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Henioglobin
Polymorphism
in White-Tailed
Subunit
By H’mitsi
I
HE
has
distortion
of the
as
first
described
In
OF
ERYTHROCYTES
the
of
shapes
similarly
contrast
racial
and
geographical
with
sickle
cell
anemia
pathological
of the
deer
which
consequences.3’4
and
been
their
to
only
the
be
all
attributed
so
than
found
the
frequently
man.5
From
Medicine,
in the
presence
bizarre
deer
was
independently
associated
the
is
a
with
a
sickled
erythro-
phenomenon
occurs
of ecological
and
geo-
of
polymorphic
nonsickling
erythrocytes
reversibly
bizarre
particular
the
population
hemoerythrocytes
virginianus).3
have
a
of
which
have
erythrocyte
hemoglobin
various
that
the
forms
common
polymorphic
hemo-
it is difficult
to
identify
or “variant.”
been
reported
cases
white-tailed
marked
moieties
or
deer
in most
The
of the
sickling
(Odocoileus
of
white-tailed
has
heterogeneity
protein
deer
to assume
in
However,
hemoglobins,
ences
such
of
Herrick2
occurrence
of deer
as “normal”
a species.
hemoglobin
morphic
the
In
heterogeneity
within
fetal
ability
to
components
Hemoglobin
other
to
in the deer
is an in vitro
phenomenon
and elevated
pH and has no apparent
sickling
number
deer.
occur
individual
condiremarkable
species
a variety
the
the
in white-tailed
this
white-tailed
globins
This
shape
later
the
represent
to
predominant
to sickle,
cannot
disc
prevalence
However,
relationship
reported
Although
propensity
laboratory
erythrocytes
in man,
graphical
areas
of the world.
Sickling
which
occurs
under
high oxygen
tension
has
TAYLOR
deer.
several
years
shaped
to the
species
globins
of
in
Seventy
JAPE
in vitro
species
biconcave
oat
in 1840.
existence
under
most
the
and
\V.
AND
in man.
associated
in most
from
crescent
Gulliver
PUTNAM
in
erythrocytes
leaf,
anemia
W.
demonstrated
by
observed
severe
cyte
FRANK
been
holly
forms
Basis
KITCHEN,
SICKLING
tions
Deer:
in many
only
two
deer,
or
mammalian
three
having
species
hemoglobins
seven
adult
exception.
This
study
will
of white-tailed
deer
is due
to structural
and
describes
the
subunit
basis
are
and
two
establish
for
that
differ-
these
poly-
hemoglobins.
the
Departments
Gainesville,
of Biochemistry
and
Medicine,
University
of Florida
College
of
Florida.
This work was supported
in part by United
States
Public
Health
Service
grants
H-5004,
H-02966-09
and HE-2T1-HE-5493.
Dr. Kitchen
is the recipient
of a Research
Career
Development
Award
(1-K3-AM
31,811-01)
from the National
Institutes
of Health.
First submitted
Oct. 14, 1966; accepted
for publication
Jan. 9, 1967.
HYRAM
KITCHEN,
D.V.M., PH.D.:
Assistant
Professor,
Departments
of Biochemistry
and
Medicine,
University
of Florida
College
of Medicine,
Gainesville,
Fla. FRAI’mx W. PUTNAM,
PH.D.: Director,
Division
of Biological
Sciences,
Indiana
University,
Bloomington,
md.
W. JAPE
TAYLOR,
M.D.: Professor,
Department
of Medicine,
Chief,
Division
of Cardiology,
University of Florida College of Medicine, Gainesville, Fla.
867
BLOOD,
VOL.
29,
No.
6
(JuNE),
1967
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868
KITCHEN,
MATERIALS
AND
PUTNAM
AND
TAYLOR
METHODS
The
majority
of the present
herd of approximately
70 penned
deer
are Florida
deer
(Odocoileus
virginianus),
representing
most
geographical
and ecological
the state. Other
white-tailed
deer from Georgia,
Maryland
and Texas were obtained
the cooperation
of the Southeastern
Cooperative
Wildlife
Disease
Study.
Deer
from the wild formed
the nucleus
of the research
animals
and represent
a random
sample.
Subsequently,
offspring
from
selected
breedings
have
been
raised
in
captivity.
Collection
of Samples.
The deer are housed
in a 2-acre
site, surrounded
by a 12-foot
fence.
within
a mile of the laboratory.
This proximity
to the laboratory
has assured
the
safe handling
of collected
samples,
as well as the opportunity
to study
animals
repeatedly
Deer.
white-tailed
areas of
through
trapped
during
various
seasons
and
needles,
blood
samples
animals.6
Saniples
were
and were
kept
on ice
specimens
were obtained
were
refrigerated
times
of
the
year.
Using
disposable
syringes
and
19-gauge
were
obtained
from the jugular
vein of restrained
or immobilized
placed
into heparinized
tubes or BD vacutainers
containing
EDTA
until
reaching
the laboratory.
Samples
from
more
than
600 field
as a result
of hunting
and trapping
operations,
and these
samples
until
arrival
at the laboratory.
Production
and Screening
of Sickled
Deer Erythrocytes.
The examination
and classification
of sickled
deer erythrocytes
was performed
on whole
blood
and buffered
blood
samples
(isotonic
potassium
phosphate
buffer,
pH 7.6). Samples
were oxygenated
for 20 minutes
in
a tonometer
with
100 per cent oxygen
at room
temperature.3
The production
of sickled
erythrocytes
by these
conditions
can be shown
to be reversible
and dependent
upon
controlled
conditions.
A gas mixture
of 95 per cent N2 and 5 per cent CO2 through
the blood
in a tonometer
restores
the cells to the normal
biconcave
discoid
shape.
Demonstration
of
reversibility
and examination
within
the shortest
possible
time
are considered
important
since
aberrant
forms,
some
of
which
become
sickle-shaped,
can
be
demonstrated
in
other
species.3
Preparation
saline,
of Hemoglobin.
hemolyzed
with
Red
distilled
blood
water
cells
and
were
frozen.
washed
three
Samples
were
times
in
thawed
1.2
and
per
peatedly
to ensure
complete
hemolysis.
The hemoglobin
solutions
were
separated
ghosts
and incompletely
hemolyzed
red blood
cells
by centrifugation
at 40,000
30 minutes.
Samples
were stored
in the carbon
monoxide
form in the freezer
at -10 C.
Electrophoresis.
Starch
gel
electrophoresis
and
agar
gel
electrophoresis
cent
frozen
were
re-
from
g for
performed
as previously
described.3
Individual
hemoglobmns
for routine
identification
were quantitated
following
electrophoresis
on starch
block,
according
to the method
of Kunkel
et al.7
The a and
chains
were separated
for qualitative
examination
by electrophoresis
in 6 M
urea starch
gel, pH 8.1, following
the method
of Chernoff
and Pettit.8
High voltage
electrophoresis
was
done
in a routine
manner
at pH
6.5, following
the technic
described
by
Putnam
and Easley.9
Specific
color reactions
for peptides
containing
histidine,
tyrosine,
arginine
and tryptophan,
as well
as ninhydrin
staining,
were
performed
following
the
methods
described
by
Easley.l#{176}
Isolation of a and 1 Chains.
Separation
of the a and I chains
by column
chromatography
was performed
as described
by Wilson
and Smith,11
modified by placing 0.025 M mercaptoethanol
in the starting
and limiting
buffers.
RESULTS
Seven
adult
identified
by
demonstrated
tinuous
buffer
have
been
deer
hemoglobins
their
and
differences
best
in
system.
arbitrarily
starch
The
in
gel
electrophoresis,
electrophoretically
identified
creasing
mobility
toward
8.6. For
example,
I
=
two
the
slowest
by
anode
adult
fetal
hemoglobins
electrophoretic
Roman
during
hemoglobin,
behavior,
pH
8.6,
distinguishable
numerals
according
starch
gel
VII
=
have
which
using
been
can
be
a disconhemoglobins
to
electrophoresis,
their
inpH
fastest adult hemo-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
DEER
HEMOGLOBIN
globin,
F1
slower
=
Hemoglobin
in Figure
1 and
However,
not
that
been
naturally
in Table
under
The
controlled
conditions
stick
form,
leaf
I-Il-Ill
sickle,
has
VII
F1,
various
can
F2.
be
related
been
in vitro
conditions,
clude
sickling.
with
the
cent
phoretic
with
light
micro-
of
morphologic
Three
animal
hemoglobins
III-V,
varying
from
one
been
to
II
not
all
the
possible
occurrence
combinations
of deer
with
only
have
III)
under
found
three.
been
the
and
been
to
pre-
identified
Seventy-nine
studied
had more
than one hemoglobin.
Although
of the varying
hemoglobins
were demonstrated
the
IIIto
morphology
(I,
have
be-
II-V,
erythrocytes
have
not
cells
under
morphology
deer
VII)
deer
will
do not
erythrocyte
of
I-
blood
aberrations
hemoglobins
(V and
adult
cells
red
of
of
that
crescent
hemoglobins
the
mixtures
relationship
propensity
associated
transformed
erythrocytes
hemoglobin
match1 ). The
in the
form
whose
hemoglobins
is
cells
deer,
Deer
It is apparent
seven
deer
as
other
sickling
(Fig.
initially
IV.
the
form
combined
aberrant
screenthe
deer
studies,
and
polymorphic
form,
whose
sickling
or
This
hemoglobins.
in a given
deer
show
have
the
two
total,
number
of the
combinations
sickling
and
In
wild
associated
laboratory
leaf
disc
percentage
do not
with
these
white-tailed
In
after
hemoglobin
specific
of the
and
during
holly
biconcave
most
shaped.
hemoglobins.
to
hemoglobin0
been
in vitro
or
singly
1 Summarizes
associated
of
observed
III.
smaller
conditions
Table
polymorphic
have
a
or which
laboratory
or
shown
identified.
in penned
have
under
the
in
found
burr
with
shaped
controlled
seen
been
In
associated
sickle
and
hemoglobin
become
are
far
A summary
occurrence
seen
appearance
has
II-IV,.
instead
been
come
II
or
but
form
“matchstick”
hemoglobin
demonstrated
crescent
form,
and
so
conditions.
traditional
I-Ill
been
morphology
forms
or star
holly
a crystalline
II,
of
burr
hemoglobins
form,
per
the
hemoglobin.
fetus
of polymorphic
demonstrated.
hemoglobins
cell
morphologic
are
or
blood
one
hemoglobins
of
various
fetal
and
are
frequency
the
laboratory
of
the
crescent
with
1. The
have
faster
=
deer
mixtures
study
that
in red
varieties
ing
occurring
and
F2
adult
polymorphic
in this
mixtures
changes
scopy
the
naturally
is listed,
and
different
all
observed
is given
reversible
nine
illustrate
occurring
deer
hemoglobin,
from
all the
hemoglobins
to
fetal
samples
have
869
POLYMORPHISM
by
observed.
a single hemoglobin
a variety
electroNon-
are rarely
observed.
The variety of red blood
of the chemical
and physical
cell forms
properties
seen initially
stimulated
of several
hemoglobins
electrophoretic
behavior.
Although
a striking difference
mobilities readily identifies F1, F2, I, II, V, VII and IV,
electrophoretic
mobilities
of
II,
#{176}Because of the degree
of hemoglobin
occurring
mixtures
of hemoglobins”
to
present in an individual
animal.
Naturally
III
and
IVa
make
further
studies
with very close
in electrophoretic
(Fig. 1), the close
it difficult to distinguish
polymorphism,
the authors
identify
the various
combinations
occurring
hemoglobin
mixtures
refer
of
reflect
to
“naturally
hemoglobin
the genetic
hemoglobin
phenotype
of the individual
animal.
The term “hemoglobin”
is used
in
reference
to the occurrence
of a single hemoglobin
found
in an animal,
whereas
“hemoglobins”
or “hemoglobin
mixture”
then refers to the occurrence
of more than a single
hemoglobin
in an
individual
animal.
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870
KITCHEN,
1.-Classification
Table
of
Hemoglobin
Electrophoretic
Frequency
Per Cent
Hemoglobin
Determined
at pH
44
I-Ill
19
11
6
6
5
4
2
3
lI-Ill
III
Ill-V
IV
I-lI-Ill
II
lI-V
I-lI
f
*
aOver
150
A
finding
second
also
8.6.
similar
at
evident
behavior
The
pH
in
6.0,
in
Figure
the
3 to
/3 polypeptide
because
North
of
American
of
the
the
in-
species.
white-tailed
illustrate
Under
deer,
was
electrophoresis
of the
or
electro-
unusual
degree
by
by
at
these
demonstrated
or
the
this
/3 chains
the
the
and
of
a difference
a difference
(a-
in
in
/3-like
of human
hemoglobin
However,
III and
of
these
IVa
have
hemoglobins
in
the
chains)
two
to
account
for
poly-
alkaline
urea
starch
both,
behavior
separation
gel,
of
chain
migrate
toward
the
upon
the comparison
hemoglobins.
imperative
could
electrophoretic
is, the
analogous
it
sub-
or
That
quite
is
electropho-
acid
The
a
hemoglobins
amino
species.
the
seen
electropho-
because
f3 chains,
conditions,
band
agar
identical
makes
different
electrophoretic
these
broad
Il-Ill.
separation
polypeptide
chains.
a or /3 chains
are
chains
be
6,
structurally
within
and
gel
between
of subunits
of deer
are
a
starch
in light
mixtures
electrophoretic
the
by
electrophoresis
position
cathode
(-),
and the /3 or ‘y chains
tion of a or /3 chains
is dependent
or
no sickling
is a subspecies
here
the
difference
peptides
heterogeneity
either
chains.
isolated
individual
no sickling
types.
2A,
subunits
a
the
the
American
can
gel
tryptic
Figure
hemoglobins
dissociated
peptide
agar
(Fig.
2B).
Hemoglobin
at p1-I 8.6 and at pH 6.0.
which
stitutions
morphic
North
in
studied
differences
on
hemoglobin
remarkable
ascertain
far
to
is essential
hemoglobin
in
contains
quite
retic
Morphology
common
a structural
soluble
corresponding
resis
the
6.0
of the
8.6
so
when
physical
pH
illustrated
pH
found
hemoglobins
and
at
characteristics
at
not
a hemoglobin
establishing
polymorphism.
As
II,
hemoglobins
hemoglobin
of the
Cell
a blood sample
taken from a wild deer.
deer,
Odocoileus
chiriquensis
virginianus,
Odocoileus
virginianus,
and
is presented
phoretically
Chemical
Blood
species.
Evidence
in behavior
Red
studied.
hemoglobin
individual
pH
6.0
Matchstick
IV1),
in this
pH
II-IVb
hemoglobin,
found
these
of
to
Type
8.6 and
deer
TAYLOR
According
Ill-VII
penned
AND
Morphology
Sickling,
crescent,
holly leaf
Matchstick
Sickling,
crescent,
holly leaf
Norma!
biconcave
discoid
shape;
Burr, “thorny
apple”
Matchstick
Matchstick
Normal
biconcave
discoid
shape;
Matchstick
Nonsickling
tOn!y
observed
from
Panama
white-tailed
North American
deer,
teresting
Deer
Penned#{176} White-Tailed
and Red Blood
Cell
Type
PUTNAM
characteristic
to those
pH
8.1,
the numerous
migrates
anode
(+).
of peptide
for
of
is shown
poly-
toward
the
Designamaps
of
peptide
maps
the respective
of
a
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DEER
HEMOGLOBIN
871
POLYMORPHISM
0
#{149}e
-
-
4
-‘‘;
1.-Top:
Fig.
Vertical
starch
gel electrophoresis
of deer
hernoglobins
at pH 8.6
( 10 v. Clii., 2 hours at 10 C. ) . Discontinuous
buffer
svsteni
I)re\’iollsl\’
described.l
Samples
1, 3, 4, 5, 6, 7, 8, 9 atid 10 are from
nine
adult
peiinecl
(leer of varaus
ages
( 1 to 12 ears ) , representing
1)0th sexes.
Samples
4 and 5, (lesignated
IV,, and IV,,,
were
identified
after
the existence
of hemoglohins
III
and
V had
been
reported.l
Therefore,
it was felt simpler
to denote
the tvo new
(liStiflCt
hemoglobin
types
b
subscript
than
to rename
types
III and
V. Sample
2 is from a newborn
fawn
( less
than
1 week
old).
In fetal
hemoglobin
F1-F,
note
as well the presence
of a small
percentage
of adult
hemoglobin
III. Bottom:
A, crescent;
B, matchstick;
C, l)urr,
an(l
D, disc,
are examples
of four
different
morphologic
forms
of deer
ervthrocvtes
01)served
in the studs’
of the sickling
phenomenon
in white-tailed
deer.
An
a
(a1)
the
other
F2.
The
(&)
component
of faster
mobility
hemoglobins.
A single
comparison
isolated
F1 and
of the
F
was
these
fetal hemoglobins
identical
electrophoretic
tween
(ffi)
hemoglohins
has
mobility
hemoglobin
is common
been
associated
is the
/3
V or
VII
hemoglobins
and
with
fetal
in
number
for
the
respectively.
hemoglobin
types
sickling
of
the
subunits
and
y-like
f3’ and
#{176}Each of the
a and
chains
has been
designated
corresponds
(lirectlv
to the Roman
numeral
designation
which
it was
first associated.
For example
& was first
which
is by far the most frequent
hemoglobin
type.
and
VII
chain
Il-V.
(/3).
erythrocytes.
with
an
for the
associated
and
of
The
the
in
nearly
differences
/3
of
chains
with
/9)
structural
a
not
F1
of
Ill-V
the
second
I, but
hemoglobiiis
A fast
hemoglobin
A
and
of a-
(/9,
9 chains
account
IV,,
the
F1
migrations
the
Three
with
hemoglobins.#{176}
is seen
to identify
associated
precludes
deer
of
electrophoretic
made
III
most
y chain
(Table
2).
mobility
II,
chain
to
is a component
l)e-
component
Of
still
faster
presence
By
of
referring
arabic
number
which
hemoglobin
tYpe with
with
hemoglobin
III.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
872
KITCHEX,
PUTNAM
,
0>
>
D>
!
0,
AND
TAYLOR
+
,
>>
W>
,
u.4
-4
-4
H
H
-
Ma;”
#{149}
#{149}.-,#{149}‘
0>
.-
.
#{149}
D>
1
‘:‘
.
0>
>
1
-
-
M
H
II
isjiiiil
1
2
3
4
5
S
I
2
#{149}
4
5
6
7
I
I
Fig. 2.-A:
Vertical
B: Agar gel electrophoresis
globin
samples
to Table
to
chain
type
A
can
see
not
have
sickle
or
ferences
a
of
This
all
of
comparison
/3 chains
their
of
the
side
by
eral
separate
conditions
results
shown
spots
while
respective
side
in
schematic
with
further
IVa
tyrosine
reactions
of such
be
samples
Further
This
illustrates
made
several
are
/3 chain
present
histidine
in
hemoglobins
of
the
II,
and
of
corresponding
II,
III
and
electrophoresis.
so that
strips
with
position
ninhydrin
and
are
ninhydrin
of hemoglobin
16 in the
/3 chain
reactions,
combined
III
under
reactions.
of the
/3 chain
staining
Sev-
made
staining
tyrosine
(Pauly)
are also shown
technic
a
electrophoresis
II and
specific
the
because
hemoglobins
in the
and
reaction,
one-dimensional
that
and
of hemoglobin
differences
of
stained
number
dif-
hemoglobins.
specific
electrophoresis
/3
peptides
structural
important
one-dimensional
by
15 spots
tryptic
patterns
deer
the
type.
for
one-dimensional
of
the
with
of
chains
hemo-
hence,
‘y chain
evidence
a
were
compared
are positive
for
(a-nitroso-/3-naphthol)
form.
on
in the
in the
the
is particularly
The
6.4
adult
/37);
distinguishable
/3 chains
at pH
in all
associated
electrophoretic
and
mobilities.
apparent;
noted
IVa.
which
spots
the
given
one-dimensional
are
18 are
for
staining
III
is
at pH
8.6.
6.0.
Hemo-
pH
or
electrophoresis
has
4. Differences
hemoglobin
cially
burrs
electrophoretically
II,
could
of the
in Figure
positive
(,8
hemoglobins,
one-dimensional
applications
identical
found
chains
are identical
when
compared
by
digest
of the respective
a chains.
run
were
III,
the
digests
IVa
The
nine
are
1
or
one-dimensional
electrophoretic
hemoglobin
of the tryptic
fetal
forms
two
matchsticks
/3 chains
the
of
these
of deer
hemoglobins
1 per cent
agar,
in
cell
of
of the
of hemoglobin
close
The
form
the
and
aberrant
one
case
of
isolated
that
to
or, in the
comparison
of the
gel electrophoresis
of deer
hemoglobins
identical
to those
in B.
do
that
failure
starch
in A are
1, one
globins
of
3
and
in
espe-
negative
Figure
with
IVa,
which
specific
have
4
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
DEER
HEMOGLOBIN
Pr.
ui.
873
I’OLYMORPHISM
4
---.
-
-
+
-
-
I-rn
Fig.
phoretic
,
S
-
-
FF.
3.-6
M urea starch
migration
of the
gel
electrophoresis,
a, /9- and
various
I0#{149}
u-v
11b
1Y
-
ut-vu
pH
y-like
i-n-rn
u.i
8.1, demonstrating
the
polvpeptide
subunits
electroof
deer
hemoglobins.TM
-
similar
electrophoretic
behavior
at pH
8.6,
have
indeed
structurally
different
/3 chains.
CONCLUSIONS
In
this
variety
study
of
hemoglobins.
found
Seven
is not
to all white-tailed
as is the
case
for
if either
one
of two
VII.
an
blood
the
that
preclude
when
geneity
exists
Proof
was
for
one
all
conclusion
deer
when
each
the
This
of
with
phenomenon,
does
occur
not
hemoglobin
sickling,
V or
is responsible
a
of
chain
kinds
on
have
are
polypeptide
and
V or
with
that
the
present
structural
of deer
also
either
with
A are
S and
gave
more
than
every
in
cell
found
in the
that
VII,
either
none
of
of
which
hemoglobins
which
hemoglobin
hetero-
finding
that
subunits
one
present
in the
human
in all cells.
hemoglobin
evidence
is
is,
observation
conclusion
is in accord
that
of hemoglobin
the
present
The
dissociation
system
of the
is based
these
cell
heterogeneity
hemoglobins;
that
combinations.
kinds
by
kind
from
hemoglobins
two
provided
electrophoresis.
than
contains
This
other
both
sickling
one
hemoglobin
this
either
been
from
a particular
However,
hemoglobin
deer
within
heterozygote
that
polymorphic
even
in
have
varying
exhibit
associated
with
sickle
sickling,
sickle
of
a
polymorphic
hemoglobins
is present,
not
deer,
to
animal
erythrocytes
in man.
IVa,
fetal
presence
of hemoglobin
deer
deer.
erythrocytes
the
anemia
related
in a given
whose
indicates
of
individual
of
two
white-tailed
and
erythrocytes.
evidence
erythrocytes
from
cell
Hemoglobin
for the burr shaped
all
by
Florida
noted
and
number
caused
kinds
penned
was
adult
the
deer
sickle
and
forms
deer,
common
Indirect
wild
different
Sickling
hemoglobin
700
erythrocyte
in white-tailed
three.
to
over
of aberrant
for
kind
(a
and
/3
in 6 M
urea
starch
existence
of
the
of
/3
chain
chains)
among
gel
more
the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
874
KITCHEN,
PUTNAM
.-o
-
.
22
.
.1;a
0
--
E
o
+
#{149}1..
+
bc
.2
.i
+
.E
-
+
+
0
++
0.
a.
+±
o
+
++++++
c#{149}
.+‘
,
+
a.
+
-c
.
bOO
o
O
.8
.
+
4)
E
0+’
+
+
a.
.O_.-
0
0
+
c)
+
++
++
4)
++
4)
‘O
_
.8
++++++++++
Cl
+
.2w
+++
.-‘-
4)
c
02
0.0
.00
-
..fl
0
AND
TAYLOR
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
DEER
HEM0(;LOEIN
875
POLYMORPHiSM
.
-
-
I
LI
L1IL
I
2[IJI
‘
-
LI
one-dimensional
/9 chains
(1 )
from
Liii I I 1EJ U
LJL1[I
-
[i
r--r r
U
4.-A
U
s/s
#{149}
-__
LTI
#{149}-
1
Fig.
isolated
J
-.
-
--
#{149}
/11/
I
electrophoresis
hemoglobin
III,
strip
of the
hemoglobin
(2)
trvptic
II, and
peptides
of the
(3) hemoglobin
Iva are shown.
Strips have been stained
with mnhydrm.
The schematic
stripes
below
the corresponding
peptide
separations
are given for comparison
of the reaction
of the
peptide
spots to specific
stains.
Slanted
bars represent
positive
reactions
for arginine.
Stippling
represents
positive
reactions
for histidine
and”or
tvrosine.
Stars represent
positive
tvrosine
reactions
reaction,
for trvptophan.
but positive
for
points
of application
of samples.
polymorphic
deer
guishable
deer
‘y chain
one
starch
have
gel
been
be
deduced;
lists
the
kinds
examined.
and
or
a
polypeptide
chains
number
multiple
and
IVa
virtually
A
different.
patterns
the
structural
were
of the
were
morphology
on
a and
of
these
were
noted.
This
these
urea
the
and
gel
of
occurred
method
and
in
number
was
also
number
spots
suggest
to
of
that
II,
III
/3 chains,
used
in
the
all
the
a,
/3
chains
were
were
structurally
confirmed
IVa
and
that
that
the
a
to
verify
y chains.
A
laboratory
amino
the
electrophoretic
and
reaction
single
limited
for
hemoglobins
a and
positive
a and
of the
with
/3 and
staining
a mere
of
in light
one-dimensional
2
hemoglobin
which
account
far studied.
whose
of ninhydrin
specific
Table
hemoglobins
of
electrophoresis,
the
the
nine
associated
hemoglobins
similarities
in the
findings
of
written.
the
unexpected
isolated
hemoglobins
combinations
total
hemoglobins,
starch
comparison
/3 chains
numerous
These
tetrameric
found,
be
of
and
of 6 M urea
of the
can
each
of erythrocytes
that
6 M
differences
of differences
tions
in
possible
is unique
results
in individual
formulas
the
the
/3 chains
electrophoresis
found
for
to
distin-
distinct
of the
different
polypeptide
chains
hemoglobins
of animals
thus
differences
number
six
present
shorthand
all
to spots
negative
broad
arrow
points
electrophoretically
study
/3 chains
chains
suggested
identical.
structural
the
one-dimensional
not
side-by-side
of the
From
the
identified
identical
chains,
y chains
y
in the
nine
a
/3 and
of structurally
and polymorphic
Differences
III
and
the
of a and
thus
of a,
Although
chains
II,
types
point
The
different
identified.
and
the
arrows
reaction.
Among
two
electrophoresis
can
/3
hemoglobins.
hemoglobins,
a, /3 or y chains,
Narrow
the Paul-
spots
for
acid
and
chains
the
amino
the
great
reacacids
substitution
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
876
KITCHEN,
does
not
account
white-tailed
for
the
remarkable
degree
PUTNAM
of hemoglobin
TAYLOR
AND
heterogeneity
in
deer.
SUMMARY
A
variety
and
two
rocyte
of
fetal
was
with
normally
even
associated
y subunit
geneity
with
of the
deer
numerous
limited
when
based
It
there
(let
(le
aherrante
seven
adult
the
eryth-
of
it was
precluded
by
other
hemoglobin
types
of the
hemoglobin
poly-
upon
a variety
of combinations
concluded
multiple
from
the
peptide
of
results
of
differences
upon
chains.
IN
formas
basis
to
sickling
type,
with
was
were
polypeptide
SUMMARIO
Un varietate
adulte
e duo
esseva
associate
glohina
V o
normalmente
hemoglobina
(‘
tm
subunitate
While
subunit
was
that
non-a
related
of a subunits,
six kinds
of /3 subunits
and
whole
hemoglobin
molecule.
The
hetero-
chains.
studies
deer.
hemoglobin
The
hemoglobins
of three
been
in combination
sickling.
polypeptide
structural
comparison
a single
presented.
Two kinds
were
related
to the
was
have
forms
in white-tailed
associated
V or VII,
morphism
these
erythrocyte
hemoglobins
not
hemoglobin
one
aberrant
INTERLINGUA
erythrocytic
esseva
relationate
a
septe
hemoglobinas
fetal
in cervos
albicaudate.
Ben que le falciformation
del erythrocytos
non
con un typo
individual
de hemoglobina,
illo esseva
excludite
per
hemohemoglobina
VII, mesmo
in combination
con altere
typos
de hemoglobina
associate
con
falciformation.
Le base
subunitari
del
polymorphismo
de
es presentate.
Duo generes
de subunitates
a, sex generes
de subunitates
/3.
y esseva
relationate
al molecula
total de hemoglobina.
Le heterogeneitate
hemoglobinas
ie
cervo
esseva
basate
catenas
polypeptidic.
Es
multiple
differentias
peptidic
esseva
indebted
to
1)olYPel)ti(lic
concludite
ab
in
le
tin
varietate
resultatos
de
del
a judicar
presente,
combinationes
restringite
per
un
de
studios
numerose
structural
comparation
que
de tres
catenas
non-,(.
ACKNOWLEDGMENTS
The
authors
assistance.
are
We
acknowledge
Department
of Veterinary
The collection
of blood
Science,
samples
through
and
the
permission
Commission.
Bronx. New
Many
Beatrice
the
Codwin
of
University
and animals
cooperation
samples
were
and Jacksonville,
York;
P.
cooperation
the
Sarah
in the
5-year
Florida
through
zoos.
L.
Martin
Agricultural
of Florida,
over this
of
possible
Florida
and
the
establishment
period
was
Game
the
for
technical
Experiment
and
cooperation
Station,
of deer pens.
made
possible
Fresh
of
Water
the
Fish
Dallas,
Texas;
REFERENCES
1.
Gulliver,
C.:
Observations
peculiarities
of
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Proc.
325-327,
2.
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its
blood
ani-
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B.:
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red
(Chicago)
H.,
J.:
relation
in
17:
elongated
blood
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Arch.
4. Undritz,
E.,
6:517-521,
Putnam,
Hemoglobin
5.
1910.
F. W.,
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Kitchen,
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Lehmann,
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1960.
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their
bearing
biochemical
Naturalist
H.:
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erythrocytes
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187:333-334,
Allison,
in
of
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Sickling
Nature
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sickling
1964.
Betke,
1237-1239,
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in
to
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H.:
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the
on
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Handling
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restraint
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HEMOGLOBiN
deer
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and
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POLYMORPHISM
other
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2:2-7,
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ma!
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F. W.,
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C. \V.:
Structural
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I. The
tryptic
peptides
of
Bence-Jones
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240:1626-1638,
1965.
10. Easley, C. W.: Combinations
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thalassemia.
J. Clin. Invest.
36:16151625, 1957.
8. Chernoff,
A. I., and Pettit,
N. M., Jr.:
11.
The amino acid composition
of hemoglobin. III. A qualitative
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tion
identifying
tide chains
globin
column
750-756,
abnormalities
of hemoglobin.
1964.
of
polypepBlood
24:
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useful
tide
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Acta 107:386-388,
Wilson,
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of
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1967 29: 867-877
Hemoglobin Polymorphism in White-Tailed Deer: Subunit Basis
HYRAM KITCHEN, FRANK W. PUTNAM and W. JAPE TAYLOR
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