Biochem.
J.
(1975)
147,435-438
Printed
in
Great
Britain
Identification
of
Amino
Acid
Thiohydantoins
Directly
by
Thin-Layer
Chromatography
and
Indirectly
by
Gas-Liquid
Chromatography
after
Hydrolysis
By
MINNIE
RANGARAJAN
and
ANDRE
DARBRE
Department
of
Biochemistry,
University
of
London
King's
College,
Strand,
London
WC2R
2LS,
U.K.
(Received
20
November
1974)
A
method
is
described
for
the
identification
of
amino
acid
thiohydantoins
by
two-
dimensional
t.l.c.
An
indirect
method
for
the
determination
of
amino
acid
thiohydantoins
is
described
by
which,
after
hydrolysis,
the
corresponding
amino
acids
are
determined
by
g.l.c.
The
sequential
degradation
of
peptides
from
the
C-terminus
by
the
method
of
Stark
(1968)
requires
the
identification
of
the
cleaved
amino
acid
thiohydantoin.
This
can
be
achieved
qualitatively
by
using
two-dimensional
t.l.c.
directly,
or,
after
hydrolysis,
by
determination
of
the
amino
acids
quantitatively
by
g.l.c.
The
preferred
method
for
determining
the
sequence
of
amino
acids
in
proteins
is
that
of
Edman
(1949),
in
which
the
N-terminal
amino
acids
are
converted
into
their
phenylthiohydantoin
deriva-
tives
(Edman
&
Begg,
1967;
Pisano
&
Bronzert,
1969;
Laursen,
1971;
Pisano
et
al.,
1972;
Peterson
et
al.,
1972).
Methods
for
sequencing
from
the
C-
terminus
are
less
successful
and
are
mostly
restricted
to
the
determination
of
the
terminal
amino
acid
by
hydrazinolysis
(Akabori
et
al.,
1956)
or
by
3H
labelling
(Matsuo
et
al.,
1966),
or
of
a
limited
number
of
residues
by
using
carboxypeptidase
(Ambler,
1967a,b).
The
method
originally
proposed
by
Schlack
&
Kumpf
(1926)
for
the
formation
of
C-terminal
peptidyl-thiohydantoins
was
shown
to
have
appli-
cation
as
a
sequencing
method
(Stark,
1968).
The
amino
acid
thiohydantoin
cleaved
from
the
C-terminus
of
the
peptide
was
not
determined
directly,
but
indirectly
by
difference
analysis
(Stark,
1968).
In
later
work
the
amino
acid
thiohydantoin
was
identified
by
t.l.c.
(Cromwell
&
Stark,
1969;
Yamashita,
1971)
and
by
g.l.c.
and
mass
spectro-
metry
(Rangarajan
etal.,
1973;
Rangarajan
&Darbre,
1974).
Because
we
were
unable
to
identify
arginine
we
developed
a
rapid
two-dimensional
t.l.c.
separation
on
pre-coated
polyamide
plates
for
all
the
common
amino
acid
thiohydantoins.
Also,
we
report
on
the
hydrolysis
of
the
thiohydantoins
to
their
constituent
amino
acids,
so
that
these
may
be
dtermind
by
g.Lc.
Vol.
147
Materials
and
Methods
Polygram
polyamide-6/UV254
and
Polygram
poly-
amide-6
pre-coated
(0.1mm)
plastic
sheets
(20cm
x
20cm)
and
a
Desaga
UVIS
lamp
with
emission
at
254nm
were
purchased
from
Camlab
(Cambridge,
U.K.).
Chromatography
was
carried
out
with
solvent
system
1,
dichloroethane-acetic
acid
(45:8,
v/v),
2,
chloroform-95
%
(v/v)
ethanol-acetic
acid
(20:10:3,
by
vol.),
3,
toluene-n-heptane-acetic
acid
(12:6:5,
by
vol.),
4,
acetic
acid-water
(7:13,
v/v),
5,
acetic
acid-water
(1:
3,
v/v).
Butyl-PBD
[5-(4-biphenylyl)-
2-(4-t-butylphenyl)-1-oxa-3,4-diazole]
was
purchased
from
Intertechnique
Ltd.,
Portslade,
Sussex,
U.K.
This
was
used
at
a
concentration
of
0.025%
(w/v)
in
solvent
systems
1,
2
and
3
when
plates
not
incor-
porating
a
fluorescent
indicator
were
used.
Amino
acid
thiohydantoins
were
prepared
as
described
by
Rangarajan
et
al.
(1973).
Amino
acids
were
determined
as
their
trifluoroacetylated
methyl
ester
derivatives
(Darbre
&
Islam,
1968;
Islam
&
Darbre,
1972).
Acid
hydrolyses
were
carried
out
in
Rotaflo
stopcocks
(Darbre,
1971).
Results
and
Discussion
Thin-layer
chromatography
One-dimensional
t.l.c.
of
amino
acid
thiohydan-
toins
on
silica-gel
plates
was
described
by
Cromwell
&
Stark
(1969)
and
Yamashita
(1971).
We
were
unable
to
resolve
unambiguously
many
of
these
compounds
and
therefore
developed
a
two-dimen-
sional
separation
on
polyamide
pre-coated
plates,
as
reported
previously
for
methylthiohydantoin
amino
acids
(Rabin
&
Darbre,
1974).
In
Table
1
the
R,
values
(x100)
are
reported
for
the
obe-dimensional
separation
of
19
amino
acid
thik-
435
M.
RANGARAJAN
AND
A.
DARBRE
Table
1.
RF
(x100)
values
for
amino
acid
thiohydantoins
in
one-dimensional
separations
on
polyamide
plates
The
solvent
systems
are
described
in
the
Materials
and
Methods
section.
Results
are
averages
of
four
determinations.
Abbreviation:
CmCys,
carboxymethylcysteine.
Amino
acid
thiohydantoin
Solvent
system
Ala
Arg
Asn
Asp
Cys
CmCys
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Ser
Thr
Trp
Tyr
Val
...
1
57
20
18
63
76
4
12
37
18
75
86
49
65
83
45
40
34
81
77
2
76
80
33
49
50
33
68
65
75
85
90
88
84
84
87
76
67
60
88
lOOxRF
3
31
5
2
1
39
4
S
16
4
49
65
9
32
51
27
25
8
1
56
4
72
90
63
70
57
43
64
75
93
56
56
83
61
45
58
57
27
45
63
5
64
87
75
59
36
21
53
68
92
41
39
75
47
29
55
44
16
28
51
hydantoins
in
five
different
solvent
systems.
The
chromatography
tank
was
used
without
equilibra-
tion
of
the
atmosphere
and
the
solvents
could
be
used
without
change
of
RF
values
for
up
to
1
week.
The
polyamide
plates
incorporating
a
fluorescent
indicator
were
most
convenient.
When
viewed
under
u.v.,
dark-purple
spots
were
seen
on
a
green
fluorescent
background.
In
the
absence
of
this
indicator,
butyl-PBD
was
used
as
an
external
indicator,
but
it
was
soluble
only
in
solvent
systems
1,
2
and
3.
With
plates
incorporating
the
fluorescent
indicator,
the
lower
third
of
the
plate
was
very
dark
when
viewed
under
u.v.
after
chromatography
in
solvent
systems
4
and
5.
The
best
two-dimensional
separation
was
obtained
with
solvent
system
4
(Kulbe,
1971)
used
for
the
first
dimension
followed
by
solvent
system
2
(Cromwell
&
Stark,
1969),
giving
a
total
running
time
of
1
h.
The
schematic
representation
of
the
separation
of
19
amino
acid
thiohydantoins
is
shown
in
Fig.
1.
The
origin
was
1
cm
from
the
edges
of
the
plate,
which
was
sectioned
to
limit
the
solvent
flow
to
7.0cm
from
the
origin,
and
the
markers
in
the
outer
zones
were
tyrosine
and
carboxymethylcysteine
(see
Rabin
&
Darbre,
1974).
Butyl-PBD
was
used
in
solvent
system
2
when
plates
did
not
include
an
indicator.
Under
u.v.,
dark-purple
spots
appeared
on
a
pale-purple
fluorescent
background.
The
limit
of
detection
was
approx.
0.5nmol
for
alanine
thio-
hydantoin.
Colour
differences
in
daylight
helped
identification.
Histidine,
tryptophan
and
tyrosine
thiohydantoin
spots
were
yellow.
On
heating
to
about
80°C,
the
thiohydantoins
of
aspartic
acid
and
aspara-
gine
turned
yellow,
those
of
glycine,
serine
and
threonine
turned
pink
and
that
of
S-carboxymethyl-
cysteine
turned
brown.
We
attempted
to
prepare
glutamine
thiohydantoin,
but
t.l.c.
with
different
solvents
failed
to
distinguish
between
glutamine
and
glutamic
acid
thiohydantoins.
It
is
suspected
that
the
strong
acid
used
during
the
preparation
of
glutamine
thiohydantoin
results
in
its
hydrolysis
to
glutamic
acid
thiohydantoin.
This
would
probably
occur
when
the
method
of
Stark
(1968)
is
applied
to
a
peptide
and
thus
glutamine
would
not
be
distinguished
from
glutamic
acid.
No
suitable
peptide
was
available
for
this
study.
Rabin
&
Darbre
(1974)
were
unable
to
distinguish
between
the
methylthiohydantoins
of
glutamine
and
glutamic
acid.
These
authors
pointed
out
that
Kulbe
(1971)
and
Summers
et
al.
(1973),
although
using
similar
solvent
systems
with
polyamide
plates,
disagreed
over
the
relative
positions
of
glutamic
acid
phenylthiohydantoin
and
glutamine
phenyl-
thiohydantoin.
Suzuki
et
al.
(1973)
reported
that
they
were
unable
to
prepare
glutamine
hydantoin.
Hydrolysis
of
thiohydantoins
In
addition
to
the
methods
for
the
direct
identifi-
cation
of
amino
acid
thiohydantoins
by
g.l.c.
and
mass
spectrometry
(Rangarajan
et
al.,
1973)
it
was
desirable
to
have
an
indirect
procedure
by
which,
after
hydrolysis,
the
amino
acids
could
be
determined
1975
436
IDENTIFICATION
OF
AMINO
ACID
THIOHYDANTOINS
c
0
c
E
-o
0
i1
o
First
dimension
Fig.
1.
Schematic
representation
of
two-dimensional
t.l.c.
of
19
amino
acid
thiohydantoins
Solvent
system
4
was
used
for
the
first
dimension
and
solvent
system
2
for
the
second
dimension.
(See
the
Materials
and
Methods
section.)
by
g.l.c.
Stark
(1968)
hydrolysed
amino
acid
thiohydantoins
under
alkaline
conditions
with
low
yields
of
amino
acids.
Turner
&
Schmerzler
(1954)
reported
severe
decomposition
of
some
derivatives
by
hydrobromic
acid.
Because
some
amino
acid
hydantoins
could
be
hydrolysed
more
successfully
than
the
corresponding
thiohydantoins,
Cromwell
&
Stark
(1969)
removed
the
sulphur
from
thiohydan-
toins
by
oxidative
desulphuration
before
hydrolysing
them
with
alkali.
We
studied
methods
of
acid
hydrolysis
and
concluded
that
low
yields
might
be
due
to
either
oxidative
destruction
or
to
incomplete
hydrolysis.
The
results
in
Table
2
were
with
6M-HCI,
containing
0.1
%
phenol
(Li
&
Yanofsky,
1972)
and
1
mM-fi-mercaptoethanol,
at
135°C
for
23
h.
Arginine
thiohydantoin
on
hydrolysis
gave
arginine
only,
and
not
ornithine
as
was
obtained
after
alkaline
hydrolysis
of
arginine
phenylthiohydantoin
(Van
Orden
&
Carpenter,
1964)
and
arginine
hydantoin
(Stark
&
Smyth,
1963).
Hydrolysis
of
serine
thiohydantoin
gave
alanine
(approx.
30
%)
by
g.l.c.
All
the
amino
acid
Vol.
147
thiohydantoins
studied
gave
single
g.l.c.
peaks,
except
those
given
special
mention.
Isoleucine
thiohydantoin
prepared
from
L-isoleucine
(allo-free)
always
yielded
a
mixture
of
L-isoleucine
and
D-allo-
isoleucine
(Rangarajan
et
al.,
1973).
Cysteine
thio-
hydantoin
gave
a
mixture
of
alanine
(approx.
20%)
and
cysteine
(approx.
13
%).
S-Carboxymethyl-
cysteine
thiohydantoin
gave
a
mixture
of
glycine
(approx.
85
%)
and
methionine
(approx.
5
%).
Tryptophan
thiohydantoin
gave
a
mixture
of
glycine
(approx.
19%)
and
alanine
(approx.
5%),
although
Inglis
et
al.
(1971),
using
hydroiodic
acid
hydrolysis,
claimed
a
total
yield
of
better
than
70%
from
the
phenylthiohydantoin.
Threonine
thiohydantoin
was
exceptional
in
giving
three
amino
acids
on
hydrolysis,
a-amino-n-
butyric
acid
(3
%),
aspartic
acid
(6
%)
and
homoserine
(64
%)
which
were
identified
and
determined
by
g.l.c.
When
threonine
thiohydantoin
was
hydrolysed
with
alkali
(Baptist
&
Bull,
1953)
the
products
detected
by
paper
chromatography
were
a-amino-n-butyric
acid
o
Origin
Cm-Cys
Tyr
'
Leu
se3abvi
ELys
Phe
lie
Met
QArs
OThr
QAIa
OHI
Q
Tp
Tyr
0G0
GyC
Tyr
OCys
Q
Asp
QCm-Cys
Q
Asn
Dcm-Cys
0
a
Origin
Origin
437
438
M.
RANGARAJAN
AND
A.
DARBRE
Table
2.
Recovery
of
amino
acids
from
their
thiohydantoin
derivatives
after
acid
hydrolysis
Hydrolysis
was
carried
out
in
Rotaflo
stopcocks
with
6M-HCI
containing
0.1%.
(w/v)
phenol
and
1
mM-,8-
mercaptoethanol
at
135°C
for
23h.
The
amino
acids
were
determined
by
g.l.c.
Results
of
duplicate
experiments
are
given.
Recovery
(%)
Amino
acid
thiohydantoin
I
II
Ala
84
88
Arg
72
74
Asn
65
65
Asp
80
82
Cys
33
33
CmCys
95
91
Glu
80
80
Gly
105
100
His
72
68
Ile
107
105
Leu
82
81
Lys
69
69
Met
66
66
Phe
90
90
Ser
29
30
Thr
75
74
Trp
24
26
Tyr
92
92
Val
102
99
andglycine.Whenthreoninephenylthiohydantoinwas
hydrolysed
with
acid,
a-amino-n-butyric
acid,
alanine
arid
glycine
were
detected
in
varying
proportions
by
Ingram
(1953)
and
Levy
(1954),
using
paper
chromatography,
and
by
Inglis
et
al.
(1971),
using
the
ion-exchange
amino
acid
analyser.
Hydrolysis
with
alkali
yielded
a-amino-n-butyric
acid,
glycine
and
threonine
(Ingram,
1953).
No
previous
reports
in
the
literature
on
the
hydrolysis
of
hydantoins,
thiohydantoins
or
their
3-alkyl
derivatives
involved
the
use
of
g.l.c.
for
qualitative
or
quantitative
determinations.
M.
R.
was
supported
by
a
Tutorial
Studentship
from
King's
College.
A.
D.
thanks
the
S.R.C.
for
support
and
Professor
H.
R.
V.
Arnstein
for
his
interest.
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