Criddle,
262,
357-386
(1968).
2847-2850
2. Mol.Biol, F. P.,
23,
444-445
Biophys.
Paris, H. J.,
J.,
H. J. and Bonner,
264,
P. C., 2.
Delafield,
Biochem.
11,
--SC. Paris,
R. E.,
10.
Rev. Microbial.,
Pathol.,
M. M., C. &. -Acad.
K. C. and Monro,
--Ann.
(1967).
(1966).
Oxford
University
(1964). Acta,
203,
in press.
249-255
(1970).
$1970
BIOCHEMICAL
SMALL POLYPEPTIDE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
COMPONENTS OF THE BACILLUS THURINGIENSIS
PARASPORAL CRYSTALLINE Vaughn
B. Sayles,
Jr.,
INCLUSION*
John N. Aronson+,
and Arthur
Rosenthal
Department of Chemistry University of New York at Albany Albany, New York 12203
State
October 10, 1970
Received
SUMMARY The bipyramidal protein crystals isolated from sporulated Bacillus thuringiensis cells can be solubilized in SM urea-0.5% dithiothreitol to yield The principal fraction of the solubicomponents of very low molecular weight. lized crystal protein elutes from Sephadex G-15 after bacitracin (mol. wt. 1.4 x 103). Sedimentation equilibrium experiments indicate a molecular weight of approximately 1.4 x 103. Electrophoresis of the solubilized crystal protein on 7% polyacrylamide gels containing 0.1% sodium dodecylsulfate reveals two major bands, one which migrates slightly behind bromophenol blue and a secondary band which moves slightly ahead of the dye front. The parasporal is
a bipyramidal
inclusion
crystalline
protein The crystal
Lepidoptera
larvae
(1,2).
composition
(1,3),
is
crystal
below
The effects cysteine
agent,
surprisingly
of alkaline (6),
solutions
and urea with
(4)
or denaturing
agent.
(a),
protein.
molecular
Reported
have
which
in
insoluble.
Protein
agent
or strong
studied.
(4),
weights
is
toxic
is
for
released
denaturing
agent
(4),
of a reducing
has been in the agent
effective protein
should
be addressed. 1126
the
is present. (5,6),
found
to increase of the
and a denaturing and guanidine-
in solubilizing
the crystal
widely.
*This work is taken in part from the thesis submitted by V. B. Sayles, Graduate School of the State University of New York at Albany in partial ment of the requirements for the M.S. degree. t To whom inquiries
from
concentration
(7),
vary
by
acid
thioglycollate
urea-S-mercaptoethanol
solubilized
amino
only
Solubility
to be quite
thuringiensis
when ingested
a normal
the pH or an increase
Combinations
proven
of Bacillus
despite
of dithiothreitol
such as urea-dithiothreitol
S-mercaptoethanol
sporulation
protein,
have been
an increase
during
substance
pH 11.8 when a reducing
proportionally reducing
formed
A study
Jr.
to the fulfill-
Vol. 41,No.
(9)
determined
crystal
uncorrected
protein
(6) with
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5, 1970
sedimentation
of 4.1s
(Svedberg
collate
G-25
column
obtained
evidence
for
corresponding from her
laboratory
dialyzed,
reducing
and then
(8) material
Varying
results
Somerville
phoresis.
(10)
reported
a single
spores
and crystals.
of 40,000-to-200,000 techniques
of both
evident
obtained
of urea
at 20°C,
In a later
study and
by a factor
using
and found
in some preparations.
have been
co-
of two,
Schlieren
pattern
+ 9,000.
et al.
concentration
M thiogly-
gave a homogeneous
been
containing
as many as twelve
equilibrium
on
S-mercaptoethanol-guanidine
also
extract
a high
in
10,000.
have
&mercaptoethanol
fractionated
of 0.83s
of 8-mercaptoethanol
of 80,000
Lecadet
sedimentation
coefficient
solubilized
by sedimentation weight
protein
of different
of approximately
the concentration
analyzed
and a molecular
constituents
weight
solubilised
0.4S-to-0.8s.
of crystal
gave a sedimentation
to a molecular
alkali
low3 M p-mercuribensoate-0.1
with
three
for
and approximately solution
equilibrated
Her fraction
efficients.
obtained
from
gel
polyacrylamide fast
moving
electro-
band from
Cooksey
a variable
gel
(11)
number
a urea-
used gels of bands
Molecular
weight
exclusion
chromatographic
with
approximations
(2). Experiments
phoresis,
and gel of g.
using
l-l.5
sedimentation,
chromatography
thuringiensis
crystal consisted
are
protein of more
polyacrylamide
reported
in this
solubilized than
gel
electro-
paper.
The major
in urea-mercaptoethanol
one small
polypeptide
of molecular
x 103. Sporulated
cultures
grown
in the
trace
metals,
7,000
x g in a Sorvall
times
by centrifugation
and the spores by Gingrich
equilibrium
exclusion
or urea-dithiothreitol weight
units)
a thioglycollate-solubilised
a Sephadex
portion
coefficients
citrate-salts
of Bacillus
medium
0.1% w/v glucose,
were (13).
of Vogel
removed
and Bonner
and 0.04% w/v
RCZ-B centrifuge through
thuringiensis
distilled
after
(12)
The pellet
water,
suspended
procedure
the spores
1127
were
thuringiensis
supplemented
acids
at 2'C.
by a floatation
The residue
casamino
var.
were
in distilled
siphoned
with
harvested
was washed
similar
(1)
to that
at
three water, described
off was pelleted
Vol. 41, No. 5, 1970
and washed
by centrifugation
was suspended vegetative
debris
crystals
x g.
washed
The washed
bv phase
contrast
at room temperature particulate
times
pellet
pH 7.2,
was removed
system (14).
through than
The crystal described
95% free
by centrifugation
crystals, were
speed
of CC14
water
each experiment at full
and
The sedimented
distilled
preparations
for
The pellet
spores
and Morrison
of greater
microscopy.
x g.
in a biphasic
by centrifugation
consisted
at 7,000
and remaining
by Pendleton
in the solutions
matter
water
by distribution
as described three
distilled
buffer,
removed
1% Na2S04
were
judged
were
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
through
in 0.2 M phosphate
and aqueous
2,500
BIOCHEMICAL
at as
incubated and residual
on an International
HN centrifuge. Preparations a Sephadex gave in
G-15
solubilized column
the same basic
the first
weight
Figure
12,400)
1:
dithiothreitol at 4'C with dithiothreitol
peak
with
with
0.2 M NaCl containing
pattern,
although
following
the void
and bacitracin
Fractionation
in
of crystal
0.2 M NaCl-0.01% at a flow
G-15
the
volume
(molecular
on a Sephadex
(w
8 M urea-0.5%
dithiothreitol rate
latter (Fig.
protein
1). about
(-o->,
protein
Cytochrome 1,400)
from
were
was present
C (molecular used as markers.
in 8 M urea-0.5%
x 45 cm).
of 6 ml/hour.
1128
case less
solubilized (1.3
and eluted
0.01% or 0.1% dithiothreitol
weight
column
dithiothreitol
or with
Samples
were
eluted
0.2 M NaCl-0.1%
BIOCHEMICAL
Vol. 41, No. 5, 1970
Use of other
gel
elution
0.2 M NaCl containing
with
columns
two peaks,
results: cytochrome
sometimes
C and the
Upon further
(Sephadex
in
being
solubilized
material
longer
as shown in Fig. The lower equilibrium with
in
shoulders,
experiments
bring
about
set
experiments
(or
further
after
as before
between
bacitracin). from
with
the first
the first
indicated
that
to minimum
higher
and
gave similar
collected
disaggregated
sub-units,
mercaptan
but
concentrations,
disaggregation.
material
a Spinco
scanner
(eluted
obtained
solution
weight
with
P-2)
in the region
of the material
completely
molecular
or Bio-Gel
appeared
These
the mercaptan
1) would
G-25,
peak of mercaptan
in size.
was not
a photoelectric
with
two peaks were
diminished
incubations
Sephadex
0.1% or 1.0% 8-mercaptoethanol
the solvent
peak and rechromatography, peak eluted
G-50,
concentrated
incubation
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Model
was studied
in sedimentation
E analytical
ultracentrifuge
at 280 nm with
the monochromator
equipped
slit
width
... . .
0.90
. .
1 .5
i
"
I
. .
1.00 t
. .
49.5
50.0
50.5
51.0
r2 (cm2)
Figure mentation
2:
Plot
of In c versus
equilibrium
in 8 M urea-0.01%
pattern.
was from
sample
was spun at 60,000rpm The cell
peak near
length
no.
obtained
from
The sample
dithiothreitol
sample
10.9Oc.
r2
was crystal
and fractionated 28 of the
cm.
1129
model
trace
protein
as shown
.Ol% dithiothreitol
on a Spinco
was 1.6
the scanner
E analytical
of sedi-
solubilized
in Fig.
1.
elution. ultracentrifuge
The The at
BIOCHEMICAL
Vol. 41, No. 5, 1970
lmm and the centered
about
(as shown radial
scanner
used
width
fraction
in Fig.
distance
The plot
slit
no.
1) is were
portrayed
in Fig. from
eluted
2.
radius
average
the formula
of analysis with
Values
the scanner
versus
the weight
into
Results
mm.
28 of the sample
of In concentration
substitution
0.06
determined
to approximate
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.01% dithiothreitol
of concentration
trace
squared,
molecular
of a portion
obtained
as shown weight
and
at equilibrium. in Fig.
2, was
of the protein
by
(15):
M
2RT
=
(1 - ;p)w2 where
M is
the weight
T = temperature protein,
in degrees
In c versus p = 1.0
concentrations
reveals
concentration with
0.75
q
gel
further
of crystal
analyzed
electrophoresis.
gels
containing
similar
Disc
to that
described
ammonium persulfate
two drops was run in
the upper
dodecyl
results,
column
by Davis
were
at 3 milliamperes/gel
the amidoschwartz-ethanol-acetic
2 at lower
the dithiothreitol
M = 1 x lo3 were
8 M urea-l%
obtained
by polyacrylamide
was performed
on 7% polyacrylamide
(SDS).
(16).
Gels
The procedure
polymerized
riboflavin
were
used
layered
with
in
above
at 4'C for acid
large
pore
2 to 3 hours. system
(17).
1130
described
either
pH 8.3,
the urea-mercaptan the
was 0.07%
and the 0.0124
buffer,
Samples
&mercaptoethanol
fractionation
sulfate
or 4.4 x lo-4%
of 50% sucrose
in
by plotting
of Fig.
had altered
in
electrophoresis
reservoir.
velocity
obtained
the plot
(2-amino-Z-(hydroxymethyl)-l,3-propanediol)-glycine 0.1% SDS in
of the
of 1.4 x lo3 was calculated in
solubilized
and after
0.1% sodium
deg.,
as well.
protein
before
weight
Similar
preparations
volume
of the line
preparation
slightly.
erg/mole
wL = the angular
The break
column
x 10'
specific
solution, the slope
used.
the
of the sample
Samples were
is
R = 8.3114
partial
molecular
were
that
S-mercaptoethanol
; = the
c)/dr'
An approximate
and v
weight,
of the protein
and d(ln r2.
molecular
Kelvin,
p = the density
radians2/second2,
when
average
gel.
The gels
M TRIS
was also
containing Electrophoresis were
by Kaplan
stained
and Criddle
BIOCHEMICAL
vol. 41, No. 5, 1970
Figure
3:
Band patterns
on 7% polyacrylamide
4.4 x lQe4% riboflavin buffer run
at 3 milliamps/gel
were: crystal G-50.
(1)
blue
crystals
protein Migration
Typical bilized major front.
and containing
at pH 8.3 was also
of bromophenol
bands
indicated
recovered
from
the lowest
results
Analyzer.
normally
as found
the same as literature
values
about
1.4 x lo3
must
contain
3).
at most
on the above gels
eluted
(2) from
solubilized Sephadex
moving
and analyzed
the normal
Since
amino of intact
a single
13 different
1131
a non-fractionated bands,
amino
solubut
by the bromophenol
in hydrolysates (1,
peak
slower
occupied
revealed
in the same proportion
wt.
3 in which
was hydrolyzed
Analysis
position
the anode.
to have several
the position sample
Samples
(usual
were
B-mercaptoethanol;
mol.
are shown in Fig.
was seen
near
towards
Samples
dye was used
in 8 M urea-l%
with
M TRIS-glycine
reservoir.
by the arrows).
was downward
A fractionated
Amino Acid
is
photopolymerized The 0.0124
0.1% SDS.
and no marker
at 4"C,
RESEARCH COMMUNICATIONS
gels
0.1% SDS in the upper
solubilized
preparation
AND BlOPHYSlCAl
blue
on a Spinco acids
(Table
crystals,
polypeptide acids,
also dye 120C 1)
essentially of mol.
it
two
must be
wt.
BIOCHEMICAL
Vol.41,No.5,1970
concluded
that
crystal
the low-molecular
proteins
is
the
allowed
crystal
The aggregation reported
in
solubilized
exhibited
protein
to stand
weight
polypeptide
fraction
from
solubilized
heterogeneous.
The aggregation from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
is
some time
by the
readily
low molecular
reversible,
and of samples
phenomenon
could
A small
peptide
crystal
appears
to retain
polypeptides
by analysis
further
the diversity
the literature. protein
as judged
incubated
explain
weight
in
the proper
of molecular
fraction
from
toxicity
of samples
chmotrypsin
solution.
weight
digests
(18).
TABLE I Amino
acid
composition
from
crystal
of low molecular protein
weight
fractionated
Sephadex
material
on
G-50
per cent by weight
per cent by weight
amino
lysine
4.0
glycine
4.0
histidine
3.0
alanine
4.8
arginine
10.2
half
12.7
valine
6.1
amino
acid
aspartic
acid
acid
cystine
0.9
threonine
5.5
methionine
0.0
serine
3.3
isoleucine
6.0
14.5
leucine
8.9
3.0
tyrosine
5.2
phenylalanine
5.4
glutamic
acid
proline
ACKNOWLEDGEMENT This
work
was supported
in part
by National
GB-7997. 1132
Science
Foundation
values
grant
of
Vol.41,No.5,
BIOCHEMICAL
1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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