From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
The
Amino
With
T
By
Mioz
the
technical
HE AMINO
of sustained
taken
on
increasing
With
types
nature
the
impetus
which
Although
one
was
C.
JEVONS
of E. Battle
LLU,
and
M.
since
the
of the
different
300
provided
by
classical
investigations
of tile
laboratory
has
tryptic
been
this
fascinating
of
observation,
in order
resulting
technic
of
investigations,
variations
peptides
interested
by
in
Ingram1’m
various
in the basic
Ingram
the
1-1gb
to
many
other
to determine
the
from
the
genetic
column
been
the
have
utilized
technic4’5”
approach
chromatography.8’9’1#{176}
genetic
tile
has
groups
fingerprint
biochemistry
the
other
and
Since
of the
it seemed
worthwhile
to review
in some
detail
by us, particularly
as they differ
in several
significant
by
subject
have
odd
amino
acids
constituting
in Hgb
S when
contrasted
have
come
under
scrutiny
the
amino
acid
abnormality
in these
including
separation
employed
Bond
of human
hemoglobin
has been
the
course
of many
years.
Such
studies
the
“fingerprinting”
employed
of study,
globins,
in use
AND
assistance
exists.
the
usually
methods
CHERNOFF
significance
that
only
molecule
of hemoglobin
and
site
of
aberration
1.
acid composition
interest
over
who
discovered
hemoglobin
half
A.”2
Acid Composition
of Hemoglobin.
II. Analytical
Technics
our
human
technics
respects
hemo-
currently
from those
Ingram.
METHODS
Hemoglobin
lected
preparation.
in
any
of
erythrocytes
with
Except
when
globin
solutions
cent
of
70
volumes
against
and
two
dialysis
cent
The
Starch
of
dialysis
the
These
studies
70
water,
Memorial
were
Institutes
Aug.
of
10,
cent
was
by
as
Research
supported
of
1.5
one
3 ml.
Center,
by
carbon
prepared
method
to
which
as
of
or
of
a CO
University
U. S. Public
step.
the
of
Tennessee,
Health
Service
accepted
for
publicaion
54
Sept.
of
the
at
per
least
discarded
hemoglobin
with
dialyzed
70
free
per
of
lyophilization.
co-workers14
was
fractions
soluition
could
Knoxville,
Grant
29,
C.)
62
dialysis
washed
Health.
1960;
of
svas
hemoglobin
hemoglobin
(4
Following
and
by
and
of
ratio
precipitate
most
hemo-
against
Cold
of
the
monoxide
Kunkle
more
all
fractionation
was
a powder
F,
manipuilation.
concentrations
time
the
previouisly.”
Hgb
soluition.
of
col-
washing
further
using
precipitate
with
the
room
amount
at
isolate
but
to
blood
of
described
to
hemoglobin
small
venous
purification
water
a cold
sulfate
when
to
out,
initial
hemoglobin
then
as
prior
an
in
the
retreated
mixtures
much
out
The
water
to be used
distilled
volume
per
hemoglobin
As
cold
sulfate,
mass.
electrophoresis
study.
From
Submitted
in
one
cent
method
carried
as
carried
from
uisuial
and
was
rarely
with
prepared
the
compound
employed
to
per
against
hemoglobin
for
National
fluid
62
toluene
CO
was
was
amorphous
puirified
the
diluted
Dialysis
of
an
block
separate
quired
cent.
dissolved
sulfate,
sulfate.
to
per
with
by
procedure’2
to
frequently
was
were
anticoagulant
lysis
hemoglobin
was
)2S04
as
and
converted
continumed
precipitated
of
denaturation
the
changes
soluitions
types
saline
solutions
(NH4
and
ten
normal
were
)2S04
saturated
Hemoglobin
several
the alkali
Crystallization
(NH4
the
1960.
No.
be
Term.
A2.956
employed
was
re-
separated
from
the
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
AMINO
ACID
COMPOSITION
OF
HEMOGLOBIN,
ANALYTICAL
55
TECHNICS
a
0
E
Ui
a:
:D
.300
45OmgmGLOBINA
25 ml FRACTIONS
-
/
-
I \
-
.200
)
___
___
AO0
___
ii
I
‘0
20
40
\
60
80
SEPARATION
of
the
on
each
1.-Separation
Also depicted
manipulations
block
block
with
Hgb
F
100
420
before
of
dimensions
water,
purified
Globin
Clobin
An
Teale16
to
buit,
separate
200
OF
Hgb
GLOBIN
A
the interrupted
molarity
during
A
by
of urea
the
gradient
course
x 0.7
The
cm.
of buffer,
modified
isolated
fractions
retreated
alkali
with
denaturation
hemoglobin
was
were
CO
technic
from
to
the
lyophilized.
previously
converted
also
eluted
and
described.12
the
CO
compound
was
prepared
attempt
in
the
was
addition
to
material
by
made
to
the
into
the
more
a
method
separate
Anson
heme
laborious
and
of
the
by
procedure
chains
proved
and
the
Mirsky’4
involved,
frumitless
as
methylethylketone
suibsequent
and
this
approach
abandoned.
Alpha
and
beta
modification
this
series.15
ment
of low
resin
in
its
varying
the
In
brief,
pH
and
of
detector
of
interest
were
of
umrea
carried
by
separates
strength.
by
2
$
‘,
chains
into
its
8.
the
By
of
of
as
column
nii.
dialyzed
repeatedly
against
diacetyl
monoxime
method.’8
out
with
an
is
F occurs.
(280
the
a
urea
was
The
water
Lyophilization
by
an
a
in
environType
of
1.9
pH
gradient,
almost
less
continuously
4 C.
by
CG-50,
somewhat
fractions
at
in
solution
while
out
publication
chains
interrupted
1).
carried
first
Amnberlite
efflument
(fig.
distilled
on
possible
The
)*
in
polypeptide
is carried
employing
were
described
component
hemoglobin
Hgb
system
separations
chain
Smith’7
from
chains
recording
the
to
and
and
Separation
elution
from
and
a and
Alpha
\Vilson
2,
and
total
distinct
monitored
containing
until
shown
freeze
drying
material
to
was
be
free
then
out.
Try ptic
were
globin
a
the
a UV
of
ionic
phase
the
by
separation.
technic
molarity
of
separation
chain
of
acid
in
separation
digestion.
subjected
trypsin
be
I
180
lyophilization.
described.15
of
attempts
I
160
text.
free
fetal
CHAINS
of
curve
the
x 35
the
the
preparatwn.
previously
method
and
in
25
by
,6 chains
approximate
dialyzed
denaturation,
dialysis
and
140
NUMBER
a AND 3
OF
a
described
distilled
was
Following
was
of the
is the
IRC-5OCOLUMN;Interruptsd
Urea Gradient
‘
FRACTION
Fig.
method.
4.9pH
carried
is
Preparations
to
incapable
out
#{176}Gilson Medical
identical
of
as
exerting
a preliminary
Electronics
of
hemoglobin,
procedures
its
for
digestive
step.
Company,
globin
action
Both
or
obtaining
heat
Middleton,
the
on
and
native
alkali
\Vis.
the
individual
tryptic
proteins,
denaturation
peptides.
peptide
chains
Inasmuch
denaturation
procedures
as
must
have
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
56
CHERNOFF
been
with
employed
our
activities
protein
to
are
The
or
solution
prepared
tap
a 2
and
carbonate.
mg.
ml.
per
a drop
as
use
an
inert
three
takes
mnay
hours
at
place
in
go
bly
indicator
into
of
two
by
is
was
made
to
determining
Such
at
which
the
of
temperature
enzyme
acts.
)9CO3
buffer
and
to
an assumed
tryptic
per
cent
of the
hemoglobin
constitutes
the “resistant
core”9),
tion of 1 mg. of tryptic
peptides
carefully
. carried
out to ensure
removed
by centrifugation
and
chromatographic
From
or
globin
sufficient
per
30
spots
in
to
by
14
are
the
by
of
of
to
saturated
with
the
into
which
dip
into
of
yellow
tempera-
tryptic
conditions
period
37
C.
No
digestion
of a pH
found
essentially
a
period
all
the
dried
to
stat
of
90
sites
at
remnove
the
peptides.
cent
of
higher
of
starting
lambda
or
result
freeze
weight
of
the
of the molecule
immnumne
water
solution
to the
is added
solution.
the
action
of trypsin
and
to yield a concentramixing
The
red)
hemoglobin
( approximately
Adequate
peptides.
(phenol
of
itself
are
respective
No.
must
insoluble
simpernatant
the
be
core
is
in
while
settling
may
five
an
out
tanks
stainless
areas.
steel
separatory
(about
two
remain.
the
aqueous
at
90
in
large
contain
cooling
papers
to
in
10
is
utiliied
for
C.
the
plastic
with
coil.
an
area
spotted
and
in
spot
organic
Nichrome-platinum
with
ml.,
are
atmosphere
descending
minutes.
described
of
the
the
papers
an
30
as
Fresh
filtering
Following
volumes
are moistened
the
discarding
approximately
inert
of
peptide
cabinets
tanks
of
The
hours.
22
houmrs)
unequmal
corner
in n-buitanol:acetic
for
phase.
for
one
A aliquots.
funnel,
The
in
is filled
The dried
a
hours
oven
the remainder
a large
buffer
These
applied
paper
cabinets
in
or its expendable
2).
is
filter
chromatography
to
dried
solution
MM
ingredients
is carried
(fig.
of
3
between
applications
to keep
Descending
chromatography
standard
three
papers
baffle,
center
1#{189}mug.)
period
of
water
which
solvent
is immersed
the
in
from
Anfinsen2#{176}
air
mixing
Electrophoresis
and
the
out
by
droplets
the
cold
suitable
a
any
El.ectrophoresis.
side of
per
Whatman
in diameter.
carried
equilibrate
chromatography,
Dreyer,
of
daily
after
free
permitted
of
1#{189}cm.
is
prepared
phase
solvent
of 80
50
1 to
sheet
a stream
1 to
(4:1:5)
is
aqueous
to
clear
A (approximately
#{189}
inch
dried
H20
solvent
60
22
range
acid:
known
remains
dc-ionized
maximnal
the
resuilting
the
)
purposes.
40
an 18
of
js
preparations
and
of
mixtuire
the
basis
somne
24
who
for
precipitate
between
under
liberation
the
a minimum
Ingram
) added
digestion
is probaoumt studies
which
houmrs
a
8.2
proceed
provided
as
utilized
the
digestion
20
bonds
the
concentrate
the
24
tempemature
oumt by
for
to
resulted
peptide
of
althoumgh
We
to
to
EtOH
to
Enzymatic
have carried
( up
any
digestion,
On
minutes
hate
carried
Following
30
cent
permitted
give
free)
is adjusted
per
Dissolution
hydroxide
been
stmflicient
technics.
and
sodium
have
20
to
qumantity
reactants.
of minutes.
added
sufficient
in
of
the
confined
salt
is
“fingerprints”
of
)2CO3
mixture
( in
red
have
crystallized,
Digestion
digestion
liberation
added
reaction
temnperature.
at
NH4
2x
is
the
phenol
of
of
(
NI
steps.
matter
digestion
quantity
cent
at this
with
0.6
we
LIU
50 to 100 mg. of the
10 mg. per ml. of water.
shaking,
cooled
to room
of
HC1,
of
period
a
periods
the
stumdies
“Fingerprinting”
or globin
or less
longer
for
room
( NH4
volatile
a
of
N
pH
shaking
within
follow
the
experimnent.
per
within
permitted
10
of 0.1
). Comparable
C.
in
simpler,
From
( Worthington,
subsequent
completely
to
volummue
1. The
occasional
is
a concentration
Trypsin
50:
in
in 90 minutes
38
of
instances
advantage
to
minutes
with
most
hours
attempt
C.,
solution
no
( up
tures
for
32
complete
indicate
ratio
former
instances.
4 to 6 minutes
half
solution
a protein
to enzyme
to 8.4 with 3N HC1 and
at
C. for
one
the
special
water
to 99
M
yield
in
in
96
water
0.2
of
as
at
Since
except
suspended
is heated
umnder
final
results.
procedure
dissolved
material
temperatumre
a
comparable
this
AND
buffer
solvent,
by
Katz,
on
each
Varsol,#{176}
electrodes
the electrophoretic
10 ml.;
glacial
acetic
acid
100
ml.; H.0, 2890
pH
3.7)
in such
a
the peptides
are washed
into a sharp
line
by the
advancing
buffer
fronts.
A
convenient
method is pictured
in figures
3 and 4. A small
qumantity of buffer
fills the shallow
pan
into which is placed a plastic
rack containing
three
movable
glass rods.
The paper
is
draped
over
the
center
rod so that the original
point
of the peptide
application
and
the
phenol
red indicator
spot
are
directly
over this rod. The remaining
two rods hold the
buffer
fashion
(pyridine,
that
#{176}StandardOil
Company
of
New
Jersey.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
AMINO
ACID
COMPOSITION
OF
Fig. 2.-Electrophoretic
are the stainless
steel
Varsol
and the plastic
paper
in
washed
the
into
is blotted
make
the
amps
solution.
a
(fig.
per
paper
draped
the
5).
buffer
which
over
buffer
60
fronts
in
along
subsequent
a plastic
while
minutes
advance’
facilitates
Electrophoresis
for
ANALYTICAL
57
TECHNICS
tanks
utilized
for high
voltage
electrophoresis.
coils, unequal
layers
of buffer,
the overlying
holding
the peptide
papers.
the
zone
and
with
buffer
As
narrow
lightly
contact
cooling
frames
HEMOGLOBIN,
rack
so
the
chromatographed
is carried
out
an
“uphill
at
mi sharp
separation.
as
to
permit
the
line
2000
fashion
of
volts
in
and
which
PAPER
the
GLASS
Visible
layer of
line,
the
The
moistened
emids
peptides
of
pe’ptides
the
is
shallow
papcr
kept
approximately
to
out
90
buffer
are
p::per
millisection
RODS
_______________________
GLASS
METHOD
into
Fig. 3.-Schematic
a sharp
line.
PLATE
OF
BUFFER
WETTING
diagramu
PAPER
of method
FOR
of “washing”
SOLUTION
ELECTROPHORESIS
chromatographed
peptide
of
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
58
CHERNOFF
AND
LIU
I
ILL
r---
-___#{248}-
1-
Fig.
serves
4.-Method
as aimode,
clri(’d
are
tle
tn
ill
first,
the
I)cPti(les
dure,
in
described
the
peptides
0.02-5
cent
The
and
reagent
omit
act
hanging
covere(l
chamber,
conteiit
of
the
hydrolysis
Amino
and,
paper
acid
over
after
so
of
the
paper.
out
analysis.
thorough
1814
such
in
the
that
the
HCI.
Ten
the
paper
The
20
The
peptide
in
mixing,
applied
by
as
off.
Elimtion
partially
off
75
15
the
diluted
in their
22#{189}
the
the
I)aI)ers
e’lectroplmore’tic
that
reverse
pepticles
s(’vcral
phase
are
identical
of
proce-
inferi:r
with
to
those
to
to
those
already
inches.
at
opened
with
entirety
Two
the
caught
tubes
are
with
the
20
minutes
tube
to
the
6
set
N
areas
“cut
chamber
(fig.
rinsed
two
of
pepti(le
HC1
6).
lightly
acid
in
cleaned
between
thc
slides
beneath
the
freely
at
a single
when
carried
remove
elute
with
the
of
tweezers,
between
being
to
stained
in a plastic’
with
covered
placed
in a high
108
end
out
filled
used
lightly
below),
one
is carried
sufficient
sealed
are
technics
have
is
6 N)
papers
test
being
oven
of
the
are
end
nun,
contact
are
vacuo,
tI
found
(essentially
with teflon
and carefully
drops
an
by
staining
troumgh
within
contents
in
(see
by
tubes
hours
hydroxide
inches
HC1
The
makes
four
elution
for
boiling
squared
occurs
use.
mnanipumlation.
handled
ninhydrin
half,
pepticle
of
details
run
have
separated
a manner
squiared
approximately
sodium
measuring
for
tips
carried
of
to
We
majority
ethanol
in
carefully
excess
emit
a siphon
Ehition
order
chromatography
are
the
actual
in
electrophoresis,
advantageous
Technical
“fingerprints”.
end
F’ollowing
cleanly
for
absolumte
other
slides
as
more
line
ninimtes,
l)een
constant
paper
to remove
pointed
rim.
dryness
the
microscope
which
the
1)eptides
acetone
glass
the
in
inclined
an
emit
on
from
pointe(l
containing
in
distilled,
30
a sharp
chromatography.
above.
change
for
has
ol)tained
ninhydrin
removed
omit”
the
Glass
it
are
described
identified
per
interest
the
for
times,
into
e’atlIO(le.-’1
C.
descending
separations
elution.
as
9()
hemoglobin
procedumre
except
Peptide
section,
At
by
of
the
the
buiffer
peptides
approxinlately
followed
although
found
at
proce(lure.
separation
tryptic
(let’1)
oven
p/lose
Reverse
of “washing”
the
temperature
point
inside
omit
entire
flame
in
the
pepticle
md
acid
C.
hydrolysis
a small
tubes
drop
of
are
triple
to sheets
of Whatman
samples
may be applied
evaporated
distilled
to
water
No. I filter
to one sheet
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ANIIN()
A(
(:o\u’osrrR)N
11)
OF
1IE1O(;LOBIN,
ANALY’FI(;AL
‘#__11
‘-----
Fig.
(if
5.-Plastic
by
I)1t1)tr
immaterial
one
placed
to
Descendimig
acetic
acid,
water
tle
which
of
30
the
the
minutes,
jars.
The
then
is
be
Staining
ethanol.
The
B.
papers
10
minutes
for
full
EtOH
tu)ml
is stable’
room
to
for
utes.
The
pear
with
in the peptide
stain
A.
chief
two
The
papers
advantage
spots
D,
Ninhmvdrin-colhidine
dissolved
in 300
ml.
standard
of
three
that
per
at
the
the
solvc’nt
place
for
are
to
cent
ascending
need
or
air.
bimtanol.
miot
one
b
hour,
the
short
heated
to
90
chromatography
pyridine:
rooni
same
dried
ap-
heated
along
air
tlmt
1))
about
chromatography
pipers
coiihne
diauu’tt’r
by
using
take
subjected
hours
to
in
applications
except
The
80
takeim
umin,
cabinets
to
electrophoresis.
is
five
between
permitted
and
care
thami
descending
line
20
temperature.
per
cent
The
air
imi
water
dined
and
papers
identification.
for
peptides:
temperature,
for
as
for
added
imp to
temperature.
in
hours.
voltage
0.5
this
1 0 minutes
for
0.025
a spray.
full
per
The
Per
ninhydrin
cent
is
preparation
color
c itt
sprayed
iisuail
in
iis(’d
al)sohlte
ac
a
dip.
(levelopmemlt.
nmnimydrin
papers
in
are
absolute
heated
ethanol.
to
90
C.
for
development.
stain
are
C.
mainly
color
which
spots
(‘liltion of peptidcs:
stain
Ninhydrin-collidine
lute
the
and
18
high
Etrcmmme
above,
stain
room
to 90
for
this
for
acid
at
line.
is
for
Ninhydrin
heate(l
stain
applied
out
center
out
during
mm greater
drying
consists
amino
A.
are
Ninhvdrin
have
the
stable
area
added
out
system
technics.2’
\Ve
C.
along
for
Moderat(’ly
wet
is
carried
stained
center
described
carried
solvent
a
an
Equilibration
solvent
cut
papers
carried
system
day.
paper
to
and
is
solvent
chromatography
may
a time
at
same
very
dimension
covered
paiX’r
chromatography
up
for
A
ai)out
the
in
two
following
C.
for holding
oriciitation
proper
being
plying
made
frames
59
I’ECHNICS
weeks
are
of
this
permitting
stain
absolute
peptides:
4.0 nil.
for
EtOH,
0.5
Cm.
collidine
if
kept
under
dipped
stain
in
lies
in
amino
100
in
sonic
acids:
ml.
ninhydnin
and
100
glacial
refrigeration
this
solution
the
instances
The
glacial
hut
and
fact
that
acetic
(Iried
at
90
consists
acid
and
C.
of
40
than
0.3
ml,
Ciii.
of
ml.
abso-
This
rolu-
rapidly
at
10
mimi-
colors
ap-
for
of
variety
identific’atiomi
solution
3(X)
acid.
deteriorates
a greater
easier
acetic
in
(liSsOlVe(l
ml.
is
possible
ninhvdrirm
collidine.
As
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
60
CHERNOFF
AND
LIU
/
“7
:
Fig.
details
I
V.
6.-Elution
of arrangement.
chamber
with
stain
C above,
ates
rapidly
at
at
80
to 90
E.
Pauly’s
stable
10
stain
as
orange
a
for
histidine
at
room
color
F.
Pauly’s
Solution
ml.
with
aqueous
histidinc’
the
orange
either
hue
B or
C.
a
cent
been
removed
we’ks
in
are
The
equal
is
to
cold
tile
hih)eraily
bring
but
sprayed
almost
tyrosine
of
slightly
damp,
is
still
(Tyrosine
stain
out
deterior-
and
dirndl
in
cent
volumes
of
dry,
immediately
he
well
quickly.
Stain
for
tyrosine
full
E
as
an
by
the
II
may
and
in!,
II
within
10
overstain
used
HO.
ml.)
is
111.
An
niinutes,
after
following
in
B or
I).
procedure).
made
up
111-10
as
a
oramige’
within
he
with
solution
apply
are
Ciii,
(10
with
The
imitcnsity
also
100
Solution
III.
and
II-0.9
and
HCI
water.
solution
reaching
)
concentrated
I and
with
to
ml.
intensity
used
in
solution
up
(50
spray
stained
S nil,
NaNO.
spray
1
prepared
( a(jiieOus).
made’
full
may
is less
dissolved
per
and
reaching
This
are
NaNO,.
HC!
acid
fades
solutions
cent
mixture
immediately,
nearly
following
per
prepared
11-0.69
Mix
of
two
to
p:p’’
concentrated
tyrosine.
histidine:
paper
appears
has
imp
acid
1-5
paper
sulfanilic
Solution
\Vhen
iii!.
the
and
for
Na.,C03.
spray.
for
amino
tyrosine:
freshly
almost
histidine
Cm.
H20.
9
A
While
stain
1-0.8
amid
in
develops
of
stal)ie
The
temperature’.
Nt,,CO:1.
spray.
presence
Cover
minutes.
elissolved
cent
i”
applied
light
for
acid
111-5
per
C.
is
pre’I)aration
tc’mpcrature,
indefinitely
sulfanilic
the
this
room
of plexiglass.
to
per
fairly
color
10
minutes.
as an
overstain
1(X)
cent
heavy
due
to
The
after
D.
Nitroso
a nitroso
$
naphthol
,6 naphthol
stain
in
acetone.
Solution
and
11-10
tryptophane22’23:
per
cent
Solution
HNO3
in
acetone.
1-0.1
Dip
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
AMINO
the
ACID
COMPOSITION
paper
solution
dip
II
as
pale
in
solution
I
and
heated
for
smooth
as
green
brown
after
The
per
Mix
equal
with
a
smooth
peak
Sakaguchi’s
I,
even
its
care
should
Sakaguchi
be
I. Ehrlich’s
I, II
and
are
in
for
The
pink
5
as
that
only
the
a few
The
overstain
after
B or
be
stain
for tryptophane
90 ml.
acetone
in
chromatographic
papers
immediately
minuites
and
after
may
after
reaches
: 24
either
to
Gm.
which
be
sprayed
preparation.
its
The
at least
two
major
maximum
intensity
color
against
yellow
as
I. When
dry
in
water.
in
absolute
into
within
fades
a
to
an
over-
a
as
before
Solution
soluition
few
a
an
EtOH.
the
within
used
minutes
few
hours.
overstain
proceeding
after
with
the
or
dipped
color
of
tryptophane
ten
concentrated
in
minutes.
this
HCI.
solution.
appears
May
be
The
Must
be
within
umsed
as
five
an
over-
Sharper,
DISCUSSION
approach
described
from
separation
precedes
those
first,
used
by Ingram
and
this communication
in
previously
a
reported.
system
of
cleaner
the
Many
heavy
bulk of the
of the
technical
touched
without
upon
saying
in the previous
that
compounds
employed
in
denaturation
such
may
a source
of
digestion,
as for
of denaturation
or urea.
The
use
since
less
separations.
result.
Nevertheless,
tion
of
peptide
globin
structure.
into
the
hemoglobin
and
are
found
chains.
of
we
rather
latter
beta,
peptides
positions
to be
seen
pitfalls
altered
to
be
certain
at pH
have
hemoglobin
chains
is based
of
somewhat
has
heme
preparation
It goes
must
Of
to keep
the
in fingerprints
many
than
been
found
to be
occasionally
in mind
of
the
rather
is quite
may
induce
on observations
some
not be
tryptic
heat
chain
preparations
eliminated
and
several
been
here.
possible
enzymes.
to use
associated
either
already
That
other
electroelimin-
6.5.20
need
step
for
than
or gamma
are almost
for
and
meaningful.
preferred
the
resolving
purification
is a necessary
have
it is important
component
conclusion
chromatographic
beta
results
denaturation
However,
patterns
its
This
the
alpha,
higher
a more
acidic
pH
of the peptides
peptides
and
in
the
power,
electrophoresis.
been
achieved.
Further-
of hemoglobin
action
of globin
with
The use of individual
sirable
since
overlapping
which
as
the
the
process
available,
advantageous
clear-cut
for
in the
inasmuch
well
neutral
problems
co-workers,
differ
performing
section
and need
not be repeated
of the highest
degree
of purity
studies
occur
concern
as
technics
so called
details,
By
somewhat
the system
of slightly
lower
resolving
peptide
separations
have,
as a result,
more,
the methods
adopted
in this study
employ
phoresis
which
leads
to more
complete
separation
alkali
( p-dimethylaminobenzaldeml.
with
within
AND
the technical
the methods
respects
chromatographic
power
alpha
the
B.
Though
similar
to
as well
as by others,
be
in
make
dirty
used
is dipped
color
Ehrlich’s
reagent
are
added
10
blue
RESULTS
ates
naphthol
paper
completely
be
diacetyl
a
appears
minutes.
a
May
cent
arginine
10
paper
pink
to
collidine.
cent
The
taken
of
hours.
or
per
given.
of
resulting
appearance
per
color
to
an
order
be
dipped
then
is
should
the
of phenol
I-.0.1
paper
Care
the
111-2.5
the
The
C.
by
presence
Solution
III
90
causes
Solution
used
to
recognized
stable
the
within
taken
dissolved
stain
water.
be
temperatumre.
80
61
TECHNICS
stain.
hyde)
used
is
in
motion.
may
at
arginine:
in
intensity
stain
roam
tryptophane
used
for
of
at
colors
stain
ANALYTICAL
minutes
Tyrosine
be
KOH
volumes
reaching
dry
while
cannot
cent
HEMOGLOBIN,
three
resulting
B but
Sakagumchi’s
11-20
and
possible.
background
color.
stain
H.
OF
some
causes
satisfactory.
is highly
deless confusing
that
the
separa-
changes
in
to be reported
tryptic
of the
peptides
individual
the
in
of
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
62
Although
many
niolecule,
I1Ofl
medium,
it has
enzymes
1)ossible
chymotryptic
lysyl
Other
of
enzymes
( for
the
and
Scrupulous
graphic
“hardware”
fresh,
and
f)ermissible
important.
relative
to
the
acid
forms
of
the
as an initial
sequence
studies,
results
are
in
this
and
several
initial
of these
proteins.
deal
of information
and tertiary
structure
studies
of
Perutz,
Ingram
and
human
hemoglobins
hemoglobin,
composed
primarily
of four
identical
half
symmetrically
and
separated
to an
advanced
heme
coiled
are
oriented
by
#{176}Fluoro2:5
alpha
each
each
about
taking
dinitrobenzene.
and
two
advantage
the
of the
has
beta,
to
chains.27’28
with
been
shown
that
of
axes
the
certain
moiety,
protein
place,
two
in the hemoglobin
present.17’28’3435
These
different
of symmetry
phenomena
Itano,
mass
amino
of
conacids,
moderate
presumably
dissimilar
in
peptide
molecule
and that
four
polypeptide
and
referred
chemistry
globin
300
exist
are
the primary,
that the classic
conditions
takes
the
the
The
Under
molecule
be
of
reasonably
approximately
axis.
must
problem
of the
It seems
the
or
information.
Braunitzer,
attached
of
still
paper
technic25
of knowledge
stage.
only
procedures
preparation
to
Schroeder,
state
groups
a central
It
variety
Stein,
best
in terms
method
definitive
in
is
peptides.
at
quantitative
FDNB*
more
be
Nevertheless,
resulting
elegant
papers
polypeptide
dissociation
fashion.29’3032
chains,
designated
two
members
of
chains
must
latitude
unavoidably
information
inaccurate
peptide,
this
approach
our
of four
about
pH,
asymmetric
and
brought
molecules,
oriented
alkaline
the
has been
gathered
concerning
of hemoglobin.
Suffice
it to say
Moore
have
successful
systems
chromatographic
for
an
differences
Kendrew,
N-
chromato-
yields
as Sanger’s
the
A great
secondary
acid
of
obtained.
on
More
required
structural
others
aminoin both
handled,
be
somewhat
a given
such
obviously
as
many
be
colummn
therefore,
of
roles
solvent
can
problem.
methods
usmg
reported
and
may
comprising
primarily,
sists
mainstays
carefully
chromatography
quantitative
degredation,26
consists
the
for
arginyl
chymotrypsin
are
useful
studies
acid
regarded
that
than
) . Leucine
serve
cleaned,
peptides
to the
studies,
cry-
was found
to be
purified
further.
cleavage
of
be
definitive
amino
approach
chromatographic
of the
one
must
the
for
information
amino
acids
acid
studies.
rigorously
is essential
dimensional
semiquantitative
detecting
all
‘The
be
in
free,
checked
other
studies
specific
enzymes
papers
composition
analysis
two
Edman
other
details
must
bonds
to
be
equilibration
must
be complete.
Somewhat
greater
with the amino
acid analyses,
but here too, details
are
Through
the judicious
use of specific
stains,
mumch
Although
and
to
Thus,
less
Salt
should
in these
( for
group
8 to 8.5.
LIIT
hemoglobin
Stable
in these
studies
not have
to be
applicability
pepsin
range
lead
the
trypsin.
reagent
utilized
and did
and
attention
fingerprinting.
serves
ready
and
the
of
of
pH
may
material
COOH-terminal
peptide
amino
find
)
core
the
and
which
carboxypeptidase,
terminal
used
digestion
usefulness
in
contamination
which
resistant
peptidase,
be
The
such
for
and
activity
broken.
tree
available
activity
must
being
virtually
are
specificity
its maximum
preparations
till!
the
has
stallized
an
AND
CHERNOFF
may
to
be
above.
readily
End
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
AMINo
01” HEMOGLOBIN,
COMPOSITION
ACID
ANALYTICAL
63
TECHNICS
#{163}9
,..
!
1”
Fig. 7.-Peptide
11gb A.
(29
pattern
of
222r\//m\
BuOH
PEPTIDE
group
its
analysis
has
the
N-terminal
chains
been
shown
may
has
recently
l)e
chain
its
ferent
second
set of peptide
chains,
is a252.40
Abnormalities
of the various
a
$ chains
or
and
can
=
a2$2;
be
and
conveniently
including
A, S, F, C, D, E, and
respects
these
studies
are incomplete,
some
reported
of
in our
these
shortly
patterns
as
well
=
have
to
as
for
serve
as
Hgb
a
and
same
a
have
as
A2 has
at
It
a2y2.
yet
a dif-
and its formula
presumably
have
been
found
in either
by
a2$;
superscript
=
purposes
of types
for
with
as
$, etc.
No. 11142).
felt worthwhile
points
reference
letters
Hgb
H
at a number
R (Durham
it was
comparative
expressed
that
Hgb C =
been
directed
a2mfl2;
the
$, which
from
be
designated
hemoglobin
in some
scribe
Hgb
I
laboratory
different
may
designated
hemoglobins
Hgb
S
studies
14r
For
A consists
of two
or a2A132(. Hgb F shares
the
Ingram
follows:
Initial
No.
Hgb
formula
Stretton
peptide
val-his-leut.35
significantly
Hence,
by
to have
sequence
to as a a2$2
chains
reported
probably
N-terminal
referred
14,3,37-W
been
A
is va1-ku.34’
polypeptide
designated
has
beta
the
sequence
and
but
Hgb
-
the
end#{176} with
$ chains
two
a
H20
PATTERN
N-terminal
chain,
:HAc,
of
Although
to de-
studies
findings
to
in
be
other
laboratories.
Hgb
A (fig.
7):
#{176}The system
and
Ingrain
and
the
peptide
work.
of
4
difficulties
numbering
inasmuch
patterns
of
not
Peptide
not
encountered
referred
to
as different
are
Ingram.
Unfortumnately,
been
of the
peptide
co-workers
fingerprint
No.
Some
all
peptides
the
amino
entirely
26
in
in
mlleTthodls
in fingerprinting
this
paper
of
separation
comparable.
the
Peptiele
present
ide’ntifie’d
in
study
our
differs
have
No.
is
also
14
No.
is
26
respective
laboratories
paper
i.s consistent
studies
from
that
been
utilized
identical
in
of
with
Ingram’s
have
yet
correlated.
The
adopted
nomenclature
by
most
for
workers
in
this field.
acids
utilized
in
this
with
that
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
64
CHERNOFF
.
Table_1.-Peptides
Comprising
the
$
and
a
Chains
of Human
Peptide
No.
a Chain
Peptide
Chain
1
3
2
4$
4cr
7
5
AND
hemoglobin
A
No.
6
10
8
12
9
13
11
15
14
17
16
18
22
19
24
20
25
21
26
23
27
(28)
(29)
(30)
(31)
Tabe
2.-Peptid
es Reacting_with
Specific
Stains
for
Amino
Acids
Stains5
Peptidt
E
F
1
+
+
G
H
I
his
Acids
2
+
+
his
3
+
+
his
5
7
+
+
his
arg
+
arg
+
9
11
+
12
+
13
+
+
14
+
+
+
his,
+
+
+
his
+
+
17
+
+
±
18
+
+
+
21
+
+
+
try
+
his,
his,
+
his
+
arg
+
E,
for
identification
not included
fPeptides
illustrated
Overlapping
fail
in
of
to
react
stain.
with
any
of
the findings
pictured
peptides,
particularly
these
interpretation
found
in the
difficult.
Those
peptides
a or $ chains
are listed
peptides,
found
$ chain,
in
the
are
still
stains.
in the
11 and
and 21 make
studies
been
all
arg
arg
+
#{176}See
“Methods”
tyr,
his
try
+
26
+
tyr
his,tyr
24
C1
tyr
tyr,arg
his
15
16
are well
drawing.
Amino
indefinite
accompanying
12, 15 and
which
in table
and
16,
schematic
and 17,
18
have
in subsequent
1. Finally,
several
are
listed
in
par-
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AMINO
ACID
COMPOSITION
OF
HEMOGLOBIN,
ANALYTICAL
65
TECHNIS
Nc?c
I
$
(rnl)
-
w
qp
10
0
I
O
a:
H
I
/Th15
/17k
7(29,_\
(181
/\WJ
Fig.
L)
(21[31)
22
U
11gb
/\
‘.,-_,.“
8.-Peptide
pattern
of
C.
29
If;\
21
BUOH:HAc:H20
.
PEPT
IDE
PATTERN
-
Hgb
C
I
-
N
‘4,
z
0.
ci)
C,)
Ui
a:
0
I
aFig. 9.-Peptide
Hgb E.
pattern
0
a:
of
H
0
Ui
-J
Ui
21
PEPTIDE
entheses.
It is very
31 in the
$ chain
probable
is due
that
to some
the
cross
apparent
contamination
BuOHHAc:H20
PATTERN-Hgb
presence
with
E
of peptides
the
a
chain,
29,
30 and
inasmuch
as their
location
corresponds
to those
of peptide
17, 18 and 21 of the a chain,
while
their
intensity
on the fingerprints
is considerably
less
than
that
of
definite
members
of the $ chain.
Peptide
28 has been inconsistent
in appearance
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
(;HERNOFF
66
Table
3.-Amino
Acid
Sequence
in Peptides
Peptide
No.
No.
14
A
Val
-
his
-
leu
-
thr
glu
-
glu
-
Hgb
S
Val
-
his
-
leu
-
thr
-
pro
-
val
-
glu
-
11gb
C
Val
-
his
-
leu
-
thr
-
pro
-
lys
t glu
pro
-
Vat
asp
-
val
-
E
Val
asp
-
vat
-
No.
asp
-
ala
11gb
lys
lys
C2
Peptide
A
26
lys
-
C1
Hgb
LIU
14#{176}
Hgb
-
and
AND
glu
-
leu
-
(asp
-
gly
-
glum
-
26#{176}
val
-
-
gly
-
gly
-
-
glu-
arg
-
val
-
gly)
-
gly
-
lys
I
E1
ala
leu
-
gly
-
arg
-
E9
tAdditional
sites at which
#{176}From data
presented
and
requires
further
table
2.
Hgb
C (fig.
4), presumably
fragments
has
3).
E (fig.
been
lysine
replaced
only
by two
in the
place
A44
(tables
of Hgb
to the
are
stains
are
listed
in
(
of a lysine
area
in table
change
E1 and
acid
specific
peptide
No. 14
Ingram’s
$ chain,
is replaced
by
the
this
listed
fragments,
2 and
to
introduction
discernible
of glutamic
peptides.
reacting
to split
stains
The
respective
43-45).
by Ingram,41
peptide
of
trypsin
Specific
9):
the
(5,
Peptides
C2 due
permitting
(table
breaks
literature
8) : As reported
the N-terminal
acid,
Hgb
trypsin
the
study.
C1 and
glutamic
locus
in
of the
residue
chain
place
of
additional
2.
relates
to peptide
E2, because
present
in
at an
No.
two
in the
No.
of the
same
26 which
introduction
position
of
of peptide
26
3).
Hgb S (fig.
10):
No additional
fragments
are found
in Hgb S, but a shift
peptide
No. 14 has occurred,
due,
as Ingram2’’
and
11111 and
Schwartz5
have
described,
to the replacement
of one of the glutamic
acid
residues
valine
in this
In order
portion
to clarify
E, fingerprints
Results
of
amino
acid
disease
(Durham
tion
have
$ chain
differences
abnormality
D
reported46)
No.
1)
in peptide
48)
in the
that
However,
Da
and
studies
changes
previous
data.
In
the tryptic
peptides,
previously
completed.
reported
peptide
in
No.
a chain
Hgb
our
the
appears
Stanleyville
of
(from
to
of Hgbs
A, S, C and
in figure
incomplete
patient
homozygous
be
in
of a sample
in either
peptide
with
peptide
No.
presumed
17
22;
of
to be
or 18. This
11.
hut
D
Hgb
R
Hgb
latter
the
Hgb
Stan-
observa-
Hgb
Da
(49, 50) has
18 (No.
23 of Ingram).
also been
reported
to
It is tempting
to con-
I are,
the
F have
technics
these
patterns
are summarized
are as yet
14 and
in fact,
been
have
findings
one
reported
and
in an
same
earlier
certain
of a uniform
errors
in the
numbering
amino
acid
will
be
the
subject
hemoglobin.
publication.29
necessitated
addition
to the adoption
we have noted
minor
and
by
3).
in the
of 11gb
I (47,
Preliminary
(table
isolated
$ chain
of each
with
other
hemoglohins
is of some
interest
in that
its abnormality
in peptide
sider
be
the
of the
studies
previously
leyville
of the
of
revisions
in
our
system
composition
for
of a report
soon
to
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
AMINO
ACID
COMPOSITION
OF
HEMOGLOBIN,
ANALYTICAL
TECHNICS
67
23
Cr
(.(j12J
Lii
I
a-
Iiil
Io
(181 1t191
l13o,/
of
Fig.
Hgb
10.-Peptide
S.
pattern
2226q(\
2b
BuOH.HAc’H20
PEPTIDE
PATTERN
-
Hgb
S
PEPTIDE
ABNORMALITIES
GLOBINS
Fig.
11.-Peptide
of $ chains
of
CandE.
A,
C,
S,
AND
/3
CHAIN
E
#{174}(4P
pattern
Hgbs
IN
5,
)
#{128}
c
l’
,-‘,
‘?
“()C
,/‘
3O
r\
#{163}
E
7’\
O’z
/28\
/27\
2
f\27
BuOH:
HAc
: P120
SUMMARY
A detailed
description
amino
acid
analysis
several
respects
from
of analyses
of several
of
our
current
of the hemoglobins
comparable
technics
human
hemoglobins
SUMMARIO
IN
methods
of
“fingerprinting”
has been
presented.
They
used
elsewhere.
Preliminary
are briefly
described.
and
of
differ
in
results
INTERLINGUA
Es presentate
un description
detaliate
de nostre
currente
methodos
acterologic”
e technicas
de analyse
amino-acidic
del hemoglobinas.
Illos
“chardif-
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
68
CHERNOFF
fere in plure
respectos
atorios.
Es describite
hemoglobinas
ab le comparabile
procedimentos
brevemente
le resuitatos
preliminari
LIU
AND
usate
in altere
labordel analyse
de plure
human.
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A
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10
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R.
Physiology
Saskatchewan,
experimental
in
60
per
thrombi
cent
in
methyl
rats
alcohol
to the jumgular vein.
This
method
produced
thrombi
in abouit
65 per
cent
of
animals
within
24 hours.
Both “depo
heparin”
and
“Treburon”
reduced
the
incidence
of thrombosis
when injected
subcutaneously
5 hours after operation. The incidence
of thrombosis
fell
with
increasing
dosage
of these
drugs,
to a minimum
of about 40 per cent when 20 to 40 mg./100
Gin,
was
given.
Many
deaths from hemorrhage
occurred
with the higher
dosages
used.
There
was a significant
negative
correlation
betuveen
the incidence
of thrombosis
and the prothrombin
time
of rats receiving
dicoumarol
or “warfarin,”
but
the
dosage
and
the
THE
these
drugs
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BmosYN-rrnsrs
GL0BIN
7. M.
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of
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was
OF
HEME
RABBIT
From
BONE
New
York.
marrow
poorly
correlated
M.
thrombosis.--R.
the
AND
THE
MARROW
IN
Department
suspended
in
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S.,
Ingram,
2:282-
M. E.:
variant
Negro.
Three
globin
I
Haggard,
haemogiobmn
1959.
Benzer,
49.
A.
Stanleyvihle
R. G. and
new
American
36:
Ada.
Vandepitte,
hemoglobin.
A
The
Blood
P.,
48. Schneider,
285,
I.:
syndromes.
difference
cell hem-
LIU
AND
phosphate
GLYCINE
J. C.
Einstein
was
INTO
Savoie
and
College
incuibated
at 37 C. in open
Erlenmeyer
flasks
with
glycine
2C14
for 4 hours
and
the
rate of glycine
incorporation
into heme and into globin was determined.
The
ratio between
the rate of incorporation
into heme and into glycine
was
found to be almost constant
for all immature
red
cells although
the
biosynthetic activity
of reticulocytes
was much
less
than
that
of nucleated
red
cells.
The addition
of cobalt changed
this constant
ratio by inhibiting
heme
synthesis
without
changing
the incorporation
into glycine.
The effects of substrate concentration,
method
of preparing
the bone
marrow
suspension1
temperature
and
dine of incubation
were
examlned.-A.
I. E.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1961 17: 54-70
The Amino Acid Composition of Hemoglobin. II. Analytical Technics
AMOZ I. CHERNOFF, JEVONS C. LIU, E. Battle and M. Bond
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