Trypsin-catalysed formation of pig des-(23-63)-proinsulin from desoctapeptide-(B23-30)-insulin

Abstract

Incubation of pig desoctapeptide-(B23-30)-insulin with trypsin in solvent systems consisting of dimethyl sulphoxide, butane-1,4-diol and Tris buffer resulted in the formation of an extra peptide bond between Arg-B22 and Gly-A1 in the DOPI molecule. This DOPI derivative can also be regarded as pig des-(23-63)-proinsulin. The structure of the new, previously unreported, proinsulin analogue was determined on the basis of amino acid analysis, dansylation and digestion with Staphylococcus aureus V8 proteinase. Receptor-binding ability of des-(23-63)-proinsulin was 20% of that of pig desoctapeptide-(B23-30)-insulin and 0.02% of that of pig insulin.

Full text

Biochem.
J.
(1986)
234,
665-670
(Printed
in
Great
Britain)
Trypsin-catalysed
formation
of
pig
des-(23-63)-proinsulin
from
desoctapeptide-(B23-30)-insulin
Teresa
KUBIAK
and
David
COWBURN
The
Rockefeller
University,
1230
York
Avenue,
New
York,
NY
10021,
U.S.A.
Incubation
of
pig
desoctapeptide-(B23-30)-insulin
with.
trypsin
in
solvent
systems
consisting
of
dimethyl
sulphoxide,
butane-
1,4-diol
and
Tris
buffer
resulted
in
the
formation
of
an
extra
peptide
bond
between
Arg-B22
and
Gly-Al
in
the
DOPI
molecule.
This
DOPI
derivative
can
also
be
regarded
as
pig
des-(23-63)-proinsulin.
The
structure
of
the
new,
previously
unreported,
proinsulin
analogue
was
determined
on
the
basis
of
amino
acid
analysis,
dansylation
and
digestion
with
Staphylococcus
aureus
V8
proteinase.
Receptor-binding
ability
of
des-(23-63)-proinsulin
was
20%
of
that
of
pig
desoctapeptide-(B23-30)-insulin
and
0.02%
of
that
of
pig
insulin.
INTRODUCTION
Desoctapeptide-(B23-30)-insulin
(DOPI)
is
an
import-
ant
substrate
for
the
enzyme-assisted
semisynthesis
of
insulin
and
insulin
analogues
by
the
method
of
Inouye
et
al.
(1979,
1981a).
In
most
of
the
cases
reported
so
far,
DOPI
was
N-protected
before
trypsin-catalysed
couplings
(Inouye
et
al.,
1979,
198
la,
b;
Gattner
et
al.,
1980;
Tager
et
al.,
1980;
Cao
et
al.,
1981;
Cui
et
al.,
1983;
Shoelson
et
al.,
1983;
Kobayashi
et
al.,
1984a,
b).
We
have
successfully
used
unprotected
DOPI
as
a
substrate
for
trypsin-catalysed
semisyntheses
(T.
Kubiak
&
D.
Cowburn,
unpublished
work),
and
observed
an
unusual
by-product
under
some
conditions.
This
product
is
shown
here
to
be
pig
des-(23-63)-proinsulin
(Dprol).
MATERIALS
AND
METHODS
Materials
and
general
methods
Zinc
pig
insulin
(lot
7NR38B)
was
purchased
from
the
Elanco
Product
Co.
(Indianapolis,
IN,
U.S.A.).
Trypsin
[L-
1
-chloro-4-phenyl-3-tosylamidobutan-2-one
('TPCK
')-treated]
was
obtained
from
Worthington
Biochemical
Corp.
Sephadex
G-50
(fine
grade)
was
from
Pharmacia.
Tris
was
from
Sigma
Chemical
Co.,
and
silica-gel
plates
HLF
from
Analtech.
Other
reagents
were
of
analytical
grade
from
Aldrich
Chemical
Co.
and
were
used
without
further
purification.
The
pH
values
reported
are
the
apparent
values,
direct
readings
obtained
with
a
glass
electrode,
uncorrected
for
the
presence
of
organic
solvents.
Hydrolyses
(6
M-HCI)
were
carried
out
in
evacuated
Pierce
hydrolysis
tubes
at
110
°C
for
24
h
in
the
presence
of
phenol
(1
drop
per
1
ml
of
6
M-HCI)
unless
otherwise
specified.
Amino
acid
analyses
were
performed
on
a
Spinco
model
MS
amino
acid
analyser.
Receptor-binding
assay
was
conducted
with
liver
plasma
membranes
(McCaleb
&
Donner,
1981).
DOPI
was
prepared
from
zinc-free
insulin
(Insulin
Research
Group,
Shanghai
Institute
of
Biochemistry,
1976)
as
described
by
Tager
et
al.
(1980).
The
crude
product
was
purified
on
Sephadex
G-50
in
3
M-acetic
acid.
DOPI
incubation
with
trypsin:
illustrative
procedure
A
sample
of
DOPI
(20
mg,
4
,mol)
was
dissolved
in
0.72
ml
ofthe
solvent
system
dimethyl
sulphoxide/butane-
1
,4-diol/0.25
M-Tris/acetate
buffer
(pH
7.5)
(5:5:4,
by
vol.)
and
the
pH
was
adjusted
to
7.00
at
25
°C
with
3
M-acetic
acid
(0.024
ml).
Then
trypsin
powder
(2
mg)
was
added.
This
dissolved
rapidly
and
the
mixture
was
incubated
at
37
°C
for
4
h.
The
DOPI
concentration
was
5.4
mm,
and
the
water
content
of
the
buffer
and
acetic
acid
was
28%
(v/v).
The
enzyme
action
was
stopped
by
the
addition
of
50%
(v/v)
acetic
acid
(0.8
ml),
and
the
mixture
was
loaded
on
to
a
Sephadex
G-50
column
(1
cm
x
90
cm),
which
was
eluted
with
3
M-acetic
acid
at
a
flow
rate
of
24
ml/h;
fractions
(1.6
ml
each)
were
monitored
at
280
nm.
Fractions
corresponding
to
the
5000-Mr
material
were
pooled
and
freeze-dried.
The
yield
was
15.2
mg.
The
products
were
isolated
from
the
5000SMr
material
by
either
preparative
h.p.l.c.
on
Zorbax
ODS
(Fig.
1)
or
on
DEAE-Sephadex
A-25
(Cui
et
al.,
1983)
as
shown
in
Fig.
2(a).
Characterization
of
the
unknown
Trypsin
digestion
of
the
unknown
(0.5
mg)
was
conducted
in
0.2
ml
of
0.1
M-N-ethylmorpholine
buffer,
pH
8,
containing
EDTA
(2
mM),
CaCl2
(20
mM)
and
trypsin
(0.05
mg).
The
mixture
was
incubated
for
3
h
at
37
'C.
After
2
h
and
3
h
small
portions
were
taken,
diluted
with
h.p.l.c.
solvent
A
and
examined
by
h.p.l.c.
on
the
Zorbax
ODS
column.
The
experiment
was
accompanied
by
a
control
that
lacked
trypsin.
Performic
acid-oxidized
(Hirs,
1967)
unknown
was
danyslated
and
hydrolysed
as
described
by
Gray
(1972).
Digestion
with
Staphylococcus
aureus
V8
proteinase
was
performed
by
the
method
of
Chance
et
al.
(1981).
After
digestion
the
mixture
was
frozen,
freeze-dried,
then
redissolved
in
h.p.l.c.
solvent
A
(0.3
ml)
and
separated
by
preparative
h.p.l.c.
on
Zorbax
C-8
(Fig.
4a).
Material
corresponding
to
peak
IA
was
collected,
freeze-dried
and
purified
by
preparative
paper
chromatography
on
Whatman
3MM
paper
(6
cm
x
12
cm)
in
propan-2-
ol/1
M-acetic
acid
(2:1,
v/v).
The
band
containing
Abbreviations
used:
DOPI,
desoctapeptide-(B23-30)-insulin;
Dprol,
I-sulphonyl.
des-(23-63)-proinsulin;
Dns
or
dansyl,
5-dimethylaminonaphthalene-
Vol.
234
665

T.
Kubiak
and
D.
Cowburn
Sakaguchi-positive
(Greenstein
&
Winitz,
1961)
and
chlorine/tolidine-positive
material
(RF
0.7)
was
cut
out
and
eluted
with
3
M-acetic
acid.
The
eluate
was
concentrated
under
reduced
pressure,
filtered
through
a
Pasteur
pipette
containing
a
plug
of
glass-fibre
paper
and
freeze-dried.
Half
of
this
material
was
hydrolysed
(6
M-HCl,
110
C,
48
h)
and
subjected
to
amino
acid
analysis.
One-fifth
of
the
remainder
was
dansylated
in
the
medium
containing
N-ethylmorpholine
(Gray,
1972).
The
N-terminal
arginine
was
identified
in
the
6
M-HCI
hydrolysate
of
the
dansylated
sample
by
comparison
with
standard
Dns-arginine
on
electrophoresis
in
8%
(v/v)
formic
acid
(Gray,
1972).
The
same
procedures
were
used
for
the
isolation
and
characterization
of
the
(Al-4)-
pentapeptide
from
the
DOPI
digest
(Fig.
4b,
peak
AB
material),
as
well
as
the
(A13-17)-pentapeptide
fragment
(Fig.
4,
peaks
2A
and
2B).
reaction
medium
without
trypsin
(Table
1,
Expt.
2).
Other
conditions
used
for
the
DOPI
incubation
with
trypsin
are
listed
in
Table
1.
Oligomerizationwasconcentration-andpH-dependent.
Generally,
at
lower
initial
DOPI
concentrations
(approx.
5
mM)
less
of
the
higher-Mr
products
was
found
(Table
1,
Expts.
3-7).
In
the
pH
range
6.4-6.7
relatively
more
oligomers
were
formed
as
compared
with
similar
experiments
conducted
at
pH
6.0
or
7.0
(Table
1,
Expts.
1,
3
and
4).
The
formation
of
the
unknown
depended
on
pH
and
the
butane-1,4-diol
content
in
the
incubation
mixtures
as
shown
in
Table
1.
It
was
observed
that
the
reaction
reached
its
equilibrium
after
4
h
of
incubation.
A
prolonged
incubation
time
led
to
more
oligomers,
RESULTS
AND
DISCUSSION
In
model
studies
on
the
use
of
trypsin
as
a
catalyst
for
peptide-bond
formation
the
optimum
pH
for
the
enzyme-catalysed
syntheses
was
found
to
be
near
6.5
(Inouye
et
al.,
1979).
The
presence
of
organic
solvents,
such
as
dimethylformamide
or
dimethyl
sulphoxide,
or
polyhydroxy
alcohols
in
the
reaction
medium
greatly
shifted
the
peptide-bond
equilibrium
towards
synthesis.
Inouye
et
al.
(1979,
1981a,
b)
successfully
pioneered
the
use
of
semisynthetic
procedures
involving
trypsin-assisted
coupling
of
the
t-butoxycarbonyl-protected
DOPI
with
synthetic
(B23-30)-octapeptide(s)
for
the
preparation
of
human
insulin
and
a
variety
of
insulin
analogues.
When
unprotected
DOPI
was
incubated
with
trypsin
under
similar
conditions
to
those
used
by
Inouye
et
al.
(1981a)
(Table
1,
Expt.
1),
h.p.l.c.
on
Zorbax
ODS
revealed
the
presence
of
an
extra
peak
at
Rt
26
min,
observed
along
with
that
of
DOPI
at
Rt
11.6
min
(Fig.
1).
The
same
unknown
product
was
present
along
with
the
DOPI
in
the
-
5000-Mr
material
after
Sephadex
G-50
chromatography.
Formation
of
the
unknown
was
accompanied
by
significant
oligomerization,
since
only
42%
of
the
-
5000
Mr
material
was
recovered
after
gel
filtration.
The
rest
of
it
was
converted
by
the
enzyme
into
higher-Mr
products,
which
were
co-eluted
with
trypsin
(Table
1,
Expt.
1).
Neither
the
unknown
nor
oligomeriza-
tion
was
observed
when
DOPI
was
incubated
in
the
3
35
z
0
I
31
o
0
c
0
27
o
0
8
16
24
32
36
Retention
time
(min)
Fig.
1.
H.p.l.c.
profile
of
the
DOPI/trypsin
incubation
mixture
on
a
DuPont
Zorbax
ODS
column
(0.94
cm
x
25
cm)
Solvent
A
consisted
of
0.125
M-NaH2PO,
adjusted
to
pH
2.45
with
H3PO4.
A
20
min
linear
gradient
from
28%
to
35%
(v/v)
acetonitrile
in
solvent
A
was
used.
The
final
solvent
composition
was
maintained
for
an
additional
15
min.
The
flow
rate
was
2
ml/min,
with
monitoring
at
220
nm.
Peaks:
1,
solvent
peak;
2,
DOPI;
3,
Dprol.
Table
1.
DOPI
incubation
with
trypsin
The
water
content
of
the
incubation
mixture
includes
0.25
M-Tris/acetate
buffer
and
the
diluted
acetic
acid
used
to
adjust
the
pH.
The
5000-Mr
material
was
obtained
by
chromatography
on
Sephadex
G-50,
and
the
DOPI
and
DproI
contents
were
determined
by
the
h.p.l.c.
peak
heights
on
Zorbax
ODS.
Initial
DOPI
Dprol
concn.
of
Me2SO/
Water
content
in
content
in
Expt.
DOPI
1,4-Bu(OH)2
content
DOPI/trypsin
Incubation
5000-Mr
5000-Mr
5000-Mr
no.
(mM)
ratio
(v/v)
(%,
v/v)
pH
ratio
(w/w)
time
(h)
material
(%)
material
(%)
material
(%)
1:1
1:1
1:1
1:1
1:1
1:2
1:2
24
6.7
10:1
24
6.7
10:0
32
6.4
10:1
32
6.4
10:1
28
7.0
10:1
28
7.0
10:1
29
6.0
10:1
19
19
19
4
4
4
4
42
91
61
65
76
80
89
49
100
68
69
77
56
82
51
0
32
31
23
44
18
1986
1
10.0
2
10.0
3
4.3
4
4.2
5
5.4
6
5.2
7
5.2
666
1
0

Semisynthetic
proinsulin
analogue
Table
2.
Amino
acid
compositions
of
DOPI
and
related
peptides
(theoretical
values
in
parentheses)
Peptides
IA,
IB,
2A
and
2B
were
isolated
by
h.p.l.c.
(Fig.
4)
followed
by
preparative
paper
chromatography.
A
hydrolysis
time
of
40
h
was
used
for
peptides
IA
and
lB.
Amino
acid
composition
(mol
of
residue/mol)
Amino
acid
DOPI
Dns-DOPI
Dprol
Dns-Dprol
Peptide
lA
Peptide
lB
Peptide
2A
Peptide
2B
Asp
Thr
Ser
Glu
Gly
Ala
Cys*
Val
Ile
Leu
Tyr
Phe
His
Arg
2.97
(3)
1.09
(1)
2.63
(3)
7.0
(7)
3.02
(3)
1.2
(1)
+
2.6t
(4)
I.lt
(2)
6.33
(6)
3.26
(3)
0.91
(1)
2.32
(2)
1.19
(1)
3.15
(3)
1.08
(1)
3.02
(3)
6.79
(7)
2.35
(2)
1.20
(1)
+
3.01t
(4)
1.23t
(2)
6.05
(6)
0:
0.32
(0)
2.10
(2)
0.99
(1)
3.22
0.93
2.89
7.00
3.48
1.28
+
2.83t
1.07t
5.81
3.14
0.99
1.95
1.02
3.3
1.06
2.93
6.77
3.33
1.30
+
2.97
1.30
6.49
0:
0.32
2.02
1.06
*
Half-cystine
values
were
not
integrated.
t
Low
values
due
to
incomplete
hydrolysis
under
the
conditions
used.
t
Tyrosine
was
converted
into
O-Dns-tyrosine.
0.25
0.05
0.17
0.92
(1)
1.00
(1)
0.10
1.12
(1)
1.07
(1)
0.40
0.04
0:30
1.2
(1)
1.00
(1)
0.10
0.80t
(1)
0.86t
(1)
0.14
0.20
1.98
(2)
1.96
(2)
2.00
(2)
1.97
(2)
0.98
(1)
1.01
(1)
1.01
(1)
0.6
0.3
(a)
2
0.4
0.2
0.20.
0
0
20
40
60
80
100
Fraction
no.
0.15
-
(b)
2
,
__
0.6
0.4
i
0.05
~~~~~~~0.2
'Z
0
0
20
40
60
Fraction
no.
Fig.
2.
Ion-exchange
chromatography
of
the
mixture
of
Dprol
and
DOPI
(a)
The
Sephadex
G-50-purified
incubation
mixture
(5000-Mr
material,
37
mg)
was
dissolved
in
the
initial
buffer
(15
ml)
and
loaded
on
to
a
DEAE-Sephadex
A-25
column
(1
cm
x
20
cm).
The
initial
buffer
was
40%
propan-2-ol
in
0.05
M-Tris
adjusted
to
pH
7.6
with
HC1
(200
ml),
and
the
final
buffer
was
0.15
M-NaCl
in
the
initial
buffer
(200
ml);
a
combination
of
linear
gradient
and
isocratic
elution
was
used
(see
the
gradient
line
------).
The
fraction
size
was
S
ml.
Pools
1
and
2
were
concentrated
under
vacuum
and
desalted
on
Sephadex
G-10.
Peaks:
1,
Dprol
(8.7
mg);
2,
DOPI
(25.5
mg).
(b)
SP
(sulphopropyl)-
Sephadex
C-25
chromatography
of
the
mixture
of
DOPI
and
DproL.
The
column
dimensions
were
2
cm
x
25
cm.
though
the
ratio
of
DOPI
to
the
unknown
remained
essentially
the
same.
Enzyme-catalysed
formation
of
the
unknown
was
reversible.
Trypsin
hydrolysed
the
unknown
to
DOPI
in
aqueous
0.1
M-N-ethylmorpholine
buffer,
pH
8,
when
no
organic
solvents
were
added.
Within
a
digestion
period
of
2
h
the
h.p.l.c.
peak
of
the
unknown
disappeared
while
that
of
DOPI
emerged.
The
amino
acid
composition
of
the
unknown
was
found
to
be
the
same
as
that
of
DOPI
(Table
2).
However,
the
unknown
was
not
a
DOPI
dimer,
judged
by
its
Sephadex
G-50
elution
volume.
The
unknown
was
also
generated
from
DOPI
by
trypsin
in
the
solvent
system
consisting
of
dimethyl
sulphoxide
and
Tris/acetate
buffer
with
no
butane-1,4-diol
added.
Therefore
the
possibility
of
ester
formation
between
butane-1,4-diol
and
the
Arg-B22
residue
was
excluded.
Since
the
unknown
was
more
hydrophobic
than
DOPI,
on
the
basis
of
its
h.p.l.c.
retention
time,
we
considered
the
case of
trypsin-catalysed
acylation
of
the
hydroxy
groups
of
tyrosine,
serine
and/or
threonine
by
the
Arg-B22
residue
in
DOPI.
O-Acyl
derivatives
of
the
three
amino
acids
mentioned
above
are
known
to
be
readily
hydrolysed
under
mild
conditions
to
generate
the
original
functional
groups
(Bodanszky
et
al.,
1976).
The
hydrolysis
proceeds
more
rapidly
in
the
presence
of
ammonia
or
amines,
and
is
complete
within
minutes
(Bodanszky
et
al.,
1976;
Riordan
&
Vallee,
1972).
No
DOPI
was
formed
from
the
unknown
on
incubation
with
2.8%
(v/v)
ammonia
or
aq.
50%
(v/v)
isopropylamine
The
initial
solvent
was
3
M-acetic
acid
(100
ml),
and
the
final
solvent
was
3
M-acetic
acid
containing
0.5
M-NaCl
(100
ml);
a
linear
gradient
of
0-0.5
M-NaCl
was
used
(------).
The
fraction
size
was
4
ml.
Pools
1
and
2
were
desalted
on
Sephadex
G-10.
Peaks:
1,
DproI
(1.1
mg);
2,
DOPI
(5.5
mg).
Vol.
234
667

T.
Kubiak
and
D.
Cowburn
Fig.
3.
Primary
structure
of
pig
proinsulin
(Show
&
Chance,
1968)
The
sequence
of
pig
insulin
is
represented
by
the
amino
acids
in
dark
circles.
Thin
unbroken
arrows
indicate
the
sequence
that
is
deleted
in
DproL.
Broken
arrows
show
expected
S.
aureus
proteinase
cleavage
sites
in
DOPI.
within
a
period
of
4
h.
Therefore
it
was
concluded
that
the
unknown
does
not
possess
any
ester
bonds.
The
unknown
was
eluted
before
DOPI
from
a
DEAE-Sephadex
column
(Fig.
2a),
as
well
as
from
an
SP-Sephadex
column
(Fig.
2b),
which
indicated
that
it
has
at
least
one
less
negative
and
one
less
positive
charge
as
compared
with
DOPI
under
the
conditions
used
for
separations.
Dansylation,
performed
on
the
performic
acid-oxidized
unknown,
followed
by
6
M-HCl
hydrolysis,
revealed
only
the
presence
of
the
N-terminal
phenylalanine
residue.
It
turned
out
that
the
Gly-Al
was
blocked
and
not
available
for
dansylation,
since
all
three
glycine
residues
were
found
in
the
dansylated
unknown
hydrolysate
as
compared
with
only
two
in
the
hydrolysate
of
Dns-DOPI
(Table
2).
The
above
results
show
that
the
trypsin-catalysed
peptide-bond
formation
between
the
Arg-B22
and
Gly-A
l
residues
took
place
leading
to
the
conversion
of
DOPI
into
the
unknown.
This
new
DOPI
derivative
can
also
be
regarded
as
a
proinsulin
analogue,
namely
Dprol
(Fig.
3).
To
prove
that
such
a
proinsulin
analogue
was
generated,
the
unknown
was
subjected
to
digestion
with
S.
aureus
V8
proteinase
under
the
conditions
described
for
insulin
by
Chance
et
al.
(1981).
S.
aureus
V8
proteinase
specifically
hydrolyses
peptide
bonds
on
the
carboxy
side
of
glutamic
acid
residues
(Houmard
&
Drapeau,
1972).
In
addition,
it
was
determined
that
in
the
insulin
molecule
an
extra
cleavage
occurred
at
the
Ser-A12-Leu-A13
bond
(Chance
et
al.,
1981).
If
trypsin
had
catalysed
formation
of
the
peptide
bond
between
the
Arg-B22
and
Gly-Al
residues,
we
should
expect
to
find
the
Arg-B22-(A1-4)-pentapeptide
in
the
S.
4ureus
V8
proteinase
digest
of
the
unknown.
As
reported
by
Chance
et
al.
(1981),
the
(A1-4)-
tetrapeptide,
deriving
from
insulin,
was
eluted
in
the
very
first
peak
from
a
Zorbax
C-8
column.
Since
the
hypothetical
Arg-B22-(A1-4)-pentapeptide
should
be
more
hydrophilic
than
the
(A1-4)-tetrapeptide,
we
expected
it
to
appear
on
h.p.l.c.
in
the
eluate
at
the
same
retention
time
as,
or
earlier
than,
the
(Al-4)-tetrapeptide.
The
Zorbax
C-8
profiles
of
the
S.
aureus
proteinase
digest
of
the
unknown
and
DOPI
are
shown
in
Fig.
4.
The
peak
lA
fraction
from
the
digest
of
the
unknown
(Fig.
4a)
was
further
analysed
on
t.l.c.
It
contained
a
chlorine/tolidine-positive
and
Sakaguchi-positive
material,
with
RF
0.30,
different
from
arginine
(RF
0.12),
and
a
minor
chlorine/tolidine-positive
Sakaguchi-nega-
tive
contaminant,
with
RF
0.8
(Table
3).
The
arginine-
containing
peptide
(Sakaguchi-positive
material)
was
isolated
and
desalted
by
preparative
paper
chromato-
graphy.
The
same
procedures
were
used
for
the
final
purification
of
the
Zorbax
C-8
fractions
IB,
2A
and
2B
(Figs.
4a
and
4b).
It
is
worth
noting
that
neither fraction
1986
668
I

Semisynthetic
proinsulin
analogue
669
1.
2
(a)
0.8
l
A
-
-------
40
0.4
2
A
--2
0
C
0
03
12
(b)
CD
CD)
0.4
20
0
0
8
16
24
32
40
48
56
Retention
time
(min)
Fig.
4.
H.p.l.c.
profiles
for
the
S.
aureus
proteinase
digest
of
3
mg
of
Dprol
(a)
and
3
mg
of
DOPI
(b)
The
chromatography
was
accomplished
with
a
DuPont
Zorbax
C-8
column
(0.46
cm
x
25
cm)
by
using
slight
modifications
of
the
conditions
of
Chance
et
al.
(1981).
Solvent
A
consisted
of
0.1
M-(NH4)2HP04
in
water/meth-
oxyethanol
(19:1,
v/v)
and
solvent
B
was
acetonitrile/
methoxyethanol
(19:1,
v/v).
A
combination
of
isocratic
and
gradient
elution
was
used
(see
the
gradient
line
------).
The
column
was
thermostatically
maintained
at
40
'C.
The
flow
rate
was
1
ml/min.
Peaks:
IA,
Arg-B22-(Al-4)-
pentapeptide;
IB,
(Al-4)-tetrapeptide;
2A
or
2B,
(A
l
3-17)-pentapeptide.
lA
nor
fraction
I
B
contained
free
arginine,
as
shown
by
t.l.c.
(Table
3).
The
amino
acid
compositions
of
peptides
isolated
from
fractions
IA,
I
B,
2A
and
2B
(Table
2),
and
their
N-terminal
amino
acids,
determined
by
dansylation,
were
consistent
with
the
sequences
Arg-B22-(A
1-4)-
pentapeptide,
(A1-4)-tetrapeptide,
(Al
3-1
7)-pentapep-
tide
and
(Al
3-17)-pentapeptide
respectively.
Other
peaks
in
the
chromatograms
of
Fig.
4
show
slight
differences
between
DOPI
and
Dprol,
presumably
because
of
incomplete
digestion
under
the
conditions
used,
similar
to
those
observed
by
Chance
et
al.
(1981).
The
presence
of
the
Arg-B22-(Al-4)-peptide
in
the
S.
aureus
V8
proteinase
digest
of
the
unknown
confirmed
the
trypsin-catalysed
conversion
of
DOPI
into
DproL.
Dprol
showed
0.02%
of
the
receptor-binding
ability
of
that
of
pig
insulin
and
20%
of
that
of
DOPI.
Note
added
in
proof
(received
18
December
1985)
After
this
paper
was
submitted,
Markussen
et
al.
(1985)
reported
the
trypsin-catalysed
formation
of
the
single-
chain
des-(B30)-insulin,
the
des-(Ala-B30)
analogue
with
a
peptide
bond
between
Lys-B29
and
Gly-Al.
The
maximum
yield
obtained
was
about
13%,
compared
with
approx.
50
%
for
Dprol
reported
here,
and
this
difference
might
possibly
reflect
closer
location
or
less
restricted
reaction
for
the
Arg-B22/Gly-Al
reaction
in
DOPI
compared
with
the
Lys-B29/Gly-A1
reaction
in
des-
(Ala-B30)-insulin.
Both
des-(Ala-B30)-insulin
and
Dprol
show
very
low
activity
compared
with
insulin,
of
the
order
of
that
of
proinsulin
(Chance
et
al.,
1968).
We
are
grateful
to
Dr.
David
Donner,
Memorial
Sloan
Kettering
Cancer
Center,
for
bioassay
data.
This
research
was
supported
by
National
Institutes
of
Health
Grant
AM-20357.
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Table
3.
Peptides
isolated
from
the
S.
aureus
V8
proteinase
digests
of
Dprol
and
DOPI
Chromatograms
were
developed
in
the
solvent
system
propan-2-ol/
1
M-acetic
acid
(2:1,
v/v).
Key:
S,
Sakaguchi-positive
material,
C,
chlorine/tolidine-positive
material.
RF
values
Paper
chromatography
Fraction
T.l.c.
on
silica-gel
on
Whatman
3MM
(see
Fig.
4)
plates
(5
cm
x
10
cm)
(6
cm
x
12
cm)
Proteinase
digest
of
Dprol
IA
0.3
(C,
S);
0.8
(C)
0.7
(C,
S);
0.88
(C)
2A
0.83
(C)
0.92
(C)
Proteinase
digest
of
DOPI
lB
0.74
(C);
0.8
(C)
0.85
(C);
0.88
(C)
2B
0.83
(C)
0.92
(C)
Standard
Arg
0.12
(C,
S)
0.32
(C,
S)
Vol.
234

670
T.
Kubiak
and
D.
Cowbun
Inouye,
K.,
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K.,
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&
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Y.
(1981a)
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Watanabe,
K.,
Tochino,
Y.,
Kanaya,
T.,
Kobayashi,
M.
&
Shigeta,
Y.
(198
1b)
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811-
813
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(1976)
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351-357
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Received
1
July
1985/13
September
1985;
accepted
7
November
1985
1986