From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Separation
of
Vitamin
Gastric
B12-Binding
Juice
and
Saliva
Cellulose
By
P.
FRANCOIS
N
ORMAL
SERUM
migrating
W.
CHESTER
W.
PRATF
contains
AND
at
as
beta-globulin.46
There
that
bolic
functions.
globulin
binder
Whereas
probably
states,
and
chronic
of
the
value
We
an
distilled
5 ml.
using
6.3,
of
free
water
is
to the
packed
a buffer
eluant
a
have
in particular,
may
implications
to
1 ml.
with
technic
based
completed
in
modification
Kochwa
are
addition
on
DEAE
as
a
metathe
betadisease
characterised
be of some
syndromes.9
in
one
other
different
diagnostic
Separation
to
its
of
serum
to
serum;
with
cellulose,
5 more
elutes
(4)
obvious
it
in
consists
B12
passing
the
1 M
globulin,
6
NaC1
3 ml.
/3k,
iso-
adding
(2)
adding
re-
2 ml.
through
(of
as eluant.
/32A,
(1 )
sample
samples
This
for
binders;
(3)
charcoal;1’
column
hours.
reported
saturate
collecting
using
gamma
2
procedure
al.1#{176}
Briefly,
hemoglobin-coated
cellulose
approximately
the
of
et
1 ml.
and
buffer
be
by
Co57B12
Co57B12
pipette
phosphate
can
method
separation
excess
B12,
the
to alpha-globulin,2
B12 carrier.6’7
Certain
binder.8
This
myeloproliferative
clinical
vitamin
and
substances
(CML)
a separation
which
agglutinin
moving
has
of
binders
alpha-globulin2’3
these
leukemia
of alpha-globulin
between
the
to describe
column”
two
an
KOCHWA,
SHAUL
HERBERT
of B12 kinetics.
chromatography
“baby
Serum,
DEAE
GOTFLIEB,
native
B12 is bound
acts as a temporary
thus
study
wish
is evidence
myeloid
B12-binders
in the
Rapid
VICTOR
least
electrophoretically
by a striking
increase
value
in differentiating
by
of
Chromatography
RETIEF,
PETER
Proteins
2 ml.
The
0.06
B12-binding
a
each)
M,
pH
/3,
and
From
the Department
of Hematology,
The
Mount
Sinai
Hospital,
and
Mount
Sinai
of Medicine,
New
York,
N. Y.
This
investigation
was supported
by USPHS
Research
Grants
AM 09062,
AM
09564
and AM 04434,
and by the Albert
A. List, Frederick
Machim,
and Anna
Ruth Lowenberg
Funds.
Dr. Retief
is a USPHS
International
Postdoctoral
Research
Fellow;
Award
#FO5TW
918.
Present
Address:
Department
of Medicine,
University
of Stellenbosch
Medical
School,
P. 0. Box 53, Beilville,
C. P., South Africa.
Dr. Herbert
is a City
of New
York Health
Research
Council
Career
Scientist,
Award
School
#1-435.
Presented
in
Clinical
Research,
part
at
Eastern
Section
Meeting
of
the American
Federation
fom
4, 1965.1
First
submitted
Aug. 9, 1966; accepted
for publication
Sept.
23, 1966.
FRANCOIS
P. RETIEF,
M.D.,
CH.B., D.PHIL.,
M.R.C.P.:
Lecturer
in Medicine,
Stellenbosch Medical
School,
So. Africa.
CHESTER
W. CornIER,
M.D.:
Instructor
in Medicine,
Mt. Sinai
School
of Medicine.
Siiwi.
KOCHWA,
Pu.D.:
Associate
Professor
of Medicine,
Mt.
Sinai
School
of Medicine.
P-rErt
W.
P1IATr,
M.D.:
Instructor
in Medicine,
Mt.
Sinai
School
of Medicine.
VIcToR
HERBERT,
M.D.:
Professor
of Medicine,
Mt.
Sinai
School of Medicine,
and Attending
Hematologist,
The Mt. Sinai Hospital.
the
December
501
BLOOD,
VOL.
29, No.
4,
PART
I
(APRIL),
1967
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
502
ET
RETIEF
the
half
albumin;
globulin
and
4 has
most
1 M
NaC1
then
macroglobulin.
of the
beta,
elutes
Fractions
and
8 has
the
remaining
5 through
most
of the
MATERIAL
albumin,
7 have
most
plus
AL.
alpha
of the
albumin,
CML
and pernicious
anemia
C. Hepaninized
plasma
gave
separation
alpha.
AND
METHODS
Specimens
Serum
frozen
froni
normal
immediately
persons
after
and
collection
patients
and
with
stored
at -20
patterns
essentially
similar
to serum.
Gastric
juice
(GJ)
from
PA and non-PA
maximal
histamine
stimulation,12
depepsirnzed
previously
described
.
subjects
was
and
stored
collected
at
-20
at
to intrinsic
37 C. for
a 50
mg.
pellet
of
hemoglobin-coated
charcoal”
to
remove
pellet,
produced
by centnifugation
of 2 ml. 2.5 per cent
charcoal
adsorbs
at least 1 mg. free B19. The B19-saturated
used as such. as a source
of IF antibody.
Preparation
of Serum,
1. One
ml.
by incubation
C. at pH
was
7.0,
after
as
I3
Saliva from normal
persons
was stored
at -20
C.
Serum
from PA patients
with high titers
of autoantibody
was supersaturated
with nonradioactive
B12, incubated
with
(PA)
GJ and
was
serum
of
added
20-40
tc./g.
0.5
ml.
B12 solutions
of
higher
sample
thus
varied
elution
patterns
with
a
#{248}mm.
in
a
to
50
serum;
B12.
A
50
iig.
of hemoglobin-coated
stored
at
-20
C.
was
Chromatography
rare
radioactive
tc./jsg.,
sera
was
scintillation
4 hours)
and
Free
radio-B12
1.75
Visking
amount
(11.3-14.4
was
lost
studies,
we
almost
as
good
(listilled
water
ml.
serum
column,
instead
column
2.
1);
eluate,
the
1
of
dialyzing
in
this
changing
serum.
modification
eluant
Specimens
to their
UBBC
0.75
ml.
removed
from
were
ng.)
this
these
IF
antibody
function.
radioserum
did
not
samples
incubation
b
at
dialyzed
affect
mixing
37
C.
radioactivity
for
counted
overnight
(or
for
a
phosphate
buffer, pH 6.3, at 4 C.
the
radioactivity
of their
contents
determined
measurable
activity
normally
diffused
into
the
M
removed
the
separations
and
coated
the
buffer
3
charcoal.
bag
(Table
he
achieved
could
applying
against
by
dialysis
ml.
prior
A
variable
1). In subsequent
by adding
2 ml.
directly
to the
of
mixture
to
applying
serum
to
the
rather
than
10 aliquots
collection
of tube
6 rather
than after tube 5.
of serum,15 0.1 ml. GJ and
saliva
were
compared
to
again
is
necessary
saturated
ml.):
was
to
added;
UBBC
when
collect
with
<
5.0,
UBBC
11
radio-B,2
0.5
ml.
was
(10
(5.0
7.5-12.4,
#{176}Visking casing,
supplied
in rolls of 100 ft length
and 8/32
inch
Carbide
Corp.,
6733
West
65th
Street,
Chicago,
Ill. 60638.
Prior
is boiled
for 20 minutes
in 0.1 M Na9CO,,
to remove
a yellow
damages
with
saturated
order
supernatant
was
0.02
from
it
after
saturated
radio-B12
following
and
previously
(in ng./0.1
(7.5
was
supernatant
overnight
the lesser UBBC
of
5.0-7.4,
cent)
that
to
Because
1.0 ml.
according
UBBC
found
(Fig.
of 2 ml.
per
were
the
of
casing#{176} against
and
No
of
pellet,
The
ng./ml.
variations
centrifugation
The
dialysis
bags
were
emptied
before
application
to the column.
buffer
because
free B1., had
been
25
volume
ml.;
charcoal
by
<
total
1.25
detector.
in
UBBC
The
removed
B,2
respectively
with
hemoglobin-coated
well-type
of
35
concentrations.
significantly.
tug.
with
and
between
Charcoal
minimum
saturated
for
unbound
suspension
serum
(Co5TB,2
or Co58B,0.
with specific
) in amounts
in excess
of the unsaturated
B1-binding
capacity
(UBBC)
as determined
by the method
of Gottlieb
et a1.1 A
working
solution
of 10 ng. radio-B12/ml.
saline
was added
to serum
as needed
to slightly
exceed
UBBC,
as follows
(UBBC
in ng./ml.):
when
UBBC
< 2.5,
0.25
ml. (2.5
ng.)
was
added;
when
UBBC
was 2.5 to 4.9, 0.5 ml. (5 ng.) was added;
when
UBBC
5.0 to 7.4. 0.75
ml. ( 7.5 ng. ) was added.
\Vith UBBC
of 7.5 to 25 ng./ml.,
1.25
ml. radio-B1
( 12.5 ng.)
activities
was
Saliva
factor
(iF antibody)
15 mm.
and treated
ng./ml.
ng.)
solution)
was
added;
1.25
ml.
(12.5
cross-section;
to use,
the
contaminant
when
ng.)
Union
casing
which
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SEPARATION
OF
B12-BINDINC
VITAMIN
Table
1.-”Loss”
of
503
PROTEINS
B12
Radioactive
from
Percentage
(a)
Test
Samples
R adioactivity
Dialysis
against
buffer*
Sample
Normal
Test
Sample
(b)
After:
Passage
through
DEAE
celluloset
87.6
±
0.7
88.0
±
1.2
serum
85.6
±
1.5
87.8
±
2.6
CML serum
Normal
GJ
PA GJ
88.7
±
1.2
87.4
±
1.3
88.7
86.8
±
0.8
81.8
±
2.1
±
1.3
81.9
±
0.9
Saliva
76.5
±
5.3
65.0
±
2.2
PA
SCtII11
#{176}Radioactivity of sample
after
buffer
dialysis
x 100
Radioactivity
of sanlple
fRadioactivity
eluted
Radioactivity
apph(’(l
beforc
x
Values
(a)
rccorde(l
All
(h)
All
column
10()
±
11S Ilicans
“lost”
B10
“lost”
was
B1.,
111111cr dialysis
stan(lard
retained
in
errors
and/or
of
on
5-17
the
observations.
dialysis
bag.
was
recOVeral)lc
from
the container
20 per
cent ) and from the column
( aI,proximat(’ly
which
(leliverecl
( approximately
thy
80
B3.
r
to the
cent).
UBBC
was 12.5-24.9.
0.25 ml. 100 ng./ml.
radio-B12
(25 ng.) was added.
the final salliple
volume
was adjusted
to 1.25-1.5
ml.
with 0.9 per cent
saline.
Specimens
were treated
with
hemoglobin-coated
charcoal
and
dialyzed.
as described
for seruni.
The effect
of IF antil)ody
pretreatment
on elution
Patterns
of CJ was tested
l)y inculiating
(;J with 0.1 nil. IF antibody
seruu
at 37 C. for 15 minutes,
l)efore
adding
ad(led.
was
In all
With
instances
radio-B1
DEAE
Cellulose
Chromatography
Procedure
The apparatus
is depicted
in Figure
2.
A 250/5.5
mm. glass column
was
prepared
the narrow
mouthpiece,
dropping
in a tiny
220
mm.
with
DEAE
cellulose0
suspended
from
plug
a 5
ml.
of glass
in 0.02
serologic
wool,
NI phosphate
and
buffer,
pipt4te
filling
pH
b
removing
to a height
6.3.
The
of
column
was connected
with 6.0 mm. rubber
tul)ing
to a 125 ml. glass separating
funnel
containing
0.02 I phosphate
buffer.
pH 6.3. The cellulose
was packed
with this buffer
at approximatelv
20 cm. hydrostatic
presstire,
and
allowed
to equilil)rate
for at least
4 hours.
The
supernatant
buffer
was reniovecl
(or permitted
to drain
away);
the sample
was tIleIl applied
to the top of the colllnln
and
collection
of eluate
started
in a series
of 7 ml. glass
tubes,
each niarked
to take 2 ml. fluid. Imlliediatelv
after
the
sample
was
adsorbed,
the column,
tubing
and funnel
were filled with 0.06 Ni phosphate
buffer,
p1-I 6.3, as initial
ehiant.
and
the flow rate set to 1 1111. per 5-8
minutes,
h’ adjusting
the height
of the funnel.
The
collecting
tubes,
each containing
2 ml eluate.
were changed
by hand ever
10-15
minutes.
After
collection
of tube
5, tile rubber
tube
was
renioved
and remaining
buffer
removed
from above
the cellulose,
and replaced
with 1 M sodium
chloride.
Ehition
with
1 M NaC1
#{176}Selectaccl ion exchange
cellulose,
#70 DEAE
Standard,
Carl Schleicher
& Schull
Co.,
Keene,
New Hampshire.
100 gm
of the dry cellulose
is added
to 2 L 1 N NaOH,
mixed,
allowed
to stand
for 1 hour,
poured
slowly
onto a Buchner
funnel,
allowed
to stand
for
5 minutes,
ai)out
in
10
2 to
2 L
2
the
liquid
L
aliquots
3 L 0.02
aliquots
to columns,
diluted
with
of
M
the
then
sucked
thru
of distilled
1)11 6.3
buffer
water
phosphate
until
pH
the
until
buffer,
6.3,
and
each
1 1111. of this stock
slurry
2 ml. of buffer
(0.02 M Ph0SPlutte
funnel,
pH
following
and
the
7. The
which
left suspended
(about
25 per
ph 6.3).
cellulose
cellulose
again
washed
with
is then
left overnight
it is washed
with al)out
10
in the final 2L. For addition
cent
cellulose
by volume)
is
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
504
RETIEF
.-.
Sivum
dyzsd
S.
Sinai
dibdid
ET
AL.
buffir
1:3
wsr
jffi
500
400
0
0
200
a.
p00’
0
123456789101
BABY
Fig.
sera
1.-Comparison
prepared
of
“baby”
DEAE
of delayed
volume,
6 tubes,
total
each
by
buffer-elution
buffer
eluate
consisted
containing
2 ml. eluate).
column
buffer
in the
ELUATES
COLUMN
DEAE
for chromatography
Because
1234567891011
latter
elution
dialysis
situation,
of 14 instead
patterns
vs. by
1:3
due
of
to 3-fold
of 12 ml.
two
dilution
(i.e.,
normal
with
larger
first
water.
sample
7 instead
of
was continued
volume
of the
for a further
5 tubes,
giving
a total eluate
of 20 ml. in 10 tubes. The void
column
is approximately
2.0 ml. Fractions
1-fl
were
referred
to as “buffer
eluates,”
and
7-10
as 1 M NaC1
eluates
or “saline
eluates.”
(In the modified
procedure,
fractions
1-7 are “buffer
eluates”
and 8-11 are “saline
eluates.”)
Eluates
were investigated
as follows:
1. Radioactivity
was determined,
and converted
to pg. radioactive
B12 per eluate,
by
comparing
the counting
rate with that of a radioactive
B,2 standard.
2. Optical
density
(OD) at 280 mt was determined
in a Beckman
DU spectrophotometer,
as an index of total protein
present.
Serum
eluates
were diluted
1:10 with saline
prior
to
testing.
but CJ and saliva were read as such.
3. B1., content of eluates from sera not presaturated
with radio-B,.,
was assayed
with the
hemoglobin-coated
charcoal radio-dilution
method of Lau et al.’3
4. Cellulose acetate
strip
electrophoresis
in veronal
buffer,
pH
8.6,
was
performed
on the full spectrum
of eluates
from representative
specimens.
In selected
instances
where
samples
had been
eluate”
and
electrophoresed
2
hours;
samples
labeled
“saline
photograph
#{176}CollodionBags
34
Gottingen,
IKodak
Co58B19,
regions
on 1 per
application
to
with
eluate”
were
the tubes
with
concentrated
maximal
tenfold
filmt
was
for
0.05
72
CB, porosity
< 5 millimicrons,
Germany.
medical
x-ray film, no screen.
size
8 ml.;
in the “buffer
ultrafiltration0
8.6 on 4” X 5” glass plates at
ml.
Autoradiographs
were produced
hours.
Shaking
with
hemoglobin-coated
cent agar gel at pH
sample
radioactivity
by
and
110
Membran-Filtergesellschaft,
by
then
volts for
exposing
charcoal
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SEPARATION
OF
VITAMIN
B12-BINDING
505
PROTEINS
L
Fig.
2.-”Baby”
DEAE
cellulose
demonstrated
no free B12 in the eluates.
that it came through in fraction 2.
column
Passage
apparatus
of free
(2 columns
Co57B19
through
shown).
the
column
showed
RESULTS
Serum
The
sera
typical
is represented
pattern
of radioactive
in
Figure
3.
B12
In
from
elution
normal
and
PA
5 normal,
serum,
PA
and
radioactivity
CML
ap-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
506
IIETIEF
ET
AL.
500
I400
I 300
900
800
700k
600
500
400
300
200
(00
2
0
3
5
4
6
NORMAL
7
8
9
0
and
3.-”Baby”
5 CML
peared
5
6
7
8
9
0
PA. SERA
DEAE
predominantly
eluted
3 and
COLUMN”
column
7, but
in
by
IM
the
NaCl.
CML
ELUATES:TUBES
radioactive
SERA
1-10
B19 elution
eluates”;
patterns
in
CML,
radioactivity
in tubes
in Figure
1 M NaC1
and
1-6)
“buffer
Maximal
occasionally
demonstrated
(tubes
4
of 5 normal,
5 PA
sera.
mally
is
3
SERA
“BABY
Fig.
2
I
4 and
4; the
eluates
8. The
total
(tubes
radio-B12
was
usually
reproducibility
radio-B,2
7-10)
in
agreed
that
1 M
was
found
maxi-
in tubes
of the
method
the
“buffer
closely
in
eluates”
duplicate
determinations.
Agar
gel
electrophoresis
(with
globulins,
maximal
but no
activity)
contained
7
y-globulin,
the
and
of
sera.
in the
demonstrated
that
only
with
presaturated
only
in
UBBC
in the
In
1 M
of a,
=
NaCl
1228
was
Figure
6,
1 M
pg./ml.),
found
OD
the
and
assay
acute
however,
in the buffer
are
from
showed
compared
a
cent
with
of
trace
(Fig.
was
eluates
with
normal
and
(B12
both
confirmed
endogenous
serum
and
showed
eluates
buffer
a2radio-
for
true
autoradiography
hepatitis
16.5 per
fractions.
and
albumin
held
but
NaC1
eluates
B,2
With
readings
in the
number
electrophoresis
eluates,
in
eluate
a2-globulins,
strip
buffer
NaCl
a,-globulins,
3 (with
maximal
This
However,
radioactive
eluates.
to
eluates.
mobility
charcoal
with
eluates,
all
radioactivity
material
addition
acetate
from
NaC1
albumin,
number
a,-globulins.
Cellulose
1 M
/3 and a2 mobility.
Hemoglobin-coated
not
in
lacked
a,-globulins
of /3-globulins
showed
contained
Buffer
eluate
apparently
CML
absence
5)
/3-globulins,
but
normal
(Fig.
radioactivity)
a-globulins.
=
5)
associated
material
CML
of
sera
B12
activity
1360
pg./mI.,
endogenous
B12
present
radioactive
B12
content
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SEPARATION
OF
VITAMIN
B1 2-BINDINC
507
PROTEINS
800
SERA
Normal
0-4
INVESTIGATED
Normal
I
0-0
IN DUPLICATE
2
0-4
Normal
0
3
C ML
:
0-4
0-4
0-
1600
885
IBuffer1,5eluates
#I’6
-glob.’)
(400
1SIj
eluates,#7-l0
(
Pg Raciso-B12
926
589
(794#{176}!)
(80.0%)
(20.6%)
814
785
494
492
(70#{176}/) (83.0#{176}/s)
(82.5#{176}6)
(16.8%)
(16.2%)
96
(25.0%
167
167
(170%) (175%)
2452
2546
(83.2%)
(83.8%)
790
(100%)
981
(100%)
232
90
(20.0%) (24.4%)
229
-glob.)
594
(75.6%)
1200
[Total,
lll4
(58
(I00#{176}k)
(l00%)
# 1-10
779
(l00%)
952
2946
(l00#{176}6)(l00%)
3038
(100%)
000
800
600
400
200
2 34
I
123456789I0
NORMAL
sera
4.-.-”Baby”
and
DEAE
a CML
serum
column
of serum,
CJ
comparable
loss
The
and
COLUMN
is summarized
decrease
was
through
the
I”
cent.
in
11.3-14.4
There
normal,
PA
without
I)rior
was
much
active
B12
The
distribution
In
Binding
B,2
average
and
between
column”
gastric
7).
unsaturated
eluates.
(;astric
(Fig.
method
has
the
serum
the
was
loss
serum
3
normal
is con-
calculated
of
froni
beta
been
alluded
to
B,2
elution
UBBC
of radioactive
B,2.
B12 by a further
Passage
12-12.6
loss
of UBBC
through
l)etween
the
column,
l)inding
presaturated
protein
with
radio-
quantitatively
original
9
by
sample
obtained
reference
procedure
mean
B,2-binding
globulin
in
the
with
proteins.
separation
cent
of
values
trace
buffer,
determined
unsaturated
and
the
been
‘as
are
measurable
capacity’4
alpha
protem
passed
of
had
protein
binding
range
in
in per
B,2,
total
against
difference
with
l)in(lers
B12-binders
stages
decreased
when
in
as
by loss
radioactive
When
than
B12
judged
significant
serum.
saturation
greater
estimating
Table
was no
CML
and
from
reproducibility
1 M NaCl.
dialysis
as
patterns
good
discrepancy
During
cellulose
elution
$-globulin
unexpected
at various
p#{128}’’
cent,
DEAE
B12
with
great
1.
NORMAL 3
ELUATES OF VARIOUS SERA
and
eluate
is not
Table
I 2 3 4 5 6 7 8 910
in duplicate;
a-globulin
The
saIi’a.
B12 contents
of radioactivity
10
radioactive
chromatographed
firmed
by coniparisoii
of total
the grd1)hs.
Saline
eltiate
means
7 89
NORMAL 2
I
DEAE
Fig.
56
using
and
and
total
capacity
and
the
baby
is
delineated
from
two
normal
and
non-IF
in
2.
Juice
Figure
juices
8, the
with
radioactive
different
binding
capacity
pttteriis
and
obtained
percentage
IF,
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
RETIEF
508
a
E
Cl
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AL.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SEPARATION
OF
VITAMIN
B12-BINDING
509
PROTEINS
(I)
w
0
globulin
.
0
I-.
:z
-globulin
3
c2-globulin
c:1 -globulin
Albumin
S I
Si
0
-globulin
-
-globulinS
3
2
-globulin
c1
-globulin
Albumin
_o
I
‘
I
I
I
CML EluateEluQteNormQl
#71
#3
I
1
Eluote Eluote
#3
#7
Fig.
5.-(a)
Agar
gel electrophoresis
of normal
and CML
sera,
accompanied
by
electrophoresis
of “baby”
column
fractions
with
maximal
B12-binder,
in buffer
(#3)
and 1 M NaCI
(#7)
eluates
obtained
from these
sera.
(b) Autoradiographs
of same
sample;
sera
presaturated
with
Co58B,2.
Radioactivity in eluates
#3 associated
with a2 and fl-globulin, and in eluates #7 with
a,-
globulin.
binder,
are
lacked
IF#{176}by
and
1 M
buffer
compured
NaCl
on the
vitro
buffer
passage
serum
(Table
#{176}Very
little
a
all
very
reduced
peak
GJ and PA GJ.
The percentage
and
of
showed
greatly
that
of
1),
samples
with
IF is occasionally
no
the
peak
and
lost
with
of
peak
from
GJ
is
difference
in otherwise
normal
non-PA
eluted
in
non-PA
GJ,
remained
during
but
had
no
IF
effect
against
to
the
in normal
that
normal
gastric
IF,
with
dialysis
between
buffer
lacking
unaffected
comparable
which
the
Pretreatment
normal
basal
juice
both
GJ,
activity.
in
column
significant
a basal
was
peak
1 M NaCl
cellulose
found
GJ
but
buffer
radioactivity
the
PA
Radioactivity
small
the
of PA GJ;
through
of one
assay.’4
eluates
eluate
antibody
with
in
juice.’3
buffer
lost
and
PA
from
GJ.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
510
RETIEF
ET
AL.
3205
1200F
1778
.2
Radio-B2
Total protein;
0-#{149}
(00
II
II
0.0. performedort
1,1
1:10 diluted serum eluates, but
undiluted G.J. and saliva eluates
000
1.0
900
0.9
800
#{163}
700
E
s
O.7
c%,J
C
600
:
500
j
a:
H
(.D
::
;
a400
:
A
300
I:\,A
200
ci
:
0.4
I
0.3
S
“,
/‘\
L
l0O
23456789(0
NORMAL SERUM
12345678910
CML PLASMA
Fig.
6.-Comparison
of “baby”
DEAE
As with
CJ
serum,
was
of
column
the
rinsaturated
12345678910
NORMAL
COLUMN
DEAE
mt)
I\
0.2
I\
h4:!;a;J4
01
:
&
radioactive
eluates
12345678910
NORMAL SALIVA
G.J.
ELUATES
B,2 and
total
protein
of normal
and CML
serum,
loss
of B,2-binder
than
when
in the
saturated
column
with
content
(O.D.
GJ, and saliva.
was
much
at
greater
280
when
B12.
Salica
The
radioactive
marked
variation
of maximal
(Fig.
In
9).
B12 distribution
in
elution
This
Table
in the
is
similar
1 it
during
buffer
more
radioactive
total
is
B,2
the
was
B,2
that
with
obtained
the
more
of
than
by
the
normal
similar
a minor
with
amount
retained
three
but
fractions
graph
slightly
was
from
capacity,
1 M NaCl
to
shown
dialysis
in eluates
binding
salivas
general
buffer
non-PA
that
lost
cellulose
eluate
GJ,
radioactivity
from
GJ
IF.
from
and
with
peak
lacking
lost
than
shows
patterns
the
saliva
serum,
and
other
two
materials.
DISCUSSION
Previous
workers
graphically
with
technics
are
column”
DEAE
a routine
procedure.
Pitney
have
separated
time-consuming
et al.2
and
cellulose
method
reported
in
B,2 had
very
little
additional
showed
by paper
electrophoresis
migrated
with
serum
DEAE-cellulose6’17
/3 or
a2-globulins
1954
or
unsuited
B,2
binding
to
a routine
here
described
may
that
the
ability
that
when
these
laboratory.
he
ratio
B,2
was
chromatoconventional
The
performed
alpha-globulin
to bind
approximately
the
proteins
CM-cellulose,18
carrying
added
70 per
in
cent
15 ng.
added
“baby
rapidly
as
endogenous
vitro.
Miller4
of added
B,2
B,2
to
1 ml.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SEPARATION
OF
VITAMIN
B12-BINDING
[-1
Co57B12
L.__J
B12 binder ( sample
with Co57B12)
511
PROTEINS
as index of
present,
presaturated
NORMAL
3
EU
UB BC
presaturated
NORMAL
(sample
with
ri[un
_
..
eluates
not
Co57 B12)
SERUM
0
5
of
[‘““
L___J
H
ufl
GASTRIC JUICE
:
[1
.
PRE-CHROMATOGRAPHY
UBBC
BUFFER ELUATE
BINDERS
812-BINDER
Fig.
7.-Comparison
when
passed
thru
column
serum.
Finkler’#{176} differentiated
H
:
SALINE ELUATE
BINDERS
BINDERS
IN#{176}BABY
COLUMN’
ELUATES
normal
serum
and
GJ
Left-hand
column
is
unsaturated
B12
binding
capacity
of an
aliquot
of sample,
determined
b
adding
Co7B12.
Other
dark colunins
represent
portions
of this aliquot
appearing
in saline,
buffer
and
total
eluate
after
passage
( saturated
with
Co5TB,2 ) through
column.
Light
columns
represent
a different
aliquot
of the same
sample,
which
was passed
thru
the column
without
added
CoTB,2.
Much
more
of the
measurable
B,2
binding
capacity
was lost from
samples
passed
through
the column
without
previous
addition
of a saturating
quantity
of B,2.
Hall
between
and
the
two
of “loss”
of B,2 binding
after
vs. before
addition
TOTAL
B,2
main
by
binders
in
capacity
of
of C&’B12.
extensive
normal
DEAE
serum.
agreement
with
our
where
approximately
rapid
“baby
column”
separation,
one-fifth
of a saturating
dose
with
alpha-globulin
(1 M
binder
(buffer
serum
ma’
M
0.06
UBBC
This
be
from
serum
method
the
and
totalling
total
value
CML
the
The
1-6).
1 M
NaC1
this
can
fact
four-fifths
amount
with
the
of radioactive
eluate
(tubes
the
show
in
in a test
B12 eluted
component
7-10);
total
fair
Figure
2,
B,2 eluted
beta-globulin
B12-binder
alpha-globulin
figure,
tables
illustrated
of radioactive
of beta-globulin
the
to
serum
on
and
amount
(tubes
B,2
is based
eluate)
total
by
buffer
is derived
in normal
NaC1
The
estimated
phosphate
endogenous
binder
eluate).
chromatography
Their
by
alpha-globulin
of
adding
by
the
the
B,2-
be calculated.
that
beta-globulins
are
maximally
eluted
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
512
RETIEF
ET
AL.
#{149} #{149}G.J.
o G.J.
o
pretreated
with
IFAb
(‘J
C
a:
a-
600
400
200
0
12345678910
I23456789I0
NORMAL
12345678910
NORMAL
DEAE
ELUATES
COLUMN
I 23456
‘7 8910
PA.
NON-PA, IF-LACK
OF GASTRIC
JUICE
Fig.
8.-”Baby”
DEAE
column
radioactive
B,2 elution
patterns
of 2 normal
GJ
specimens
(with
very
different
UBBC),
a non-PA
basal
GJ with
IF lack,
and a PA
GJ.
The
typical
effect
of pretreatment
with
IF antibody
(IFAb)
is shown
on a
normal
and a PA GJ.
from
cellulose
a much
0.06
at
higher
M
buffer
proteins
also,
only
non-IF
B,2
PA
with
IF
1 M
NaC1
IF
(Fig.
was
8).
and
and
non-IF
others
GJ
and
have
fractions
the
(792
Although
our
of 852
elution
of
pg.
to
R, sufficient
each
5, 1058
of
these
of 1078
for
it may
sent
for
a run
three
pg.
not
us
I, 1099
be
the
B12-binder
(i.e.,
the
yield
beta-
discrete
similarity
of which
same
aliquots
through
of 1274
chroma-
in-
between
are
found
in
serum
binder
was
eluted
of 1 M NaCI,
and therefore,
small
fractions
the
lacking
was
main
not
both
the beta-globulin
M buffer
instead
kindly
the
B,2
to confirm
a major
GJ,
of GJ
does
B,2-binder,
separation
produced
to
IF
pretreatment
non-PA
endogenous
tend
salivary
indicated,20
Gr#{228}sbeck
Saliva
the
containing
useful
but
that
technic
latter
all
this
IF.
similar
patterns
However,
by 0.06
for
“R-binder”
carrying
with
the
eluate
radioactivity,
non-IF
separation
the
containing
basis
of
similar
the
not
our
and
also
Dr.
S, I, and
Co57B,2
binder
eluate.
column”
saliva.
that
and
a 1 M NaC1
contained
rather
washing
manner.
eluate
and
peaks
neither
pattern
serum
binder
the 1 M NaC1
from the “baby
as
that
a
buffer
at
elute
Initial
alpha-globulins,
immunoelectrophoretically
the
proteins,
GJ
two
M.’T
in nonspecific
physicochemical
Simons’#{176} reported
binder).
dividual
the
and
a
alpha-globulins
0.15
from
as
The
while
of
of non-IF),
produced
showed
peak
saliva
globulin
binders.
antibody
tographically
of
fractionated
also
0.06,
range
by 1 M NaC1
amount
GJ
than
the
beta-globulins
eluted
a significant
is unclear.
less
in
separate
subsequently
juice
( and
of
strength,
thus
are
Gastric
a molarity
ionic
the
was
pg.
baby
eluted
R).
binder
of
as
those
Co57B12
column.”
by
0.06
in
tagged
Most
M
buffer
of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SEPARATION
OF
B12-BINDING
VITAMIN
513
PROTEINS
CJ
a:
a-
12345678910
COLUMN
DEAE
Fig.
9.-”Baby”
from 3 persons.
During
DEAE
dialysis
represented
column
against
ELUATES
radioactive
buffer,
primarily
adsorption
the
cellulose
column
resulted
activity
from
normal,
PA
CML
er,
are
to
of beta-globulin
per
(18.2
is
normally
in
buffer
and CML
serum,
in the 1 M NaCl
bound
and
1 M
to
an
NaCl
elution
lost
Visking
an
and
per
from
normal
CML
serum
the
B,2 activity
originating
eluates.
This
is the
alpha,
globulin
eluates
of
hepatitis
in
serum,
mean
As
cent);
of
normal
GJ,
Passage
equal
and
of activity
SALIVA
patterns
casing.
in
alpha-globulin
Mean
loss
cent)
and
PA GJ
(18.1
from saliva
(35.0 per cent).
With
normal
found
only
B,2
serum.
binder
these
findings
suggest
that
retained
in random
manner.
occurred
was
and
OF NORMAL
radioactivity
through
predominantly
12345678910
12345678910
of
and
test
saliva
samples
decrease
serum
saliva
of
UBBC
radioconsists
of alpha-globulin
beta-globulin
was similar
greatest
from
expected
serum.2’8
The
serum,
however,
bind-
B,2
from
loss
of
endogenous
finding,
presence
suggests
binders
normal
binder
B,2
as B,2
of
B12
that
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
514
IIETIEF
endogenous
B,2
globulin.
The
with
liver
atypical
It
is
in this
clear
why
passage
B,2
from
cludes
the
possil)ility
saturate
the
binders
in
GJ22
“protection”
and
changed
cluding
by one
exposure
The
ferential
diagnosis
as
and
gen
in
the
Previous
labeled
type
in
CML
baby
an
is
in
the
in
that
B,2
B,2
binders
with
B,2
it allows
of
rapid
distribution
of
myeloid
metaplasia.#{176}
of alpha-
anti-hemophilic
a similar
knowledge
may
not
have
been
chromatography,
routine
B,2
when
“protect”
but
of
that
UBBC
degradation,
cellulose
might
may
to our
the
pre-
cellulose
decrease
known
enzymatic
protein
of demontubes7’8
B,2
apparent
It
separation
one
“R”
by
by
source
of
in-
evaluation
binders
as
It
also
may
from
beta-globulins
globulin
from
and
of
an
aid
large
dif-
in
prove
useful
may
be
clottable
almost
and
since
B,,
Since
of
fibrino-
beta
evidence
relatively
would
in the
globulin
as well
greater
in leukocytosis
not due to chronic
rnyeloid
leukemia;
the myeloid
leukemia
granulocyte
gives
rise
to
globulin
do
B1.
binding
beta
globulin
than
the
supplied
that
fraction
B,,
binding
appears
reduced
syndrome
all
(
are
as of B,, binding
alpha
in B,, binding
beta than
rise
the
of
globulin
2)
granulocytes
that
less
beta
leukemia
and
of Di Guglielmo
that
that
protein,
R-binder,
( Table
found
suggest
suggests
binding
labeled
exclusively
it was
elevated
in chronic
myeloid
as well as in three
cases
we
Weber,
vitamin
Simons.
to be
system,
of B1, binding
is the
Simons
serum
Gr#{228}sbeck and
leukopenia),
and
binding
absence
has
summarized
are
column
had
The
.
globulin.24
beta
globulin
is also
in chronic
leukopenia
of whom
suggested
B,2
)
7
cellulose
vs.
Gr#{228}sbeck, appears
Dr.
in our
“protects”
( Fig.
“cold”
against
for
rapid
work,
granulocytes
B,2
of the conditions
of
pH and ionic strength.
separation
gamma
patients
reticulocytes,
by
specimens
where
from
of binder
reactivity
of
alphai-
and
serum
alpha-globulin-containing
produce
milk23
a gap
serum
in situations
value,
sow’s
fills
of
the
radio-B,2.
or more
to varying
technic
numbers
thus
retention
The
with
contaminating
with
against
described.
been
by
and
that
to
column
except
that
tested
beta-globulin
al.2’
inefficiently
cellulose
eluates
binder
subsequently
B,2
et
AL.
condition.
the
all
to both
Herbert
presaturation
through
strable
bound
was
of
transferred
binding
not
specimen
finding
disease,
B,2
during
in this
previous
liT
one
globulin.
in alpha
this finding
more
B1,
normal
source
Further
globulin
also suggests
binding
alpha
granulocytes.
SUMMARY
A method
main
into
of
vitamin
two
B,2
distinct
containing
pernicious
in
fractions,
(IF)
one
almost
gastric
containing
in less
beta-globulin
(%
beta;
binder
exclusively
(as
and
of non-IF
as
and
described
saliva
alpha-globulin
than
Chromatography
material,
well
is
juice
B,2 binders
% alpha),
sera
are presented.
peaks
of B,2-binding
B,2
chromatography
serum,
binder,
and
anemia
(#{190}alpha)
duced
two
consisting
cellulose
binders
beta-globulin
of alpha-globulin
factor
DEAE
some
B,2
2 hours.
from
one
myeloid
of normal
containing
non-IF
binders.
binder
Typical
normal
chronic
whereby
may
binder),
Pernicious
be
and
elution
(%
the
separated
the
other
patterns
beta;
Y alpha),
leukemia
(CML)
gastric
juice
proall the
intrinsic
and
anemia
the
other
gastric
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SEPARATION
juice
OF
also
VITAMIN
produced
of B,2-hinding
normal
B1 2-BINDINC
two
peaks,
material
gastric
l)ut
which
juices;
1)0th
IF
seemed
of these
Es
describite
un
le
ligatores
methodo
(lual
to
de
vitamina
typic
alpha
e a globulina
jectos
con
leucemia
myeloide
produceva
duo
intrinsec
de
etiam
un
Ic
altere
qUC
vitamina
con
B,2
ligar
de
certe
non
del
absente.
Saliva
vitamina
intrinsec
etiam
B12.
in normal
pareva
succos
gastric.
de
un
sol
hinder
of
(beta
%),
a
B,2
%;
alpha
del
Le
duo
succo
gastric
B,2. Un
do
do factor
factor
intrinsec,
sed
culmine
corresponder
de
al
de
gastric
culmines,
ligatores
ab
patientes
factor
iiitrinscc
material
ligator
Sill)-
succo
exclusivemente
intrinsec.
ab
chronic
typo
quo
globulina
con
vitamina
altere
pote
1/5),
normal
B,2
major
contine
presentate
a
subjectos
ligar
ligator
le
separation
fractiones
beta.
Es
e ab
quasi
in
Le
de
major
diethylainino-
vitamina
vitamina
de
produceva
habeva
JIb
de
alpha
consisteva
de factor
typo
e saliva.
capace
quantitate
peak
fraction.
cellulosa
Un del resultante
ligator
globulina
ligator
culmine
maui
non-IF
fractiones
chromatographia
material
del
perniciose
anemia
esseva
#{190}).Le
a
distincte
normal
%;
the
one
alpha-globulin
gastric,
subjectos
totalitate
to
the
ligatores
(beta
(alpha
Ic
e
durante
ab
perniciose
culmines
contincva
duo
dcl
had
INTEBLINCUA
succo
elution
beta
anemia
Saliva
correspond
with
que
2 horas.
Ic altere
le
do
configurationes
illos
in sero,
minus
alpha,
abseiit.
chromatographia
separar
B,2
esser
compute
in
le ligator
globulina
IN
do
permitte
was
eluted
SuIIA1uo
ethanolic
515
PROTEINS
capace
altere
a
quo
factor
John
Farrell
ACKNOWLEDGMENTS
The
for
art’ indebted
authors
in this
assistance
to
Misses
Le
Teng
Co and
Nlelodv
Lee
Mr.
and
studs’.
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1967 29: 501-516
Separation of Vitamin B12-Binding Proteins of Serum, Gastric Juice and
Saliva by Rapid DEAE Cellulose Chromatography
FRANCOIS P. RETIEF, CHESTER W. GOTTLIEB, SHAUL KOCHWA, PETER W. PRATT and VICTOR
HERBERT
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