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Red
Cell
Human
Proteins.
I. Two-Dimensional
of Human
Erythrocyte
By J. J. Edwards,
N. G. Anderson,
erythrocyte
lysate
proteins
Mapping
Lysate
S. L. Nance,
were
major
Proteins
and
proteins
N. L. Anderson
are
removed
by adsorption
resolved
into over 250 discrete
spots by
two-dimensional
electrophoresis
using
isoelectric
focusing
in the first dimension
and electrophoresis
in the presence
of
to DEAE-cellulose,
additional
minor components are seen. giving a total of over 275.
With the use of purified
preparations,
the
map positions
of five red cell enzymes
or
sodium
their
dodecyl
sulfate
(SDS)
in
the
subunits
were
determined:
pyruvate
second.
The
overwhelming
hemoglobin
has made such
excess
of
analyses
diffi-
kinase,
catalase.
glucose-6-phosphate
dohydrogenase,
hypoxanthine
phosphoribo-
cult
with
syltransferase,
in
the
DALT
past.
However,
two-dimensional
the
ISO-
electrophoresis
The
system,
large numbers
of red cell proteins
can be mapped in the presence
of hemoglobin.
When
‘ hemoglobin
and several
other
N
UMEROUS
cell proteins
from
the
from suggestions
function
and that
association
of specific
carbonic
anhydrase.
techniques
described
complement
and extend
those traditionally
used to find human
red cell protein
vanants.
electrophoretic
or kinetic
have been described.
The
arises
with
and
mapping
variants
variants
clinical
with
of human
nonhemoglobin
interest
in these variants
disease,
that the number
of variants
intracellular
proteins
may
and
the
theoretical
In kinetic
studies,
a separate
measurement
for
activity
are
are not
stained
resolved
and
electropherograms.
used
locating
are
enzyme
therefore
Neither
or series
of analyses
is employed,
positions
electrophoresis
is applicable
numbers
of different
proteins,
unknown,
analysis
The
protein
in one
dodecyl
From
the
National
sulfate
Molecular
Anatomy
Laboratory,
Submitted
January
by the U.S.
Address
reprint
The submitted
Government
purposes.
©
Blood,
1979
accepted
by Grune
Division
February
III.
to the
including
proteins
by position
for kinetic
problems
those
of
whose
present
in fixed
variants
and
nor
searching
functions
for
are
presently
electrophoresis
of
gels.
Biological
available
for proteins
in the presence
The
and
theoretical
Medical
or
of
limits
Research.
of
Argonne
the published
1979
Inc.
8, 1979.
Ph.D.,
the U.S.
form
Division
of
Biological
and
Medical
Research,
60439.
has been authored
& Stratton,
Vol. 53, No. 6 (June),
all of the
solely
assay
of Energy.
Argonne,
Accordingly,
or reproduce
be made.
for enzyme
in acrylamide
to J. J. Edwards,
manuscript
to publish
both
Department
Laboratory.
W-31-l09-ENG-38.
is required
Ill.
4, 1979;
requests
National
is
in
work
technique.
Program.
Argonne.
Supported
Argonne
(SDS),
in
it
of measurements
because
two highest-resolution
analytical
methods
subunits
are isoelectric
focusing
and
sodium
No.
by one
studies
that can conveniently
histochemical
tests
not identifiable
kinetic
enzyme
one-dimensional
variants
of large
interest
of a protein
may vary markedly
exhibit
many
fewer
variants
general
than
extracellular
ones.
For both
clinical
and theoretical
desirable
to be able to examine
large numbers
of samples.
Unfortunately,
this area has been limited
by the analytical
techniques
available.
for each enzyme,
limiting
the number
When
one-dimensional
electrophoresis
red
stems
by a contractor
Government
of this
of the U.S.
retains
contribution,
Government
a nonexclusive,
or allow
others
under
contract
royalty-free
to do so. for
license
U.S.
0006-4971/79/5306-0012$02.00/0
1121
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1122
EDWARDS
resolution
of these two methods
have
they appear
to lie at present
between
not been
100 and
fully
200
ET
AL.
explored;
however,
in practice
entities
for each and therefore
are not capable
of yielding
separately
sufficient
resolution
to allow a large number
of proteins
to be identified
by position
rather
than by activity.
Isoelectric
focusing
and
SDS
electrophoresis
depend
on two different
and
unrelated
parameters:
isoelectric
point and molecular
weight.
The former
is the most interesting
from a
genetic
viewpoint,
tions
alter
positional
example,
hence
If
approximately
one-third
of all single
the charge
of a protein
variants
are of interest
since
sufficiently
even
when
to be easily
their
functions
their
linkages
to known
their chromosomal
the techniques
two-dimensionally,
individual
resolutions
variants
may
gene locations
may
isoelectric
focusing
of
theoretically
of the two
be determined
be found.
and SDS
modified
analyses
by
to be done
reduction
systems
In this
have
series
Anderson
in parallel
been
of reports
and
(so-called
and
continue
we explore
substitu-
electrophoresis
that
For
and
are
the product
and 40,000
used
of the
proteins
subunits
can be seen by Coomassie
analysis
was first
introduced
by
technique
by O’Farrell,3
and was
Anderson4’5
to
ISO-DALT
system).
allow
to be developed
the
acid
experimentally,
the resolution
should
be
methods,
i.e., between
10,000
or protein
subunits.
A practical
limit of 1000
blue
staining.
This
form
of two-dimensional
Stegemann,2
was perfected
as a high-resolution
extensively
amino
detected.’
Note
are unknown.
use
of the
large
numbers
of
Computerized
in this
data
laboratory.
ISO-DALT
two-dimensional
analytical
system
for analysis
of red cell proteins.
The initial
problem
to be solved
concerns
the large
amount
of hemoglobin
present
(over 95% of the total protein
mass)
and whether
or not the minor
proteins
known
to be present
can be separated
and
seen
in the presence
of such
an overwhelming
amount
of one protein.
Additional
problems
considered
are the reproducibility
of patterns
and the identification
of spots
be concerned
with applicable
in
Among
each
with
the
major
known
enzymes.
with additional
data analysis.
Subsequent
enzyme
the alterations
in red cell enzymes
previously
of seven
enzymes
of the
Embden-Meyerhoff
deficiencies
and
associated
with specific
variants,
aspects
of computerized
in the
hexose
monophosphate
shunt,
over 80 variants
of glucose-6-phosphate
Whereas
hemoglobin
has been
removed
described
pathway,
four
will
deficiencies
five enzyme
deficiencies,
lysates
by
the
of
extraction7
are denatured
or removed
by these methods.
It is therefore
initially,
methods
for resolving
as many
proteins
as possible
hemoglobin
removal
and
nents by hemoglobin
lost in the process.
removal,
In this
suggesting
study we find over
that
the majority
mapping
of
red
cell
structural
and functional
the detection
of possible
conditions.
A preliminary
then
realizing
275
soluble
of
spots
red
proteins
gain
greater
that
some
report
of this
sensitivity
nonhemoglobin
on two-dimensional
cell proteins
remain
provides
relationships
of the
molecular
aberrations
work
exchangers,8
use
other
than
important
without
of ion
and
chloroform-methanol
hemoglobin
to develop,
use
are
nonglycolytic
dehydrogenase.6
from
red cell
or by selective
reports
identifications,
a
basis
maps
to
for
lysate
molecules
associated
with
has
appeared.9
for
minor
proteins
proteins
compomay
be
of red cell lysates,
be described.
The
understanding
the
and is essential
some dysfunctional
for
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
RED
CELL
LYSATE
MAPPING
1123
MATERIALS
Fresh
blood
samples
from
acid
(EDTA)
aminetetraacetic
intermediates
and
sulfonic
acid
acid
related
(MES),
(CHES)
were
were
4B
and
from
fitted
syringes
procedures
with
were
a filter
Preparation
Packed
erythrocytes
of
were
the
and
containing
buffy
most
EDTA
at
result
and
from
support.
(DE-52)
were
were
constructed
Ampholines
model
UA-5
were
reaction
2-(N-morpholino)ethane
sulfonic
P-L
Biochemicals.
and
DNAse
the
CNBrI
was
microgranular
from
purchased
absorbance
ethylenedi-
including
cyclohexylaminoethane
from
Pharmacia,
columns
-
five times
and
changes
were
from
form
disposable
from
LKB.
and
20-mI
All
column
monitor.
volumes
for
protein
of
by adding
I hr
20 mm.
of phosphate-buffered
70%-90%
lysed
gently
g for
in the
equal
removed
cells
stirring
at 50,000
No
80#{176}C.
with
supernatant
The
and
by centrifugation
stored
were
Co.
was
DEAE-cellulose
ISCO
washed
coat
reticulocytes.
2.5-mM
removed
bed
an
Chemical
(MOPS),
containing
biochemicals
of Lysate
Aspiration
platelets
G- 1 50
tubes
Special
Sigma
acid
Chromatographic
with
evacuated
Agarose-NADP
and
paper
monitored
into
from
sulfonic
Sephadex
Whatman.
drawn
obtained
Calbiochem.
(CM-52)
METHODS
as anticoagulant.
were
from
CM-cellulose
obtained
were
morpholinopropane
Sepharose
Worthington.
donors
at I .4 mg/mI
compounds
buffers
activated
healthy
AND
two
volumes
at 4#{176}C.
Stroma
Samples
not
patterns
have
pH
any
and
water
unlysed
to be used
thus
7.4.
leukocytes
of glass-distilled
and
of lysate
electrophoretic
saline,
contaminating
cells
were
immediately
far
been
were
observed
as a
of storage.
Hemoglobin-free
samples
DEAE-cellulose
pH
6.2.
column
The
hemoglobin
nonhemoglobin
buffer,
pH
5.8,
saline,
Diaflow
PM
(vide
the
infra).
ferase)
were
volumes
with
chloride.
The
5-mM
a lysate
sodium
of equilibration
two volumes
samples
subsequently
buffer.
of 200-mM
were
to a
buffer,
The
remaining
sodium
dialyzed
concentrated
sample
phosphate
phosphate
overnight
against
by ultrafiltration
(Amicon,
volume.
or their
(with
with
with
Analyses
activities
Summaries
column
at 4#{176}C
and
of enzymes
measured
Nucleonics).
by Yoshida’#{176} by applying
equilibrated
the column
sodium
7.4,
and
locations
Enzyme
two
from
10) to the original
Purification
map
previously
with
stripped
pH
as described
cm)
250-mM
phosphate-buffered
To
7.0
eluted
were
containing
membrane
prepared
X
was
proteins
Enzyme
were
(2.0
the
of the
the
aid
subunits,
each
exception
of a computerized
purification
enzyme
of catalase
was
and
GeMSAEC
procedures
and
purified
from
hypoxanthine
centrifugal
measurement
red
cell
lysates
phosphoribosyltransfast
analyzer
of activities
(Electro
for each
enzyme
follow:
Carbonic
raphy
anhydrase
as
extraction.
the
(E.C.
described
by
Measurements
p-nitrophenol
anhydrase
.1).
et
al.”
of carbonic
procedure
of
Carbonic
anhydrase
following
the
anhydrase
Tashia&2
was
removal
reaction
with
purified
of
rates
by DEAE-cellulose
hemoglobin
were
acetazolamide
by
calculated
as
a
chromatog-
chloroform-methanol
using
specific
a modification
inhibitor
of
of
carbonic
activity.
Glucose-6-phosphate
DEAE-cellulose
the
4.2.1
Tanis
dehydrogenase
chromatography,
enzyme
to an
(E.C.
the
agarose-NADP
affinity
column.
1.1.1 .49).
Following
absorption-elution
Elution
the
method
of the
enzyme
removal
of
of Yoshida’3
was
effected
hemoglobin
was
with
by
used
1-mM
to bind
NADP.
Enzyme
activity
was calculated
from the rate of NADPH
formation
observed
at 340 nm by the
procedure
of Lohr and WaIler.’4
Pyruvate
kinase (E.C. 2.7.1.40).
Pyruvate
kinase was isolated
from a hemoglobin-free
lysate by
ammonium
sulfate precipitation
and CM-cellulose
and DEAE-cellulose
chromatography,
as described
by Chern et al.’ Pyruvate
kinase activity was measured
by a coupled reaction
with lactate dehydrogenase in which the rate of oxidation
of NADH
was measured
at 340 nm by the procedure
of Beeson
and
Black.’5
Lactate
dehydrogenase
DEAE-cellulose
washed
phosphate
column
(E.C.
column
with
equilibration
buffer,
equilibrated
pH
1.1.1.27).
equilibrated
buffer,
6.8.
with
The
sample
MES/KCI
Samples
with
and
75-mM
then
was
buffer,
lactate
hemoglobin-free
phosphate
dehydrogenase
concentrated
pH
of
sodium
was
by ultrafiltration
6.5. Elution
lysate
buffer,
eluted
and
of the enzyme
were
pH
6.4.
with
applied
was
The
applied
to
column
150-mM
a
was
sodium
to a CM-cellulose
accomplished
by the
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1124
EDWARDS
affinity
elution
activity
was
procedure
from
et al”
Samples
Catalase
procedure
was
Hypoxanthine
out
essentially
free
lysate
described
was
used
and
ammonium
water,
to a final
in
Eppendorf
mm,
the
through
the
column.
tube
used
Preparation
Prior
(vide
and
containing
tube.
The
enzymes
Isoelectric
Two-dimensional
the
used
ISO
cross-linker,
9-M
600
SDS
enzyme
exception
that
column.
according
treatments,
to
the
was
carried
hemoglobin-
chromatographic
by
those
authors.
The
reaction
was
measured
as
spectrophotomet-
of Jorgensen.22
sample
then
was
dialyzed
resuspended
preparations
overnight
against
in phosphate-buffered
4B
the
was
from
The
for electrophoretic
into
column
by
of
DNAse
into
culture
three
was
10
with
of buffer
aliquots
and
centrifugation
actin
1
an
tube
times
volumes
I -ml
the
following
placed
a glass
l-ml
in
in
the
mg
washed
with
containing
and
forced
of 40
collected
Ml of
in a clean
analysis.
Analysis
or hemoglobin-free
SDS/5%
for
10
were
was
column
the
eluate
a marker
to
with
placed
bed
to the
as
Sepharose
column
Sepharose
applied
removed
lysate
dithiothreitol
5 mm
except
coupled
the
microcolumn
The
serve
according
was
.sl of
briefly.
column.
to
chromatography
50
This
was
heated
were
(D1’T)/
at 95#{176}C,then
that
separation
mapping
Isoelectric
the
focusing
The
gels
2% NP-40,
20
.tl of both
or
10-15
1 hr.
of dissociated
samples.
(first
Some
mixed
with
an equal
10% glycerol,
rapidly
sample
cooled.
and
4%
pH
9.2,
were
volume
in a 2-mI
Samples
buffer
as
was
in the
described
relating
performed
of purified
mixed.
with
pH
range
enzyme:buffer
by
to
Anderson
the
simultaneously
bis-acrylamide
of 3.5-10.
solution)
The
were
and
electrophoretic
on 20 samples
polyacrylamide
5% ampholines
Ml of purified
proteins
information
dimension)
contained
and
for
(5.6%)
samples
as
(20-30
electrophoresed
for
15
Electrophoresis
on
at
changes
was
using
a 5500-A
using
nitrogen
gels
(second
permitted
mA
constant
acetic
The
narrow-band
burst
agitation.
per
in the
to be run
with
current
acid/0.2%
of a mixture
complete.
dimension)
10 gels
of polyacrylamide
120
methanol/12%
destaining
slab
which
gradients
out
several
was
solution
apparatus,
l0%-20%
lysate
Two-Dimensional
V. then at 700V
Gel
carried
50%
for
urea,
Electrophoresis
DALT
of this
the
described
mixture
was
affinity
then
the
identically,
apparatus.
Ml of lysate:buffer
hrat
sample
whole
was
Ml of lysate
electrophoretic
was
is restated.
using
CM-cellulose
Focusing
Anderson4
technique
Each
plug.
CHES/2%
treated
as
the
Sepharose
actin
for
sample
were
procedure
by
as a sample
50-mM
to the
centrifuged
through
250
reaction
by overlayering
bound
directly
in the
Approximately
lysate
ofSamples
microfuge
lysate
7.4,
infra)
to electrofocusing,
buffer
chromatographic
procedure
of Aebi
substrate
heat
exactly
enzyme,
from
was
Whole
The
buffer
culture
the
et al.’7
as
purification
remaining
each
isolated
a cotton
pH
The
were
CNBr-activated
with
saline,
to dryness.
CHES/SDS
dehydrogenase
of Gay
hypoxanthine:phosphoribosylpyrophosphate
for
NaHCO3.
apparatus
phosphate-buffered
centrifuging
Lactate
of I mg/mI.
was
of
tip fitted
and
on
perborate
Milman,#{176} with
The
according
purified
Actin
of 0.l-M
pipette
75
and
to dryness.
also
milliliter
5 ml
2.4.2.8).
the
procedure
concentration
map.
One
dissolved
reaction
was
(E.C.
of Olsen
hypoxanthine
lyophilized
actin
procedure.
7.2.
method
whole
lysate
by successive
G-150
according
to the
with
material.
by
residual
purification
two-dimensional
pH
on the
rechromatographed
precipitations
determined
then
7.4,
starting
sulfate
oxidase
the
pH
were
procedures
the
The
by a xanthine
Erythrocyte
of
as
was
Following
saline,
activity
at
based
from
Sephadex
spectrophotometrically
to the
by Flaks.22
distilled
prepared
and
phosphoribosyltransferase
activity
buffer
of NAD,
AL.
et al.’9
according
procedures,
enzyme
MOPS/KOH
of reduction
catalase
assayed
of Thomson
using
rate
Catalase
was
CM-cellulose,
containing
activity
rically
the
(E.C.
1 . 1 1 . 1 .6).
on DEAE-cellulose,
Catalase
procedures
x
of Scopes”
calculated
ET
2.7%
gel.
Coomassie
filter
patterns
(Baird-Atomic)
of SDS
The
bis-acrylamide
The
proteins
brilliant
of methanol:acetic
protein
presence
simultaneously.
acid
were
ranging
photographed
and
as cross-linker.
were
blue
were
was
visualized
R250
from
and
with
40%:6%
gels
Electrophoresis
by overnight
subsequent
on Kodak
routinely
performed
second-dimension
initially
developed
was
staining
destaining
Process
in
in
with
to 5%:7%
Contrast
the
were
when
Pan
film
D- I I developer
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
RED
CELL
LYSATE
1 . Human
Table
1 125
MAPPING
Erythrocyte
Enzyme
Hemoglobin
Enzyme’
MDH
1 3.000
LDH
6,400
TIM
0-6-P
Lysatet
(Hb-free)
3,000
Removal
After
of
=
Yield
(%)
42.5
3.4
-
23.1
zmoles/min/liter
2,200
34.4
Mmoles/min/liter
22.5
90 Mmoles/min/liter
izmoles/min/liter
1 6,250
lhmoles/min/liter
27.1
Mmoles/min/Iiter
1,306
lLmoles/min/liter
55.5
Mmoles/min/liter
73.0
Mmoles/min/Iiter
750
DH
Slope
- 8.0
fLmoles/min/liter
2,350
and Lysate
(1:2)
f.Lmoles/min/liter
60,000
PK
Lysate
Chromatography
zmoles/min/liter
400
GOT
of Whole
Lysate
Slope
Catalase
Activities
by DEAE-Cellulose
548
PGI
4,800
zmoles/min/liter
0 Mmoles/min/Iiter
0
CA
1 .380
Mmoles/min/liter
0 Mmoles/min/liter
0
6-PG-DH
750
izmoles/min/liter
0 Mmoles/min/liter
0
Aldolase
395
Mmoles/min/liter
0 lLmoles/min/liter
0
4, 1 50
AK
GPT
Mmoles/min/liter
1 25 iLmoles/min/liter
HPRT
‘Abbreviations:
oxaloacetate
MDH
Spots
PGI
lysate
ofEnzymes
in the
purified
samples
optically
and
lysate
with
isomerase;
phosphoglucose
AK
0.9
19 lLmoles/min/liter
15.2
smoles/mn/liter
lactate
dehydrogenase;
PK
pyruvate
isomerase;
adenylate
=
357.3
kinase;
CA
100.0
GOT
kinase;
carbonic
GPT
glutamate-
G-6-P
anhydrase;
glutamate-pyruvate
DH
glu-
6-PG-DH
transaminase;
phosphoribosyltransferase.
of hemoglobin-free
Identification
LDH
triosephosphate
dehydrogenase;
hypoxanthine
tSamples
TIM
dehydrogenase;
6-phosphogluconate
dehydrogenase;
malate
=
transaminase;
cose-6-phosphate
HPRT
Mmoles/mn/liter
357.3
lLmoles/min/liter
38
maps
in Lysate
that
lysate
determine
concentrated
the
correspond
and
by the
to the original
Maps
to purified
proteins
use of an optical
localization
volume.
of the
purified
were
determined
comparator
that
by co-electrophoresis
enabled
of
us to superimpose
gels
proteins.
RESULTS
Enzyme
Analysis
The
activities
of Whole
Lysate
and
of 14 erythrocyte
Hemoglobin-Free
enzymes
were
Lysate
determined
on pooled
fresh
lysate
and lysate
from which
the hemoglobin
was removed
by DEAE-cellulose
chromatography
using a centrifugal
fast analyzer.23
These
activities
are presented
in Table
I Activity
values
are expressed
as micromoles
per minute
per liter (international
.
a 1 :2 dilution
volume.
Of the
units)
for
original
enzymes,
other
but
of lysate
I 4 enzymes
the chromatographic
or hemoglobin-free
tested,
acceptable
lysate
concentrated
recovery
was possible
procedures
in total
resulted
loss of activity
for 5
enzymes.
Table
2.
Summary
of Purifications
of Six Human
Erythrocyte
Lysate
Catalase
Slope/mg
Carbonic
G-6-P
0.009
anhy&ase
DH
HPRT
LDH
Pyruvate
kinase
protein
of lysate
based
Enzymes
Overall
Final
(1 :2)
Enzyme
‘Total
to the
for 9
Product
0.053
moles/min/mg
Slope/mg
1 .75
Purification
51.3
2.72
moles/min/mg
194.4
0.005
moles/min/mg
39.66
moIes/min/mg
7932.0
0.0024
Mmoles/min/mg
23.33
moles/min/mg
9720.8
0.043
moles/min/mg
15.69
moles/min/mg
0.0 16
moles/min/mg
79.38
Mmoles/min/mg
on a hemoglobin
concentration
of 300
mg/mI
of packed
364.9
4961.3
cells.
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1126
EDWARDS
Purification
of Six
In order
Enzymes
to determine
enzymes
were
Table
2. Most
starting
sample.
identify
the
the
purified
of the
Two-Dimensional
Erythrocyte
locations
case
the
Separation
enzyme
components
of the
lysate,
procedures,
and their activities
are
recovered
with high activities
relative
increase
in a two-dimensional
achieved
by purification
electrophoretic
of Lysate
AL.
Lysate
of some
by conventional
enzymes
were
In each
enzyme
From
ET
was
six
listed in
to the
sufficient
to
pattern.
Proteins
A typical
two-dimensional
map of lysate
proteins
and protein
in Fig. 1. A total of 250 spots representing either monomeric
subunits
is shown
proteins or protein
BASE
CA
__
,
-.
.,
-
.
#{149}-‘
-_______
-
-- ‘.4
1II
#{149}
4
HEMOGLOBIN
-
Fig. 1. Two-dimensional
separation
of human
erythrocyte
lysate
proteins.
The sample was 12.5
uI of fresh lysate denatured
in 12.5 I of buffer
containing
2 % SOS /5%
DTT. Isoelectric
focusing
was from right to left and molecular
weight
separation
from top to bottom.
The map locations
of
known
erythrocyte
enzymes
are labeled. CAT = catalase;
PK =
pyruvate
kinase;
G-6-P
DH =
glucose-6-phosphate
dehydrogenase;
HPRT
=
hypoxanthine
phosphoribosyltransferase;
CA =
carbonic
anhydrase.
Hemoglobin
and actin
are also
labeled.
Molecular
weight
values
were
determined
by SDS electrophoresis
of known
proteins.
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RED
CELL
LYSATE
subunits
following
MAPPING
1127
ranging
in molecular
electrophoresis
of
approximately
other erythrocyte
The
1 .9 mg of total
lysate proteins
localization
purified
weight
from
25 s1 of the
protein.
Thus,
can be readily
of enzymes
samples
is also
10,000
to 100,000
d can
lysate:buffer
preparation
or enzyme
shown.
Of the
be visualized
containing
even in the presence
of hemoglobin,
resolved
by this procedure.
subunits
enzymes
identified
identified,
by electrophoresis
only
lactate
of
dehydrogen-
ase is of such
small
quantity
as to make
visualization
difficult.
Several
of the
proteins
appear
as rows of small spots along
the horizontal
axis, indicating
charge
heterogeneity,
which
may be due to deamidation
or the addition
of charged
groups
such as sialic acid residues.
Over 900 two-dimensional
gels of lysates
have been run
to date.
Based
on this
experience
conclude that the charge
due to artifacts
introduced
The
The diagrammatic
diagram
gives
and
the highly
representation
a better
notion
of a typical
of the number
reproducibly
present
in a map of whole
lysate.
map of lysate
proteins
following
the removal
chromatography.
All
reproducible
patterns
obtained,
we
heterogeneity is characteristic of the proteins and is not
by sample
preparation
or electrophoretic
procedures.
enzymes
identified
pattern
of spots
and distribution
is shown
in Fig. 2.
of spots that are
Figure
3 shows
a two-dimensional
of hemoglobin
by DEAE-cellulose
to date,
with
the
exception
ACID
of carbonic
BASE
-
68,000
-;oHoJcAT
-
#{149}
lF>’
40,000
-
30,000
-
]ACTIN($)
.
“0,,
.
-
...
o
_
e
,
6,000
Fig.
actin
2.
and
Oiagrammatic
hemoglobin
representation
are
labeled
along
of protein
with
identified
spots
drawn
enzymes
from
the
as in FIg. 1.
gel in Fig.
1. Erythrocyte
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1128
EDWARDS
ET
BASE
-
AL.
I
__
I
‘-CAT
______
.
4!
0,
,.#{149}:
.t:
HEMOGLOBIN
__
Fig.
3.
hemoglobin
that lysate
Two-dimensional
anhydrase,
this
separation
of human
erythrocyte
by DEAE-cellulose
chromatography.
Proteins
lactate
dehydrogenase
(LDH) is also labeled.
are
present.
Lactate
are
dehydrogenase
lysate
labeled
proteins
as in Fig.
is in sufficient
following
1
.
removal
with
quantity
the
of
exception
to be seen
in
preparation.
Figure
4 shows
the
subunit
patterns
of the
purified
proteins.
The
patterns
of the
purified
fractions
appear
identical
to the patterns
seen in the whole-lysate
map,
with two exceptions.
Catalase
is shown
as distinct
subunits
in the purified
form,
whereas it generally streaks in maps of whole lysate, possibly because of its high
concentration.
Actin
preparations,
whereas
appears
to
be
in the whole
in
both
the
a and
fi
forms
in the purified
lysate only the a form is shown.
DISCUSSION
With
the
use
of two-dimensional
gene
products
kinase,
catalase,
syltransferase,
from
red cell lysates
glucose-6-phosphate
carbonic
anhydrase,
methods
(vide
used
infra)
enable
electrophoresis,
over
250
individual
protein
can be mapped.
The
positions
of pyruvate
dehydrogenase,
hypoxanthine
phosphoriboactin,
and hemoglobin
are located.
Since
the
relatively
large
numbers
of samples
to be
run
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RED
CELL
LYSATE
MAPPING
1129
A
B
#{149}..
.
C
D
S.
.
F
E
a
G
Fig. 4.
purified
Sections
erythrocyte
dehydrogenase;
anhydrase;
(over
blood
100
cell
Although
from
lysate
0: lactate
two-dimensional
proteins.
A:
dehydrogenase;
gels showing the protein subunit patterns of seven
pyruvate
kinase; B: catalase;
C: glucose-6-phosphate
E: hypoxanthine
phosphoribosyltransferase:
F: carbonic
G: actin.
per
day),
it is now
feasible
protein variants.
individual
proteins
to search
purified
from
systematically
lysate
samples
for
have
new
been
human
red
successfully
characterized,
attempts
to characterize
the non-hemoglobin-soluble
proteins
of the
erythrocyte
have
been complicated
by the vast amount
of hemoglobin
present.
However,
the techniques
for resolving
all or a large fraction
of the proteins
are now
available. The high-resolution
protein
mapping
technique
employing
two-dimensional
electrophoresis
makes
this
resolution
feasible.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1130
EDWARDS
The
removal
dimensional
of
seen in the. whole
remove
some other
lysates
hemoglobin
electrophoresis
results
lysate.
proteins
electrophoretic
lysate
unmasking
preparations
analysis,
carefully
controlled
that are identical
in protein
have been demonstrated
for
forms
followed
of a few
new
of
some
enzymes
have
been
AL.
two-
spots
not
hemoglobin
hemoglobin-free
also
procedures
composition.
a number
by
protein
However,
the techniques
for removing
in varying
amounts.
In order to obtain
for two-dimensional
to prepare
samples
True
isozymes
from
in the
ET
must
of enzymes,
be used
and
demonstrated
in
multiple
the
human
erythrocyte.’8’24’25
The existence
of multiple
electrophoretic
forms of subunits
for a
number
of enzymes
is also apparent
from inspection
of the electrophoretic
patterns
shown
here. The origin
of these multiple
forms remains
to be discovered.
Isozymes
may be due to multiple
genetic
loci,
isozymes
that arise by posttranslational
Crude
a pattern
the study
multiple
alleles
modification.27
lysates
or partially
of 7-1
reported
1 bands
of activity
following
here show that the enzyme
purified
at a single
glucose-6-phosphate
had
similar
molecular
protein
is therefore
were observed
in the
as compared
with that
analysis
shows
four electrophoretilysate.
The purified
enzyme
gave an
in the mature
erythrocyte
in humans
there are at least seven isozymes
that arise by posttranslational
enzyme
and are functions
of in vivo aging.26’27
In our study
was
Kelley,24
approximately
34,000
but
significantly
Milman.2#{176}
The pattern
exhibited
of whole
lysate
of overloading
frequently
shows
or to co-focusing
purified
samples
higher
by catalase
of catalase
are
into
three
fractions
because
of equal
of oxidation
that following
denaturation
remains,
resulting
in the
57,000 d.
Erythrocyte
pyruvate
the
the
same
value
subunits
value
following
some horizontal
of the enzyme
specific
shows
some
lysate
they
The
erythrocyte
isozyme
may
be
a
and
and
separation
loading
differ
results
is decreased,
of subunits
can be
can be separated
in electrophoretic
reported
here
show
of electrophoretic
heterogeneity
subunits
of molecular
weight
degree
of charge
its subunits
following
two-dimensional
separation.
This
many
to be similar
to the L-type
isozyme
of hepatocytes.
kinase
may be a hybrid
between
L-type
(liver)
and M-type
the
Arnold
by Olsen
two-dimensional
but
groups.
and reduction
a degree
separation
of five distinct
also
by
streaking,
possibly
due to effects
with hemoglobin.
However,
when
when
activities,
modification
of the
subunit
molecular
d reported
is electrophoresed,
a number
shown
that erythrocyte
catalase
of sulfhydryl
kinase
the
reported
of 26,000
electrophoresed,
or when
hemoglobin-free
lysate
distinguished.
Aebi et al.’8 have
mobility
d,
than
revealed
focusing.25
The results
of
of at least four subunits
demonstrating
charge
heterogeneity.
All of the subunits
weights
of 56,000
d. The charge
heterogeneity
of undenatured
greater
than that of the subunits,
as expected.
No differences
pattern
of the enzyme
purified
by affinity
chromatography,
weight
or secondary
dehydrogenase
isoelectric
is composed
observed
in the whole-lysate
preparation.
Our hypoxanthine
phosphoribosyltransferase
cally distinct
subunits
present
in the whole
identical
pattern.
It has been reported
that
locus,
heterotetramer
molecular
weight
of the L2’ subunits
being
slightly
subunits.3#{176} Alternatively,
interconversion
of isozymes
Although
these conflicting ideas are not reconciled
heterogeneity
of
enzyme
is considered
by
Erythrocyte
pyruvate
(muscle)
subunits,28’29or
designated
L2L21,
with
the
greater
than
that of the L2
may occur
during
isolation.3’
here, our results with whole
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RED
CELL
lysate
excess,
LYSATE
MAPPING
1131
show two electrophoretic
subunit
types,
as determined
by staining
intensity.
subunit
molecular
weights,
as both
types
with the more
No differences
migrated
in SDS
basic
were
form being
in
found
in the
as 66,000-d
proteins.
Analysis
of the pattern
of pyruvate
kinase
in hemoglobin-free
lysate
shows
the
appearance
of a third distinct
and more acidic
spot in relatively
low concentration.
The appearance
of this new spot may be due to modification
of the enzyme
during
the
procedures
used to remove
hemoglobin.
Lactate
dehydrogenase
appears
as only
electrophoresed,
and
exact
However,
in hemoglobin-free
have
has
a molecular
been
weight
identified
localization
lysates
the
of approximately
in the
a minor
34,000
two-dimensional
isozyme
of human
red cells.
cally distinct
bands,
possibly
when
is
The
lysate
is
to determine.
seen, and they
one
of the
form
LDH-
of the enzyme
the LDH-l
whole
difficult
are easily
d. Only
maps.
Purification
representing
spot
in the map
enzyme
subunits
1 form
enzyme
is the
major
yielded
two electrophoretiform and the minor
LDH-2
isozyme.
The resolving
power
and the applicability
of two-dimensional
the study of isozymes
are demonstrated
by the separation
achieved
carbonic
anhydrase.
This enzyme
has
designated
CA I and CA 11.12 Isozymes
map of whole lysate at molecular
weight
I form and 30,000 d for the CA
CA
basic. Removal
of hemoglobin
removes
all carbonic
anhydrase
The
use
of
II form,
with
the CA
to identify
more
I form
being
from lysate by DEAE-cellulose
isozymes.
high-resolution
mapping
enzyme
to
been shown
to exist in two isozymic
forms
of this monomeric
enzyme
appear
in the
positions
of approximately
29,000
d for the
techniques
analysis
of a large number
of erythrocyte
protein
way in this laboratory
to use the multiple-sample
system
electrophoresis
with erythrocyte
subunits
and
provides
a
gene products.
capabilities
to analyze
slightly
more
chromatography
method
for
the
Work is now under
of the ISO-DALT
known
mutant
proteins.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1979 53: 1121-1132
Red cell proteins. I. Two-dimensional mapping of human erythrocyte lysate
proteins
JJ Edwards, NG Anderson, SL Nance and NL Anderson
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