THE
JOURNAL
Copyright
OF HISTOCHEMISTRY
© 1976
by The
Vol.
AND CYTOCHEMISTRY
Histochemical
CONCANAVALIN
Society.
PEROXIDASE
SEPARATED
BY
BRIDGE
Department
of
C. ALLEN,
Pathology,
Received
Medical
for publication
October
S. SPICER of
21,
OF
FOCUSING
a-i
ON
GEL
SAMUEL
University
1976
in U.S.A.
STAINING
ISOELECTRIC
POLYACRYLAMIDE ROBERT
908-914,
Printed
A-HORSERADISH
GLYCOPROTEINS
24, No. 8, pp.
Inc.
South
1975
Carolina,
and
DAVID
AND
Charleston,
revised
in
ZEHR
form
South
Carolina
February
26,
29401
1976
The Coomassie Blue protein stain and the periodic acid-Schiff stain for glycoproteins are compared to a new method of staining glycoproteins resolved electrophoretically. The method utilizes a Concanavalin A-horseradish peroxidase sequence to visualize selectively glycoproteins with terminal or internal mannose or terminal N-acetylglucosamine. The method applied to characterization of M and Z allele products of a-l-antitrypsins separated by isoelectric focusing of polyacrylamide gel slabs has revealed differences in carbohydrate content of various components that were previously undetected.
Electrophoresis
and
polyacrylamide soluble
gel proteins
ous
histochemical
to
characterize
with
periodic
and
on of
can methods
(PAS) and
acid-Schiff
blue
for
The property glycoproteins
be
stain
acid
not sugar
according
mucosubstances
to
their
resolved
Concanavalin
sugar has
been
the
terminal
residues proteins.
position
chemically horseradish
Con
A-reactive
by staining peroxidase (5)
in the diaminobenzidine for HRP. This method
presence
and
the capacity of Con two glycoproteins.
with
first
content,
adaptation
of Con
A-reactive
of this
sugars
A to combine The present method
are
as the method.
known
on pH genetic
type
of side
to
3.5-5.0 type,
chain
in in
second
and
three
type
source
$),
two
cyto-
(4).
The
then in-
and been
infantile reported
subon
glycine instead
in HRP
For
divalently report con-
shown
including
to preparations
At
proteinase by isoelec-
a single carbohytypes. The
of four
sialic
three two
associated
the
residues
those
a minimum,
of on with two
908
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
The of
of terminal
units M protein have and
and
three
chains
(4).
this report, results selected standard
Pi types sera
each
1
sialic
emphysema
chain side
N-
(2 a and
with
polypeptide
of
acid.
on the other hand, one more arginine
carbohydrate
purpose staining
(of
three
of mannose
and
cirrhosis, to contain
in
residues
mannose
two types of oligosaccharide in equal amounts in the
of four
(1). The a molec-
of
of
of terminal of
the Thus,
a configuration),
three
Z proteins
the of
under
alleles.
gradients protein has
three
of galactose
acid. These are present
mateso-called
be
consists
consists
acetylglucosamine,
around activ-
The
of 54,000 and consists chain with four branching chains of two structural
galactose
to glucose
(DAB) depended
the
of N-acetylglucosamine, which two are in the
reported
Con A and and, finally,
focus-
of microheterogeneic
utilized
evaluating
ular weight polypeptide drate side
sugars char-
glycoproteins with (HRP)
the
cerns
in addition
for
tric focusing M, or normal
provide moieties
which occur rarely, if ever, in glycoA method was developed recently (2)
for localizing
cubating strate
(6)
group was
control of at least 25 codominant a variety of genetically determined inhibitor (Pi) patterns are separable
(3)
to react with internal or terminal mannose the a configuration and N-acetylglucosamine
particular proteins
a-1-antitrypsins
electrophoretically.
A, for example,
this
rial
mucosubstances do
of lectins to bind specific offers a means of further
acterizing,
ity,
vicinal
Blue
isoelectric
Since much interest has centered a- i-glycoproteins with antiproteinase
serum
stained such as
for
Alcian
(9). These stains, however, information concerning specific present in the macromolecules. in
the
or The
gel
ing.
Vari-
by
by polyacrylamide
resolved
adapted
separated
components various
with
been
proteins
glycoproteins
toluidine
have
gel electrophoresis isoelectric focusing.
the
carbohydrate-rich in these gels glycolrich
focusing
enable separation high resolution.
methods
either polyacrylamide polyacrylamide gel
the
isoelectric
slabs
MM, from
MZ
are sera,
and
a different
ZZ.
CON-A-STAINED subject
were
tested
for
each
of’
GLYCOPROTEIN
the
Protein in distilled
aforemen-
Pi types.
tioned
MATERIALS
AND
METHODS
Isoelectric focusing: The a-1-antiproteinases MM, MZ, ZZ, SZ, FZ and MS sera were separated 1-mm
thick
gel slabs
previously
LKB
constant
power 35
watts
for
in
washed ered
supply
the
of
was
last
12.5%
trichloracetic changes
that
10 mm,
with
gel
a gel
for
peroxidase
final
rinse
(5) with
tended
strate
to produce
resolution
All
substrate
solution
M Tris
surface
osmium
consisted
buffer
sub-
sheen
that
treatment.
DAB
of 6 mg g Tris/100
(0.61
with
ml
a
in 20 ml of to pH
with 1.0 N HC1). Just before use, 6 drops of 3% H,O, were added. Additional enhancement of the peroxidase stain was achieved by reacting the developed stain with 1.0% osmium tetroxide for 5 mm. Periodic acid-Schi.ff staining: For periodic acidSchiff (PAS) staining, the fixed gels were reacted with a 1% periodic acid solution for 30 mm, washed three 7.6
times
for
mm
5 mm
in each
each
in distilled
of two
alternative,
more
water
changes
rapid
and
stained
of 1% Schiff
method
was
5
base.
also
An
employed
and
and
20
Schiff
base
turned
of
decanted
and
and
used
fresh
but
such
mm, was
expiration does
date
not
The
necessarily
gel
bands
of caution, stored
gel was
added.
a note be
the
solution
PAS-positive
As
5 parts
point
the
the
should
decanted
and
base
base
reagent.
was
acid at which
10
and
a precaution
an acceptable
added,
Schiff
to the
acid
periodic
apparent. Schiff
prior
periodic
After
rapidly
readily
commercial
was
brown.
cleared
became
The 1 part
(Fisher)
dark
then
mm.
in
the
the
on the
cold
bottle,
guarantee
PAS
and
destained
acid
to 6 mm
at 65#{176}C. were placed quantitative
methods
and
common
the
Coomas-
the
Con
A-HRP-
of Pi types
samples
type
of a Pi
more
with
Examples
technique, was carried
ZZ, the
of
quantitative out on
MM
MZ
1-3. Con mireplicate
reference
standard.
The total amount of antitrypsin present was between 46.3 and 56.1 tg, as determined either by cellulose acetate electrophoresis in conjunction
with
total
biuret
protein
radial immunodiffusion, intensively stained Blue
3.62%
was
the
M-1
total
M
column tained
above,
based on a-1-antitrypsin. has
been
nose
a
an
mannose
actually larger
contained (Table
M-1 on the
pmoles
band two
of allele
theoretically
should
be
of man-
approximate and
in
equally How-
distribution of the Con-Aon microdensitometric
that predicted some 824.2 has
of
present
the residues are all M allele products.
present
for MM
pmoles
indicated almost double the M-1 to M-3 (Table I, column
be
product
12 moles 412.2
I,
conassay
of 54,000 of Pi type
of some
value
density
the
weight each mole
the percentage reacted bands
bands
which
to contain
assuming between
The least Coomas-
products
average
band M-1, distributed
the
of 34.4 molecular Since
reported
(4),
analysis
band, allele
1). Accordingly, an average, based
methods
or by quantitative
respectively. protein band with
of the
pared to Therefore,
for mixture
0.2%
changes,
of the
Z stained
A-HRP bridge crodensitometry
solution a
two
MM are shown in Figures To determine the sensitivity
ever, HRP
on the
Hoch-Ligeti
and
then
ml
alcohol
storage.
of each
5, F and
sequence.
procedure described by McManus and (8). In the latter instance, the trichloroacetic acid-fixed gels were washed rapidly with running distilled water and placed in a 1.0% periodic acid
based
were
in 10% acetic
and
M,
DAB
sie
The
adjusted
Gels
by the above procedures acid for photography,
Pi products
alleles
to
45
RESULTS
and
prior
of
employing
ethanol
of 25%
sie Blue
agitation
a metallic
background,
surface mm
incubation
following
acid.
were
10
consisting
45 ml absolute
Blue,
microdensitometry’
period.
Longer
minimize
solution
acetic
Gels stained in 10% acetic
or phosphate-buff-
constant
PBS.
obscured 0.05
with
glacial
each,
Concanavalin A-horseradish peroxidase technique: The fixed gels were covered for 90 mm with a 0.1 mg/100 ml Concanavalin A (Con-A) solution (5 mg Con-A; Sigma grade IV; salt-free in 50 ml PBS) with constant agitation at room temperature. The gel was washed three times each for 5 mm in PBS and then was flooded with a solution of 1 mg horseradish peroxidase (HRP) in 100 ml of PBS for 90 mm with constant agitation. The gel was again given three 5mm rinses in PBS and then was covered for 10 mm with the diaminobenzidine (DAB) substrate medium for
Brilliant
an
35 mm
separations
acid
a 10 mm
next
Blue
Coomassie
on as
25 watts
at
for the
of water
over
gradient
exception
employed
16-18#{176}C. The
(PBS)
3.5-5.0
the
30 watts
in several saline
a pH with
20 mm,
first
temperature fixed
(I),
power
for the
and
utilizing
described
staining: The fixed gels were washed once water and stained 12 mm at 65#{176}C with a
Coomassie 10 ml
from
909
SEPARATION
by pmoles
in the been sensitivity
corresponding
protein staining. of mannose may
M-1
utilized
amount in 2) com-
band, in
of the
and
this
determining method.
integrated
The area
under the peak is about seven times above the practical level for accurate integration, which ‘ORTEC Model 4300 integrating microdensitometer.
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
910
ALLEN,
SPICER
AND
ZEHR
-‘‘a a a
1
M$
1$
Mi
a CON
a
A
V
a
I
I
‘C
3
\
H
H
COOMASSII
‘.
/
MM
U
U
3.95 FIG.
gradient aligned
1. Pi
some
MM
serum
separated
by
stained with Coomassie Blue, using band M-6 as a reference.
corresponds staining practical
type
to in Table purposes 116
pmoles
the
M-1
value
shown
isoelectric
PAS
and
by
I, column 3, and Figure of accurate quantification, of
4.62.
pH
mannose/lO
mm
focusing
the
PAS 1. For
Con
on a polyacrylamide
A-HRP
or
are
quantified.
readily Of
would
note
about
suffice
are
gel
technique.
application
amount
wide
bridge
two
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
slab
in a pH
Desitometric
12
pmoles/mm2
for
to 5.0
area
Perhaps
one-half
visual
regions
3.5
traces
of gel
of this
detection.
of
are
glycoproteins
CON-A-STAINED
GLYCOPROTEIN
911
SEPARATION
t
CONAHRP
C
M6
PAS
P1
11,PiZZ
MZ
L
COO
-r
.
ILUE
MASSIE
14
I
4.62
pH 462
FIG.
2.
Pi
MZ and
types
ZZ separated
gradient stained with Coomassie Blue, made on the M-6 band and in the ZZ
by isoelectric
focusing
PAS and the Con on the Z-4 band.
A-HRP
TABLE Quantit
ative
Micro
densitometry
of Pi T,ype A-Horseradish
Peak
MM
bridge
gel slab
technique.
in a pH 3.5 to 5.0 in the Pi MZ was
Alignment
I
Serum
Stained
Peroxidase
by Coomas
Bridge
Ratio
Area’
on a polyacrylamide
of Each
sie Blue
PAS
and
Concanavalin
Technique Band
to
pmoles
Its Successor
Mannose/gg#{176}
Band
Coomassie
Con A
PAS
Coomassie
Con A
PAS
Coomassie
Blue
Con
A-HRP
theoretical
bridge
8.04
15.57
1
12.64
20.16
3.00
2 3
15.12 18.60
21.31
2.82
1.19
1.06
0.94
9.60
16.44
27.38
3.15
1.2:3
1.28
1.12
11.88
21.19
4
51.70
37.48
8.79
2.78
1.37
2.79
32.88
29.04
5
62.64
41.56
19.96
1.24
1.11
2.27
39.85
32.20
6
64.94
42.60
22.85
1.04
1.02
1.14
41.31
33.00
7
49.42
35.86
7.23
0.76
0.84
0.32
31.44
27.77
8
74.18
60.40
7.42
1.50
1.68
1.03
47.11
46.63
‘Integrator ‘Determined results
(2.93
counts from
x
102.
average
protein
value
obtained
from
radial
immunodiffusion
g/l).
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
and
biuret-electrophoresis
912
ALLEN,
SPICER
AND
ZEHR
- f______
----
It
I
-
jr1--.4
-
-.‘
3.
FIG.
Separation
and
C are Pi ZZ
MZ
similarly
of stained
Pi
types
with
with
ing
in pH
from
to 4.44,
four
or
five in
2).
higher based
4.20
former
and Con
number on
region
Coomassie A-HRP apparently
comparative
of
first
isoelectric and
bands, depending pH between 3.83
The
with PAS with the glycoproteins
ZZ
on
a polyacrylamide
bridge,
The
six bands
and
and
A-HRP
a-1-antitrypsins.
displayed
ranging
MZ
Con
PAS
and
F
E
gel
Coomassie
slab
in a pH
Blue,
gradient
respectively.
of 3.5
D, E and
to 5.0.
A, B
F are Pi type
stained.
to the
anodal
D
BC
A
points other on
and stained
of these
the
rang-
tive
contained the
4.09
not
serum, (Figs.
only
1
lightly
Blue but very strongly bridge technique. These possess a relatively
groups
reactive microdensitometry.
for
Con-A On
other
hand,
proteins
react
ZZ
and
up
to
with
strongly
reacting
the
second
of these
Con-A.
manifesting double
positive, types
the in
infantile
or
Con MM
Individuals
more
the
A-negative
or MZ
(Fig.
cirrhosis amount bands
PAS-posidid
regions
with
Pi type may
have
of the
PAS-
found
in
3).
DISCUSSION
The strate
results obtained the applicability
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in this study demonof the Con A-HRP-DAB
Pi
CON-A-STAINED
sequence to sera resolved
identification of by electrophoresis.
GLYCOPROTEIN
glycoproteins Quantitative
in
913
SEPARATION
dues. what
Further studies these glycoprotein
are
in progress to define zones are and why the
data were obtained primarily to determine the approximate sensitivity of the lectin technique for detecting glycoproteins of the various Pi
Pi ZZ cirrhotic often shows a much increased PAS staining as compared to other phenotypes.
allele
Con pH
products.
amount
However,
of binding
Coomassie
allele of
of
almost
residues
that
the
repeated
in additional and
allele The the ucts
M
allele
of the
made
here
ences
between
the
be
made,
(23)
other
The
A-positive
points
between
partially shown technique
by
gradient grate
the
gel
forward
separation M and
observed
to the focusing column ure
to
that
column, at
pH
3.82
of Con
that
the
nose
or
A-HRP
pH terminal
3.82-4.09
into
of this
Z allele
products
in
isoelectric only been
Pi
allele
albumin to
staining
(7).
region
from
following been It
products and
4.09
cific
lectins
pass
the
front. concurs in gels
components fi N-acetylglucosamine
rehas bind
proteins
through
the
This failwith the and lack
protein
mannose
must
have
me
in the
have
dase
again
of the
a config-
not
has
been
not
stud-
been
demonstrates
Since
the
affirms a manresi-
the
at-
proteins
focusing
with
sugars
in
present
to
study
by isoelectric are present should be
different sugar to characterize
separated
or might
by
lectins,
could with
and
molecular
properties and indeweight of the lectins to
These
HRP,
in
sequence
of histo-
gel slabs this method
glycoproteins gels.
spe-
peroxi-
the
separated
by diffusion molecular
on
carbo-
with
usefulness
extended
Other lectins with could be employed
the
tric
the
horseradish
on polyacrylamide surface of the gel,
unencumbered pendent of the
ing
with
techniques
focusing on the
further glycoproteins
coupled
isoelecreactive alterna-
if
be employed this
enzyme
as
be conjugated employed
in
in
the
to HRP
by
a single
stain-
step.
ACKNOWLEDGMENTS
The efficient Mr.
authors would technical and
Price
Oulla
and
like to acknowledge secretarial assistance Ms.
Dot
LITERATURE
mi-
albumin
have laboratory.
between to be a-
N-acetylglucosam
to characterize
moieties
isoelectric and the six
electrophoresis, of the
of
of
strong
points appear
classification
glutaraldehyde
former have been electrophoretic which, in pore
with the breakthrough bind to a Con A column
lack
with 4.09
unit
components
their
ability
tively
approach
insight
of eluates
the
while
and
be employed. specificities
contains an
apparent 4.41 have
edge
These
ied,
further
glycoproteins
Con A affinity chromatography peatedly performed in our been
differ-
identified.
bands 3.83 and
bands with pH 4.19 and
Pi type
less was
be assessed
presently
acrylamide
Further both
prod-
25%
further
characterized. The the two-dimensional to be a-i-globulins
with
with
product
PAS-positive
A-negative between pH
uration.
demonstrate
isoelectric do not
per
or internal
level. of
attempt
Such
Pi allelles
additional
Con
allele
to gain
Con
points
the
products
could
mannose.
be applied
also
each
reported
types
as each
internal
four
if these
allele
but
or terminal
The
than
contain no
which
with 4.41 amounts
hydrate
with the Con A-HRP method, suggest that such a comparison
does
sufficient may
which
Although
1-globulins,
been
sufficiently
these
proteins
considerable
chemical
compare
Z allele,
six
A binding 4.19 and
ZZ and
entity. appears
to determine
quantitatively this study could
rather
The
tempted.
to study
the
(4).
sugin the
MZ,
individually and
Pi
differ
need
of the
of Con
to M-3
MM,
the
technique
carbohydrate
the
Pi type
to study
of
that conformational observation has
as a single
present
Pi
M-1
individually,
antitrypsin
sensitive
This
unit
indication
chains or
per three
number
bands
emphasizes
product
total
expected in
changes sera
in
in the
The
carbohydrate
allele products are present.
several
A-HRP
unexpected. the
double
differences
protein
were
A-reactive
SZ
Con
Blue-stained
products
gests
the
the of
Smith. CITED
1. Allen RC, Harley RA, Talamo RC: A new method for determination of alpha-1-antitrypsin phenotypes using isolectric focusing on polyacrylamide gel slabs. Am J Clin Pathol 62:732, 1974 2. Bernhard W, Avrameas 5: Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp Cell Res 64:232, 1971
3. Caldwell RC, human saliva chem Biophys 4.
Chan
SK,
1-protease
Rees
Pigma W: Disc in polyacrylamide 110:91, 1965 DC: Molecular
inhibitor
deficiency.
electrophoresis gels. Arch basis
for
Nature
the
of Bio-
alpha225:240,
1975
5. Graham RC Jr, Karnovsky MJ: The absorption of injected horseradish
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
early stages peroxidase
of in
914
ALLEN,
the proximal tubules of mouse tural cytochemistry by a new chem Cytochem 14:291, 1966 6. Komfeld R, Ferris C: Interaction lin
glycopeptides
Chem 7. Maurer
with
kidney: technique.
SPICER
ultrastrucJ Histo-
of immunoglobu-
concanavalin
250:2614, 1975 HR, Allen RC: Polyacrylamide
AND
A.
J
phoresis
8. Biol
ZEHR
9. Rennert gel electro-
in clinical
chemistry:
dardization and performance. 40:359, 1972 McManus JFA, Hoch-Ligeti acid spot test for 1,2 glycols. ysaccharides.
OM:
Disc
problems Clin C: Lab
electrophoresis
Nature
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213:1133,
of stanChem
Modified Invest
of acid 1967
1:19,
Acta periodic 1952
mucopol-
JOURNAL
Copyright
OF HISTOCHEMISTRY
© 1976
by The
Vol.
AND CYTOCHEMISTRY
Histochemical
CONCANAVALIN
Society.
PEROXIDASE
SEPARATED
BY
BRIDGE
Department
of
C. ALLEN,
Pathology,
Received
Medical
for publication
October
S. SPICER of
21,
OF
FOCUSING
a-i
ON
GEL
SAMUEL
University
1976
in U.S.A.
STAINING
ISOELECTRIC
POLYACRYLAMIDE ROBERT
908-914,
Printed
A-HORSERADISH
GLYCOPROTEINS
24, No. 8, pp.
Inc.
South
1975
Carolina,
and
DAVID
AND
Charleston,
revised
in
ZEHR
form
South
Carolina
February
26,
29401
1976
The Coomassie Blue protein stain and the periodic acid-Schiff stain for glycoproteins are compared to a new method of staining glycoproteins resolved electrophoretically. The method utilizes a Concanavalin A-horseradish peroxidase sequence to visualize selectively glycoproteins with terminal or internal mannose or terminal N-acetylglucosamine. The method applied to characterization of M and Z allele products of a-l-antitrypsins separated by isoelectric focusing of polyacrylamide gel slabs has revealed differences in carbohydrate content of various components that were previously undetected.
Electrophoresis
and
polyacrylamide soluble
gel proteins
ous
histochemical
to
characterize
with
periodic
and
on of
can methods
(PAS) and
acid-Schiff
blue
for
The property glycoproteins
be
stain
acid
not sugar
according
mucosubstances
to
their
resolved
Concanavalin
sugar has
been
the
terminal
residues proteins.
position
chemically horseradish
Con
A-reactive
by staining peroxidase (5)
in the diaminobenzidine for HRP. This method
presence
and
the capacity of Con two glycoproteins.
with
first
content,
adaptation
of Con
A-reactive
of this
sugars
A to combine The present method
are
as the method.
known
on pH genetic
type
of side
to
3.5-5.0 type,
chain
in in
second
and
three
type
source
$),
two
cyto-
(4).
The
then in-
and been
infantile reported
subon
glycine instead
in HRP
For
divalently report con-
shown
including
to preparations
At
proteinase by isoelec-
a single carbohytypes. The
of four
sialic
three two
associated
the
residues
those
a minimum,
of on with two
908
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
The of
of terminal
units M protein have and
and
three
chains
(4).
this report, results selected standard
Pi types sera
each
1
sialic
emphysema
chain side
N-
(2 a and
with
polypeptide
of
acid.
on the other hand, one more arginine
carbohydrate
purpose staining
(of
three
of mannose
and
cirrhosis, to contain
in
residues
mannose
two types of oligosaccharide in equal amounts in the
of four
(1). The a molec-
of
of
of terminal of
the Thus,
a configuration),
three
Z proteins
the of
under
alleles.
gradients protein has
three
of galactose
acid. These are present
mateso-called
be
consists
consists
acetylglucosamine,
around activ-
The
of 54,000 and consists chain with four branching chains of two structural
galactose
to glucose
(DAB) depended
the
of N-acetylglucosamine, which two are in the
reported
Con A and and, finally,
focus-
of microheterogeneic
utilized
evaluating
ular weight polypeptide drate side
sugars char-
glycoproteins with (HRP)
the
cerns
in addition
for
tric focusing M, or normal
provide moieties
which occur rarely, if ever, in glycoA method was developed recently (2)
for localizing
cubating strate
(6)
group was
control of at least 25 codominant a variety of genetically determined inhibitor (Pi) patterns are separable
(3)
to react with internal or terminal mannose the a configuration and N-acetylglucosamine
particular proteins
a-1-antitrypsins
electrophoretically.
A, for example,
this
rial
mucosubstances do
of lectins to bind specific offers a means of further
acterizing,
ity,
vicinal
Blue
isoelectric
Since much interest has centered a- i-glycoproteins with antiproteinase
serum
stained such as
for
Alcian
(9). These stains, however, information concerning specific present in the macromolecules. in
the
or The
gel
ing.
Vari-
by
by polyacrylamide
resolved
adapted
separated
components various
with
been
proteins
glycoproteins
toluidine
have
gel electrophoresis isoelectric focusing.
the
carbohydrate-rich in these gels glycolrich
focusing
enable separation high resolution.
methods
either polyacrylamide polyacrylamide gel
the
isoelectric
slabs
MM, from
MZ
are sera,
and
a different
ZZ.
CON-A-STAINED subject
were
tested
for
each
of’
GLYCOPROTEIN
the
Protein in distilled
aforemen-
Pi types.
tioned
MATERIALS
AND
METHODS
Isoelectric focusing: The a-1-antiproteinases MM, MZ, ZZ, SZ, FZ and MS sera were separated 1-mm
thick
gel slabs
previously
LKB
constant
power 35
watts
for
in
washed ered
supply
the
of
was
last
12.5%
trichloracetic changes
that
10 mm,
with
gel
a gel
for
peroxidase
final
rinse
(5) with
tended
strate
to produce
resolution
All
substrate
solution
M Tris
surface
osmium
consisted
buffer
sub-
sheen
that
treatment.
DAB
of 6 mg g Tris/100
(0.61
with
ml
a
in 20 ml of to pH
with 1.0 N HC1). Just before use, 6 drops of 3% H,O, were added. Additional enhancement of the peroxidase stain was achieved by reacting the developed stain with 1.0% osmium tetroxide for 5 mm. Periodic acid-Schi.ff staining: For periodic acidSchiff (PAS) staining, the fixed gels were reacted with a 1% periodic acid solution for 30 mm, washed three 7.6
times
for
mm
5 mm
in each
each
in distilled
of two
alternative,
more
water
changes
rapid
and
stained
of 1% Schiff
method
was
5
base.
also
An
employed
and
and
20
Schiff
base
turned
of
decanted
and
and
used
fresh
but
such
mm, was
expiration does
date
not
The
necessarily
gel
bands
of caution, stored
gel was
added.
a note be
the
solution
PAS-positive
As
5 parts
point
the
the
should
decanted
and
base
base
reagent.
was
acid at which
10
and
a precaution
an acceptable
added,
Schiff
to the
acid
periodic
apparent. Schiff
prior
periodic
After
rapidly
readily
commercial
was
brown.
cleared
became
The 1 part
(Fisher)
dark
then
mm.
in
the
the
on the
cold
bottle,
guarantee
PAS
and
destained
acid
to 6 mm
at 65#{176}C. were placed quantitative
methods
and
common
the
Coomas-
the
Con
A-HRP-
of Pi types
samples
type
of a Pi
more
with
Examples
technique, was carried
ZZ, the
of
quantitative out on
MM
MZ
1-3. Con mireplicate
reference
standard.
The total amount of antitrypsin present was between 46.3 and 56.1 tg, as determined either by cellulose acetate electrophoresis in conjunction
with
total
biuret
protein
radial immunodiffusion, intensively stained Blue
3.62%
was
the
M-1
total
M
column tained
above,
based on a-1-antitrypsin. has
been
nose
a
an
mannose
actually larger
contained (Table
M-1 on the
pmoles
band two
of allele
theoretically
should
be
of man-
approximate and
in
equally How-
distribution of the Con-Aon microdensitometric
that predicted some 824.2 has
of
present
the residues are all M allele products.
present
for MM
pmoles
indicated almost double the M-1 to M-3 (Table I, column
be
product
12 moles 412.2
I,
conassay
of 54,000 of Pi type
of some
value
density
the
weight each mole
the percentage reacted bands
bands
which
to contain
assuming between
The least Coomas-
products
average
band M-1, distributed
the
of 34.4 molecular Since
reported
(4),
analysis
band, allele
1). Accordingly, an average, based
methods
or by quantitative
respectively. protein band with
of the
pared to Therefore,
for mixture
0.2%
changes,
of the
Z stained
A-HRP bridge crodensitometry
solution a
two
MM are shown in Figures To determine the sensitivity
ever, HRP
on the
Hoch-Ligeti
and
then
ml
alcohol
storage.
of each
5, F and
sequence.
procedure described by McManus and (8). In the latter instance, the trichloroacetic acid-fixed gels were washed rapidly with running distilled water and placed in a 1.0% periodic acid
based
were
in 10% acetic
and
M,
DAB
sie
The
adjusted
Gels
by the above procedures acid for photography,
Pi products
alleles
to
45
RESULTS
and
prior
of
employing
ethanol
of 25%
sie Blue
agitation
a metallic
background,
surface mm
incubation
following
acid.
were
10
consisting
45 ml absolute
Blue,
microdensitometry’
period.
Longer
minimize
solution
acetic
Gels stained in 10% acetic
or phosphate-buff-
constant
PBS.
obscured 0.05
with
glacial
each,
Concanavalin A-horseradish peroxidase technique: The fixed gels were covered for 90 mm with a 0.1 mg/100 ml Concanavalin A (Con-A) solution (5 mg Con-A; Sigma grade IV; salt-free in 50 ml PBS) with constant agitation at room temperature. The gel was washed three times each for 5 mm in PBS and then was flooded with a solution of 1 mg horseradish peroxidase (HRP) in 100 ml of PBS for 90 mm with constant agitation. The gel was again given three 5mm rinses in PBS and then was covered for 10 mm with the diaminobenzidine (DAB) substrate medium for
Brilliant
an
35 mm
separations
acid
a 10 mm
next
Blue
Coomassie
on as
25 watts
at
for the
of water
over
gradient
exception
employed
16-18#{176}C. The
(PBS)
3.5-5.0
the
30 watts
in several saline
a pH with
20 mm,
first
temperature fixed
(I),
power
for the
and
utilizing
described
staining: The fixed gels were washed once water and stained 12 mm at 65#{176}C with a
Coomassie 10 ml
from
909
SEPARATION
by pmoles
in the been sensitivity
corresponding
protein staining. of mannose may
M-1
utilized
amount in 2) com-
band, in
of the
and
this
determining method.
integrated
The area
under the peak is about seven times above the practical level for accurate integration, which ‘ORTEC Model 4300 integrating microdensitometer.
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
910
ALLEN,
SPICER
AND
ZEHR
-‘‘a a a
1
M$
1$
Mi
a CON
a
A
V
a
I
I
‘C
3
\
H
H
COOMASSII
‘.
/
MM
U
U
3.95 FIG.
gradient aligned
1. Pi
some
MM
serum
separated
by
stained with Coomassie Blue, using band M-6 as a reference.
corresponds staining practical
type
to in Table purposes 116
pmoles
the
M-1
value
shown
isoelectric
PAS
and
by
I, column 3, and Figure of accurate quantification, of
4.62.
pH
mannose/lO
mm
focusing
the
PAS 1. For
Con
on a polyacrylamide
A-HRP
or
are
quantified.
readily Of
would
note
about
suffice
are
gel
technique.
application
amount
wide
bridge
two
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
slab
in a pH
Desitometric
12
pmoles/mm2
for
to 5.0
area
Perhaps
one-half
visual
regions
3.5
traces
of gel
of this
detection.
of
are
glycoproteins
CON-A-STAINED
GLYCOPROTEIN
911
SEPARATION
t
CONAHRP
C
M6
PAS
P1
11,PiZZ
MZ
L
COO
-r
.
ILUE
MASSIE
14
I
4.62
pH 462
FIG.
2.
Pi
MZ and
types
ZZ separated
gradient stained with Coomassie Blue, made on the M-6 band and in the ZZ
by isoelectric
focusing
PAS and the Con on the Z-4 band.
A-HRP
TABLE Quantit
ative
Micro
densitometry
of Pi T,ype A-Horseradish
Peak
MM
bridge
gel slab
technique.
in a pH 3.5 to 5.0 in the Pi MZ was
Alignment
I
Serum
Stained
Peroxidase
by Coomas
Bridge
Ratio
Area’
on a polyacrylamide
of Each
sie Blue
PAS
and
Concanavalin
Technique Band
to
pmoles
Its Successor
Mannose/gg#{176}
Band
Coomassie
Con A
PAS
Coomassie
Con A
PAS
Coomassie
Blue
Con
A-HRP
theoretical
bridge
8.04
15.57
1
12.64
20.16
3.00
2 3
15.12 18.60
21.31
2.82
1.19
1.06
0.94
9.60
16.44
27.38
3.15
1.2:3
1.28
1.12
11.88
21.19
4
51.70
37.48
8.79
2.78
1.37
2.79
32.88
29.04
5
62.64
41.56
19.96
1.24
1.11
2.27
39.85
32.20
6
64.94
42.60
22.85
1.04
1.02
1.14
41.31
33.00
7
49.42
35.86
7.23
0.76
0.84
0.32
31.44
27.77
8
74.18
60.40
7.42
1.50
1.68
1.03
47.11
46.63
‘Integrator ‘Determined results
(2.93
counts from
x
102.
average
protein
value
obtained
from
radial
immunodiffusion
g/l).
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
and
biuret-electrophoresis
912
ALLEN,
SPICER
AND
ZEHR
- f______
----
It
I
-
jr1--.4
-
-.‘
3.
FIG.
Separation
and
C are Pi ZZ
MZ
similarly
of stained
Pi
types
with
with
ing
in pH
from
to 4.44,
four
or
five in
2).
higher based
4.20
former
and Con
number on
region
Coomassie A-HRP apparently
comparative
of
first
isoelectric and
bands, depending pH between 3.83
The
with PAS with the glycoproteins
ZZ
on
a polyacrylamide
bridge,
The
six bands
and
and
A-HRP
a-1-antitrypsins.
displayed
ranging
MZ
Con
PAS
and
F
E
gel
Coomassie
slab
in a pH
Blue,
gradient
respectively.
of 3.5
D, E and
to 5.0.
A, B
F are Pi type
stained.
to the
anodal
D
BC
A
points other on
and stained
of these
the
rang-
tive
contained the
4.09
not
serum, (Figs.
only
1
lightly
Blue but very strongly bridge technique. These possess a relatively
groups
reactive microdensitometry.
for
Con-A On
other
hand,
proteins
react
ZZ
and
up
to
with
strongly
reacting
the
second
of these
Con-A.
manifesting double
positive, types
the in
infantile
or
Con MM
Individuals
more
the
A-negative
or MZ
(Fig.
cirrhosis amount bands
PAS-posidid
regions
with
Pi type may
have
of the
PAS-
found
in
3).
DISCUSSION
The strate
results obtained the applicability
Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 18, 2015
in this study demonof the Con A-HRP-DAB
Pi
CON-A-STAINED
sequence to sera resolved
identification of by electrophoresis.
GLYCOPROTEIN
glycoproteins Quantitative
in
913
SEPARATION
dues. what
Further studies these glycoprotein
are
in progress to define zones are and why the
data were obtained primarily to determine the approximate sensitivity of the lectin technique for detecting glycoproteins of the various Pi
Pi ZZ cirrhotic often shows a much increased PAS staining as compared to other phenotypes.
allele
Con pH
products.
amount
However,
of binding
Coomassie
allele of
of
almost
residues
that
the
repeated
in additional and
allele The the ucts
M
allele
of the
made
here
ences
between
the
be
made,
(23)
other
The
A-positive
points
between
partially shown technique
by
gradient grate
the
gel
forward
separation M and
observed
to the focusing column ure
to
that
column, at
pH
3.82
of Con
that
the
nose
or
A-HRP
pH terminal
3.82-4.09
into
of this
Z allele
products
in
isoelectric only been
Pi
allele
albumin to
staining
(7).
region
from
following been It
products and
4.09
cific
lectins
pass
the
front. concurs in gels
components fi N-acetylglucosamine
rehas bind
proteins
through
the
This failwith the and lack
protein
mannose
must
have
me
in the
have
dase
again
of the
a config-
not
has
been
not
stud-
been
demonstrates
Since
the
affirms a manresi-
the
at-
proteins
focusing
with
sugars
in
present
to
study
by isoelectric are present should be
different sugar to characterize
separated
or might
by
lectins,
could with
and
molecular
properties and indeweight of the lectins to
These
HRP,
in
sequence
of histo-
gel slabs this method
glycoproteins gels.
spe-
peroxi-
the
separated
by diffusion molecular
on
carbo-
with
usefulness
extended
Other lectins with could be employed
the
tric
the
horseradish
on polyacrylamide surface of the gel,
unencumbered pendent of the
ing
with
techniques
focusing on the
further glycoproteins
coupled
isoelecreactive alterna-
if
be employed this
enzyme
as
be conjugated employed
in
in
the
to HRP
by
a single
stain-
step.
ACKNOWLEDGMENTS
The efficient Mr.
authors would technical and
Price
Oulla
and
like to acknowledge secretarial assistance Ms.
Dot
LITERATURE
mi-
albumin
have laboratory.
between to be a-
N-acetylglucosam
to characterize
moieties
isoelectric and the six
electrophoresis, of the
of
of
strong
points appear
classification
glutaraldehyde
former have been electrophoretic which, in pore
with the breakthrough bind to a Con A column
lack
with 4.09
unit
components
their
ability
tively
approach
insight
of eluates
the
while
and
be employed. specificities
contains an
apparent 4.41 have
edge
These
ied,
further
glycoproteins
Con A affinity chromatography peatedly performed in our been
differ-
identified.
bands 3.83 and
bands with pH 4.19 and
Pi type
less was
be assessed
presently
acrylamide
Further both
prod-
25%
further
characterized. The the two-dimensional to be a-i-globulins
with
with
product
PAS-positive
A-negative between pH
uration.
demonstrate
isoelectric do not
per
or internal
level. of
attempt
Such
Pi allelles
additional
Con
allele
to gain
Con
points
the
products
could
mannose.
be applied
also
each
reported
types
as each
internal
four
if these
allele
but
or terminal
The
than
contain no
which
with 4.41 amounts
hydrate
with the Con A-HRP method, suggest that such a comparison
does
sufficient may
which
Although
1-globulins,
been
sufficiently
these
proteins
considerable
chemical
compare
Z allele,
six
A binding 4.19 and
ZZ and
entity. appears
to determine
quantitatively this study could
rather
The
tempted.
to study
the
(4).
sugin the
MZ,
individually and
Pi
differ
need
of the
of Con
to M-3
MM,
the
technique
carbohydrate
the
Pi type
to study
of
that conformational observation has
as a single
present
Pi
M-1
individually,
antitrypsin
sensitive
This
unit
indication
chains or
per three
number
bands
emphasizes
product
total
expected in
changes sera
in
in the
The
carbohydrate
allele products are present.
several
A-HRP
unexpected. the
double
differences
protein
were
A-reactive
SZ
Con
Blue-stained
products
gests
the
the of
Smith. CITED
1. Allen RC, Harley RA, Talamo RC: A new method for determination of alpha-1-antitrypsin phenotypes using isolectric focusing on polyacrylamide gel slabs. Am J Clin Pathol 62:732, 1974 2. Bernhard W, Avrameas 5: Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp Cell Res 64:232, 1971
3. Caldwell RC, human saliva chem Biophys 4.
Chan
SK,
1-protease
Rees
Pigma W: Disc in polyacrylamide 110:91, 1965 DC: Molecular
inhibitor
deficiency.
electrophoresis gels. Arch basis
for
Nature
the
of Bio-
alpha225:240,
1975
5. Graham RC Jr, Karnovsky MJ: The absorption of injected horseradish
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early stages peroxidase
of in
914
ALLEN,
the proximal tubules of mouse tural cytochemistry by a new chem Cytochem 14:291, 1966 6. Komfeld R, Ferris C: Interaction lin
glycopeptides
Chem 7. Maurer
with
kidney: technique.
SPICER
ultrastrucJ Histo-
of immunoglobu-
concanavalin
250:2614, 1975 HR, Allen RC: Polyacrylamide
AND
A.
J
phoresis
8. Biol
ZEHR
9. Rennert gel electro-
in clinical
chemistry:
dardization and performance. 40:359, 1972 McManus JFA, Hoch-Ligeti acid spot test for 1,2 glycols. ysaccharides.
OM:
Disc
problems Clin C: Lab
electrophoresis
Nature
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Modified Invest
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mucopol-
