Vol. 64, No. 2,1975
BIOCHEMICAL
IMMOBILIZATION MATRICES Garfield
P.
OF ENZYMES REDUCTIVE
BY
Royer,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Frederick
A.
Department The Ohio Columbus,
Received
March
ON ALDEHYDIC ALKYLATIONY Liberatore,
and
Gail
M.
Green
of Biochemistry State University Ohio 43210
21,1975
SUMMARY. We report here a convenient and inexpensive method of attaching enzymes to solid supports which contain diols. Dextran coated porous glass, Sepharose and glass coated with a glyceryl silane were oxidized with NaI04. Trypsin, carboxypeptidase A, and carboxypeptidase B were bound to the oxidized supports by treatment with NaBl&. The pH dependence of the coupling reaction and loss of lysine in bound trypsin indicate that the immobilization occurs activity
via reductive alkylation. against synthetic substrates
Reductive troduce
alkyl
bases
to sodium
couple
coated
goal
aldehyde
and
(NaB&)
or to
carriers
our without
This work was (GM-19605) and the t Abbreviations benzoyl-L-arginine
the
is
to develop
the
amino
of group
Sepharose The
good
to
may
(l-4). be We
have
had
been
neutral,
Glycophase-CPG
Schiff’s
reduced
controlled-pore are
in-
with used
this
oxidized glass
hydrophilic is
sup-
porous
glass
Si(CH2)30CH&HOHCHaOH. a method
introduction
of
charge.
to
activate A neutral
neutral support
supported by grants from the National Institutes Pierce Chemical Co. used: CPG-controlled-pore glass; Bz-L-argOEt, ethylester; Hip-L-phe, hippuryl-L-phenylalanine:
hippuryl-L-arginine.
Copyright o 1975 b-v Academic Press, Inc. All rights of reproduction in any form reserved.
catalytic
specifically
lysine
which
Dextran-coated
structure.
work
groups
supports
and
Glass;
method
cyanoborohydride.
carbohydrate
silane:
display
gentle
E-amino
sodium
(NaIO.+).
porous
a glyceryl
:k
arg,
an
metaperiodate
of
and at
enzymes
a highly
with One
a convenient
Glycophase-CPG,
with
bound enzymes and proteins.
proteins
between
(Dextran-CPG),
drate
into
borohydride
reaction
ports
is
groups
formed
sodium
with
alkylation
The
47%
carbohyis
ad-
of Health N-oHip-L-
Vol. 64, No. 2, 1975
vantageous
in
cases nor
where product
that no
is
ling
in
is
advantage
limitations
this nor
lower.
to
a neutral
with
expensive
in
applications
our are
of bound
of
(7-S).
Even
method
operating and
the
in
substrate carbohydrate with
seems and
stability
changed
neither of
involved
enzymes
be
Activation
dimensional
variety
not
Also,
bromide
reagents
a wide
(5-6).
technique,
the
RESEARCH COMMUNICATIONS
should
support.
cyanogen
activation
under
enzyme
occur
Moreover,
carriers,
siderable
of the
optimum
done
unpleasant
drate-glass
raphy
(9)
reaction
pH
adsorb
commonly
improvements
AND BIOPHYSICAL
diffusional
should
supports
Neither
the
BIOCHEMICAL
recent
preferable. pH
of the
of
the
coup-
carbohy-
conditions,
is
a con-
affinity
chromatog-
. MATERIALS
AND
METHODS
Glycophase -CPG( 550 A, lot 500) and Dextran-CPG (550 A, lot 100) were obtained from the Pierce Chemical Co. Sepharose-4B was purchased from Pharmacia. Trypsin (EC 3.4.4.4, lot 2RL2DA) and carboxypeptidase B (EC3.4.2.2, lot 54A345) were supplied by Worthington. Carboxypeptidase A was the product of Miles-Seravac (EC 3.4.2. l., batch 5-B). Zinc-free insulin was prepared by the method of Carpenter (10). N, N-dimethyl BSA was prepared as described by Lin, et al. (11). Oxidation of the Supports. Glycophase -CPG (lg) was placed in a 20 ml vi al with 10 ml of NATO* solution (6mM). A vacuum was applied for about 2 min to remove air from within the matrix. The glass was rotated slowly on a constant torque stirrer for 1 hr at 25’. Dextran-CPG and Sepharose (1 ml) were oxidized in the same way but with 0.1 M NaI04. The materials were washed with 200 ml of distilled water. Enzyme Coupling. For trypsin immobilization the buffers were 0.1 M borate (pH 8.5 and 8.0) and 0.1 M HEPES (pH 7.5 and 7.0); both buffers contained 1mM benzamidine. Carboxypeptidase A and carboxypeptidase B were coupled at pH 8.5 in 0. 1 M borate buffer, 1 M NaCl. Enzyme solution (5m1, lmg/ ml) and 150 mg of oxidized glass or 1 ml of oxidized Sepharose were rotated in a 20 ml vial at 4’. At timed intervals aliquots were withdrawn and assayed. At t = 20 min and t = 40 min NaB&( h 0.5 mg) was added. The reaction was terminated after 1 hr and the bound enzymes were washed with 200 ml of distilled water, 200 ml of 2M KCl, and an additional 200 ml of distilled water. The amount of protein bound to the supports was determined as previously described (13). Enzyme Assays. The catalytic activities of trypsin, carboxypeptidase A, and carboxypeptidase B were determined with Bz-L-arg-OEt, Hip-L-phe, and Hip-L-arg respectively. For the bound enzymes assays were performed in columns at a flow rate of 2.3 ml/min. The free volume, V was determined with a 1 mh4 tryptophan solution under conditions of assay (13). The appearance of product as a result of passage of substrate solution through the col-
479
Vol. 64, No. 2, 1975
BfOCHEMfCAL AND BfOPHYSfCAL RESEARCH COMMUNICATIONS
umn was the feed
determined by measuring the substrate solution as reference. Digestion of N, N-dimethyl BSA by
was
followed
Moore
by
( 14).
treating
Conditions
the
column
for
proteolysis
absorbance the
(254nrr$of
various
effluent
bound
with
were
the
as
the
effluent
trypsin
with
derivatives
ninhydrin
reagent
follows:
0. 1 M
of
N-ethyl
mor-
pholine-acetate
buffer pH8,. 025 M CaCl,; protein, 1 mg/ml; 37’; flow-rate, 0.23 ml/min. After about 1 hr of column operation, 1 ml of effluent was collected and mixed with 1 ml of ninhydrin reagent. Samples were boiled for 10 min and diluted to 5 ml with 50Fethanol. The absorbance (570 nm)of the samples was determined. Complete digestion was accomplished by recirculating the protein solution through a column of Dextran-CPG-trypsin. The column was prepared by mixing 50 mg of the damp enzyme conjugate with 750 mg of damp unmodified Dextran-CPG. The column diameter was 0.8 cm. RESULTS The -CPG,
courses
Dextran-CPG
itial
rapid
formation ose
time
and
termined
depletion
for
the
and
Sepharose
the
before
addition
of the
pH
8.5
solution
and and
of trypsin
at from
support
Glycophase-CPG,
DISCUSSION
immobilization
of enzyme
between
AND
enzyme,
a slight damp
are
be
adsorption the
carrier.
0 0 0 A
shown
may
dilution;
on
Control
oxidized in
Figure
1.
to
Schiff’s
case
of
ascribed in
the
activity
Glycophase
at
experiments
t =
The
inbase
Sephar-
0 was were
deper-
CONTROL DEXTRAN - CPG GLYCOPHASE - CPG SEPHAROSE
01 ’
1
I
1
I
2
IO
20
40
60
TIME (min) Time Figure 1. of Glycopshase-G done at 4 and pH
course CPG, 8.5.
of trypsin Dextran-coated
attachment CPG
480
of periodate-oxidized and Sepharose.
The
derivatives coupling was
BIOCHEMICAL
Vol. 64, No. 2, 1975
formed the
with pH
at pH be
unoxidized
range 8 and
In
with No
pH
was
loosely
bound.
acid
reduced
of trypsin
to
Glycophase-CPG
and
trypsin
could
at pH
Treatment the
over
of trypsin
bound
adsorbed
strongly
trypsin
amounts
very
808acetic
adsorption
no
8
of the
adsorption
Gly-
of trypsin
occurred
at pH
7.0
7.5.
When
the
occurred.
solid
support
this
experiment
In
mg,
portions
of
mal
coupling
experiment
suggests
that
duced
to In
The
or
was
Glycophase-CPG
enzyme
NaB&
adsorbed significant
however,
The
the
Dextran-CPG adsorbed
enzyme,
KCl.
case
slightly.
or
2M
this
cophase-CPG only
The
with
The
Sepharose
8.5.
off
8.5.
7.0-8.5.
pH
washed
and
of
supports.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NaBH.,
any
2 the
decrease
in
a result
of the
formation
(eq.
over
a 40
as
with
reduced
prior
oxidized
aldehyde
alcohols
Figure
was
This
Figure
which
1, are
of enzyme,no was
period.
in
groups
addition
Dextran-CPG
min
shown
to
with
material,
bound
not
treated used
no
bonded
binding
in
enzyme. to
the
two,
0.5
a nor-
This enzyme
result are
re-
NaB%.
4
pH-dependences
amount
of the
of enzyme
instability
bound
of NaBQ
immobilization with
and
the
pH
reactions
decreasing
pH
is
dependence
of
the
are most
shown.
probably
Schiff’s
base
1) CHO
t
aHN-Prot
2
k
CH
= N-Prot
) 1 NaBK,
IF+ 0
(1) H,N-Prot
CHZ-NH-Prot >
In
the
ines
cases could
amino
result
from
and reaction
Sepharose of two
it
is
adjacent
conceivable
that
aldehyde
groups
tertiary
am-
with
the
same
the
bound
group. If
reaction
zymes tice
of Dextran-CPG
are that
the
(1) subjected number
applies,one to
acid
of points
would
expect
hydrolysis of
and
attachment
481
a loss
in
aen.0
acid
correlates
lysine
when
analysis(Table with
the
1). amount
enNo-
of pro-
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100 9080 :5
70-
5 u
60-
g
50-
x
40-
9 -0 o\
30D GLYCOPHASE - CPG A SEPHAROSE 0 DEXTRAN -CPG
1
20
t
/
01
* 7.0
A 7.5
I 8.0
1 8.5
pH OF COUPLING Figure 2. cophase-CPG, zyme bound
tein
bound.
the
amount
the
pore
The
pH-dependence Dextran-CPG, at pH 8.5 is taken
Also, bound size
The
the to
as
kcat
values
Vf/Q
in
which
and
Q is
the
flow
rate.
in
catalyzes these
been
the
arylamine
in
and
ations
(16-17).
crease
these The
Vf
bound
one
free
from
to
Glythe
less
en-
than
reduction
of
equation,
AP=
amount
f
10%)
as
the
enzyme
native
of a bound
kcat
doubt
trypsin
value
for by
bound
used
enzyme
has
M bound
reduced
of enzyme
column
Dextran-CPG-try-
aminopeptidase
The no
( well
the
of the
activity
i.e.
glass(15).
the
oxidized
activity
is due
volume
of
are in
probably
as
case,
Sepharose-trypsin
the
Dextran-CPG
error
retention
of porous
on
case
calculated
Bz-arg-OEt
other
to is
experimental
Complete at least
1 were the
of
each
coating.
represents
Within
Reduction k,,t
dextran
Table
hydrolysis
derivative
CPG-trypsin
in
of trypsin In
bound This
of the
experiments.
reported
of protein
Glycophase-CPG.
shown
E,
psin
amount
a result
k cat
of the immobilization and Sepharose. as 100%.
to
an
Glycophasediffusional
would
limit-
probably
in-
values.
trypsins
are
also
active
against
482
protein
(Table
1).
In
this
case
BIOCHEMICAL
Vol. 64, No. 2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
el T rypsin
Derivative
Protein (Mg/lOO
6;
a
bound mg sup-
*set
Moles moles
(%a~~:~CoEt)~%A)
lys lost of trypsin
port) Dextran-CPG
0.13
14.3
0.45
2.0
Sepharose
0.76
6.6
0.19
4.1
2.21
3.8
0.04
7.6
0.60’
-
Glycophase
-CPG
-
Native a.
Determined with ImM Bz-Arg-OEt at pH 8.0, 0.025 M CaCla, 0.05 TRfS, 25’. Determined with 1 mg/ml N, N dimethyl BSA, 0.1 M, N-ethyl morpholineacetate, pH 8, 37’; 0.025 M CaCla. Estimated considerable curvature was seen in the assays of soluble trypsin with N, N-dimethyl BSA. Lin et al (11) observed this in trypsin assays with other protein substrates.
b. c.
also,
the
catalytic
efficiency
tran-CPG-trypsin 16
14
hr
Complete
would
be
useful
digest
by
trypsin does
in
.
A.
In
peptidase chain
enzyme
suggests
products
they
cases
an show
the
for
applications
the
also
been
Dex-
be
BSA
in
a
Dextran-CPG-trypsin
sequencing. not
increases.
of N, N-dimethyl
that
would
B have
extensive
Contamination a problem,
of the
since
from
against
ammo
hydrolysis
which
the
acids
of polylysine C-terminal
alanine
carbohydrate-glass in
which
were
supports
quantitive
recovery
483
by of these
Hip-L-phe A is
the
the
immobilized
characterization
activity
expected
B catalyzes
conclusion,
loading
digestion
Glycophase-CPG-carboxypeptidase both
enzyme
the
bound
autolyze.
Although
of insulin In
as
peptides
A and
complete,
tively
complete
preparing autolysis
not
here.
not
the hydrolysis
Carboxypeptidases cribed
down
catalyzed
digest.
enzyme
goes
and
had
been
appear of products
is
insulin Bound
the
des-
respec-
against
released.
method
derivatives
Hip-L-arg,
active
and
the
and
BS-
carboxy-
aminoethylated
B-
removed. very is
promising
for
essential.
In
Vol. 64, No. 2, 1975
addition, rates.
these
BIOCHEMICAL
materials
are
rigid
Use of the carbohydrate-glass
ing techniques
described
here
should
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and may be used materials also
with
be valuable
in columns the activation in affinity
at high
flow
and bindchromatog-
raphy. REFERENCES 1 Means, G. E. and Feeney, R. E. (1971) Ch emical Modification of Proteins, p. 130-135, Holden-Day, San Francisco. G. E. and Feeney, R.E. (1968) Biochemistry 7-, 2192-2201. 2. Means, W. B. and Snell, E.E. (1963) BiochemistryL, 1414-1419. 3. Dempsey, M., Williams, D. L., and Masri, M.S. (1974) --Int. J. Peptide 4. Friedman, Protein e, L, 183-185. G.P. and Meyers, W.E. (1974) in Bonded Stationary in 5. Royer, --Phases Chromatography , ed. by E. Gruska, p. 93-112, Ann Arbor Sci., Mich. G. P. (1974) Chemtech 4, 694-700. 6. Royer, J. and Ernbach, S. (1967) Nature 214, 1302-1304. 7. Axen, R., Porath, J., Axen, R. and Ernbach, S.(1967) 1491-1492. 8. Porath, --Nx215, S. C., Parikh, I., and Cuatrecasas, Biochem. (1974) 2, 9. March, --P. Anal. 149-152. 10. Carpenter, F. H. (1958) Arch. Biochem. Biophys. 78,539-545. G.E., and Feeney, R.E. (1969) --J. Biol. Chem.e,78911. Lin, Y. , Means, 793. G. P. and Uy, R. (1973) J. Biol. Chem. 248, 2627-2629. 12. Royer, M.D., Hornby, W. E., and Crook, E.M., (1966) Biochem. J. 100, 13. Lilly, 718-723. S. (1968) --J. Biol. Chem., -,243 6281-6283. 14. Moore, G.P. and Andrews, J.P. (1973) ---J. Biol. Chem. 248,1807-1812. 15. Royer, R., Goldstein, L. and Katchalski, E. (1971) in “Biochemical 16. Goldman, Aspects of Reactions on Solid Supports” ed. by G. R. Stark, Academic Press, N.Y. p. 1. 17. Royer, G. P. in “Immobilized Enzymes, Antibodies, Antigens, and PepTides” ed. by H. H. Weetall, Marcel Dekker, N. Y. (in press).
484
BIOCHEMICAL
IMMOBILIZATION MATRICES Garfield
P.
OF ENZYMES REDUCTIVE
BY
Royer,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Frederick
A.
Department The Ohio Columbus,
Received
March
ON ALDEHYDIC ALKYLATIONY Liberatore,
and
Gail
M.
Green
of Biochemistry State University Ohio 43210
21,1975
SUMMARY. We report here a convenient and inexpensive method of attaching enzymes to solid supports which contain diols. Dextran coated porous glass, Sepharose and glass coated with a glyceryl silane were oxidized with NaI04. Trypsin, carboxypeptidase A, and carboxypeptidase B were bound to the oxidized supports by treatment with NaBl&. The pH dependence of the coupling reaction and loss of lysine in bound trypsin indicate that the immobilization occurs activity
via reductive alkylation. against synthetic substrates
Reductive troduce
alkyl
bases
to sodium
couple
coated
goal
aldehyde
and
(NaB&)
or to
carriers
our without
This work was (GM-19605) and the t Abbreviations benzoyl-L-arginine
the
is
to develop
the
amino
of group
Sepharose The
good
to
may
(l-4). be We
have
had
been
neutral,
Glycophase-CPG
Schiff’s
reduced
controlled-pore are
in-
with used
this
oxidized glass
hydrophilic is
sup-
porous
glass
Si(CH2)30CH&HOHCHaOH. a method
introduction
of
charge.
to
activate A neutral
neutral support
supported by grants from the National Institutes Pierce Chemical Co. used: CPG-controlled-pore glass; Bz-L-argOEt, ethylester; Hip-L-phe, hippuryl-L-phenylalanine:
hippuryl-L-arginine.
Copyright o 1975 b-v Academic Press, Inc. All rights of reproduction in any form reserved.
catalytic
specifically
lysine
which
Dextran-coated
structure.
work
groups
supports
and
Glass;
method
cyanoborohydride.
carbohydrate
silane:
display
gentle
E-amino
sodium
(NaIO.+).
porous
a glyceryl
:k
arg,
an
metaperiodate
of
and at
enzymes
a highly
with One
a convenient
Glycophase-CPG,
with
bound enzymes and proteins.
proteins
between
(Dextran-CPG),
drate
into
borohydride
reaction
ports
is
groups
formed
sodium
with
alkylation
The
47%
carbohyis
ad-
of Health N-oHip-L-
Vol. 64, No. 2, 1975
vantageous
in
cases nor
where product
that no
is
ling
in
is
advantage
limitations
this nor
lower.
to
a neutral
with
expensive
in
applications
our are
of bound
of
(7-S).
Even
method
operating and
the
in
substrate carbohydrate with
seems and
stability
changed
neither of
involved
enzymes
be
Activation
dimensional
variety
not
Also,
bromide
reagents
a wide
(5-6).
technique,
the
RESEARCH COMMUNICATIONS
should
support.
cyanogen
activation
under
enzyme
occur
Moreover,
carriers,
siderable
of the
optimum
done
unpleasant
drate-glass
raphy
(9)
reaction
pH
adsorb
commonly
improvements
AND BIOPHYSICAL
diffusional
should
supports
Neither
the
BIOCHEMICAL
recent
preferable. pH
of the
of
the
coup-
carbohy-
conditions,
is
a con-
affinity
chromatog-
. MATERIALS
AND
METHODS
Glycophase -CPG( 550 A, lot 500) and Dextran-CPG (550 A, lot 100) were obtained from the Pierce Chemical Co. Sepharose-4B was purchased from Pharmacia. Trypsin (EC 3.4.4.4, lot 2RL2DA) and carboxypeptidase B (EC3.4.2.2, lot 54A345) were supplied by Worthington. Carboxypeptidase A was the product of Miles-Seravac (EC 3.4.2. l., batch 5-B). Zinc-free insulin was prepared by the method of Carpenter (10). N, N-dimethyl BSA was prepared as described by Lin, et al. (11). Oxidation of the Supports. Glycophase -CPG (lg) was placed in a 20 ml vi al with 10 ml of NATO* solution (6mM). A vacuum was applied for about 2 min to remove air from within the matrix. The glass was rotated slowly on a constant torque stirrer for 1 hr at 25’. Dextran-CPG and Sepharose (1 ml) were oxidized in the same way but with 0.1 M NaI04. The materials were washed with 200 ml of distilled water. Enzyme Coupling. For trypsin immobilization the buffers were 0.1 M borate (pH 8.5 and 8.0) and 0.1 M HEPES (pH 7.5 and 7.0); both buffers contained 1mM benzamidine. Carboxypeptidase A and carboxypeptidase B were coupled at pH 8.5 in 0. 1 M borate buffer, 1 M NaCl. Enzyme solution (5m1, lmg/ ml) and 150 mg of oxidized glass or 1 ml of oxidized Sepharose were rotated in a 20 ml vial at 4’. At timed intervals aliquots were withdrawn and assayed. At t = 20 min and t = 40 min NaB&( h 0.5 mg) was added. The reaction was terminated after 1 hr and the bound enzymes were washed with 200 ml of distilled water, 200 ml of 2M KCl, and an additional 200 ml of distilled water. The amount of protein bound to the supports was determined as previously described (13). Enzyme Assays. The catalytic activities of trypsin, carboxypeptidase A, and carboxypeptidase B were determined with Bz-L-arg-OEt, Hip-L-phe, and Hip-L-arg respectively. For the bound enzymes assays were performed in columns at a flow rate of 2.3 ml/min. The free volume, V was determined with a 1 mh4 tryptophan solution under conditions of assay (13). The appearance of product as a result of passage of substrate solution through the col-
479
Vol. 64, No. 2, 1975
BfOCHEMfCAL AND BfOPHYSfCAL RESEARCH COMMUNICATIONS
umn was the feed
determined by measuring the substrate solution as reference. Digestion of N, N-dimethyl BSA by
was
followed
Moore
by
( 14).
treating
Conditions
the
column
for
proteolysis
absorbance the
(254nrr$of
various
effluent
bound
with
were
the
as
the
effluent
trypsin
with
derivatives
ninhydrin
reagent
follows:
0. 1 M
of
N-ethyl
mor-
pholine-acetate
buffer pH8,. 025 M CaCl,; protein, 1 mg/ml; 37’; flow-rate, 0.23 ml/min. After about 1 hr of column operation, 1 ml of effluent was collected and mixed with 1 ml of ninhydrin reagent. Samples were boiled for 10 min and diluted to 5 ml with 50Fethanol. The absorbance (570 nm)of the samples was determined. Complete digestion was accomplished by recirculating the protein solution through a column of Dextran-CPG-trypsin. The column was prepared by mixing 50 mg of the damp enzyme conjugate with 750 mg of damp unmodified Dextran-CPG. The column diameter was 0.8 cm. RESULTS The -CPG,
courses
Dextran-CPG
itial
rapid
formation ose
time
and
termined
depletion
for
the
and
Sepharose
the
before
addition
of the
pH
8.5
solution
and and
of trypsin
at from
support
Glycophase-CPG,
DISCUSSION
immobilization
of enzyme
between
AND
enzyme,
a slight damp
are
be
adsorption the
carrier.
0 0 0 A
shown
may
dilution;
on
Control
oxidized in
Figure
1.
to
Schiff’s
case
of
ascribed in
the
activity
Glycophase
at
experiments
t =
The
inbase
Sephar-
0 was were
deper-
CONTROL DEXTRAN - CPG GLYCOPHASE - CPG SEPHAROSE
01 ’
1
I
1
I
2
IO
20
40
60
TIME (min) Time Figure 1. of Glycopshase-G done at 4 and pH
course CPG, 8.5.
of trypsin Dextran-coated
attachment CPG
480
of periodate-oxidized and Sepharose.
The
derivatives coupling was
BIOCHEMICAL
Vol. 64, No. 2, 1975
formed the
with pH
at pH be
unoxidized
range 8 and
In
with No
pH
was
loosely
bound.
acid
reduced
of trypsin
to
Glycophase-CPG
and
trypsin
could
at pH
Treatment the
over
of trypsin
bound
adsorbed
strongly
trypsin
amounts
very
808acetic
adsorption
no
8
of the
adsorption
Gly-
of trypsin
occurred
at pH
7.0
7.5.
When
the
occurred.
solid
support
this
experiment
In
mg,
portions
of
mal
coupling
experiment
suggests
that
duced
to In
The
or
was
Glycophase-CPG
enzyme
NaB&
adsorbed significant
however,
The
the
Dextran-CPG adsorbed
enzyme,
KCl.
case
slightly.
or
2M
this
cophase-CPG only
The
with
The
Sepharose
8.5.
off
8.5.
7.0-8.5.
pH
washed
and
of
supports.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NaBH.,
any
2 the
decrease
in
a result
of the
formation
(eq.
over
a 40
as
with
reduced
prior
oxidized
aldehyde
alcohols
Figure
was
This
Figure
which
1, are
of enzyme,no was
period.
in
groups
addition
Dextran-CPG
min
shown
to
with
material,
bound
not
treated used
no
bonded
binding
in
enzyme. to
the
two,
0.5
a nor-
This enzyme
result are
re-
NaB%.
4
pH-dependences
amount
of the
of enzyme
instability
bound
of NaBQ
immobilization with
and
the
pH
reactions
decreasing
pH
is
dependence
of
the
are most
shown.
probably
Schiff’s
base
1) CHO
t
aHN-Prot
2
k
CH
= N-Prot
) 1 NaBK,
IF+ 0
(1) H,N-Prot
CHZ-NH-Prot >
In
the
ines
cases could
amino
result
from
and reaction
Sepharose of two
it
is
adjacent
conceivable
that
aldehyde
groups
tertiary
am-
with
the
same
the
bound
group. If
reaction
zymes tice
of Dextran-CPG
are that
the
(1) subjected number
applies,one to
acid
of points
would
expect
hydrolysis of
and
attachment
481
a loss
in
aen.0
acid
correlates
lysine
when
analysis(Table with
the
1). amount
enNo-
of pro-
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100 9080 :5
70-
5 u
60-
g
50-
x
40-
9 -0 o\
30D GLYCOPHASE - CPG A SEPHAROSE 0 DEXTRAN -CPG
1
20
t
/
01
* 7.0
A 7.5
I 8.0
1 8.5
pH OF COUPLING Figure 2. cophase-CPG, zyme bound
tein
bound.
the
amount
the
pore
The
pH-dependence Dextran-CPG, at pH 8.5 is taken
Also, bound size
The
the to
as
kcat
values
Vf/Q
in
which
and
Q is
the
flow
rate.
in
catalyzes these
been
the
arylamine
in
and
ations
(16-17).
crease
these The
Vf
bound
one
free
from
to
Glythe
less
en-
than
reduction
of
equation,
AP=
amount
f
10%)
as
the
enzyme
native
of a bound
kcat
doubt
trypsin
value
for by
bound
used
enzyme
has
M bound
reduced
of enzyme
column
Dextran-CPG-try-
aminopeptidase
The no
( well
the
of the
activity
i.e.
glass(15).
the
oxidized
activity
is due
volume
of
are in
probably
as
case,
Sepharose-trypsin
the
Dextran-CPG
error
retention
of porous
on
case
calculated
Bz-arg-OEt
other
to is
experimental
Complete at least
1 were the
of
each
coating.
represents
Within
Reduction k,,t
dextran
Table
hydrolysis
derivative
CPG-trypsin
in
of trypsin In
bound This
of the
experiments.
reported
of protein
Glycophase-CPG.
shown
E,
psin
amount
a result
k cat
of the immobilization and Sepharose. as 100%.
to
an
Glycophasediffusional
would
limit-
probably
in-
values.
trypsins
are
also
active
against
482
protein
(Table
1).
In
this
case
BIOCHEMICAL
Vol. 64, No. 2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
el T rypsin
Derivative
Protein (Mg/lOO
6;
a
bound mg sup-
*set
Moles moles
(%a~~:~CoEt)~%A)
lys lost of trypsin
port) Dextran-CPG
0.13
14.3
0.45
2.0
Sepharose
0.76
6.6
0.19
4.1
2.21
3.8
0.04
7.6
0.60’
-
Glycophase
-CPG
-
Native a.
Determined with ImM Bz-Arg-OEt at pH 8.0, 0.025 M CaCla, 0.05 TRfS, 25’. Determined with 1 mg/ml N, N dimethyl BSA, 0.1 M, N-ethyl morpholineacetate, pH 8, 37’; 0.025 M CaCla. Estimated considerable curvature was seen in the assays of soluble trypsin with N, N-dimethyl BSA. Lin et al (11) observed this in trypsin assays with other protein substrates.
b. c.
also,
the
catalytic
efficiency
tran-CPG-trypsin 16
14
hr
Complete
would
be
useful
digest
by
trypsin does
in
.
A.
In
peptidase chain
enzyme
suggests
products
they
cases
an show
the
for
applications
the
also
been
Dex-
be
BSA
in
a
Dextran-CPG-trypsin
sequencing. not
increases.
of N, N-dimethyl
that
would
B have
extensive
Contamination a problem,
of the
since
from
against
ammo
hydrolysis
which
the
acids
of polylysine C-terminal
alanine
carbohydrate-glass in
which
were
supports
quantitive
recovery
483
by of these
Hip-L-phe A is
the
the
immobilized
characterization
activity
expected
B catalyzes
conclusion,
loading
digestion
Glycophase-CPG-carboxypeptidase both
enzyme
the
bound
autolyze.
Although
of insulin In
as
peptides
A and
complete,
tively
complete
preparing autolysis
not
here.
not
the hydrolysis
Carboxypeptidases cribed
down
catalyzed
digest.
enzyme
goes
and
had
been
appear of products
is
insulin Bound
the
des-
respec-
against
released.
method
derivatives
Hip-L-arg,
active
and
the
and
BS-
carboxy-
aminoethylated
B-
removed. very is
promising
for
essential.
In
Vol. 64, No. 2, 1975
addition, rates.
these
BIOCHEMICAL
materials
are
rigid
Use of the carbohydrate-glass
ing techniques
described
here
should
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and may be used materials also
with
be valuable
in columns the activation in affinity
at high
flow
and bindchromatog-
raphy. REFERENCES 1 Means, G. E. and Feeney, R. E. (1971) Ch emical Modification of Proteins, p. 130-135, Holden-Day, San Francisco. G. E. and Feeney, R.E. (1968) Biochemistry 7-, 2192-2201. 2. Means, W. B. and Snell, E.E. (1963) BiochemistryL, 1414-1419. 3. Dempsey, M., Williams, D. L., and Masri, M.S. (1974) --Int. J. Peptide 4. Friedman, Protein e, L, 183-185. G.P. and Meyers, W.E. (1974) in Bonded Stationary in 5. Royer, --Phases Chromatography , ed. by E. Gruska, p. 93-112, Ann Arbor Sci., Mich. G. P. (1974) Chemtech 4, 694-700. 6. Royer, J. and Ernbach, S. (1967) Nature 214, 1302-1304. 7. Axen, R., Porath, J., Axen, R. and Ernbach, S.(1967) 1491-1492. 8. Porath, --Nx215, S. C., Parikh, I., and Cuatrecasas, Biochem. (1974) 2, 9. March, --P. Anal. 149-152. 10. Carpenter, F. H. (1958) Arch. Biochem. Biophys. 78,539-545. G.E., and Feeney, R.E. (1969) --J. Biol. Chem.e,78911. Lin, Y. , Means, 793. G. P. and Uy, R. (1973) J. Biol. Chem. 248, 2627-2629. 12. Royer, M.D., Hornby, W. E., and Crook, E.M., (1966) Biochem. J. 100, 13. Lilly, 718-723. S. (1968) --J. Biol. Chem., -,243 6281-6283. 14. Moore, G.P. and Andrews, J.P. (1973) ---J. Biol. Chem. 248,1807-1812. 15. Royer, R., Goldstein, L. and Katchalski, E. (1971) in “Biochemical 16. Goldman, Aspects of Reactions on Solid Supports” ed. by G. R. Stark, Academic Press, N.Y. p. 1. 17. Royer, G. P. in “Immobilized Enzymes, Antibodies, Antigens, and PepTides” ed. by H. H. Weetall, Marcel Dekker, N. Y. (in press).
484
