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QUANTIFICATION
OF REDUCED GLUTATHIONE AND GLUTATHIONE DISULPHIDE
USING GLUTATHIONE REDUCTASE AND 2-VINYLPYRIDINE.
Rhonda Moeller and A. Z. Mason, Ph.D.
Department
of Biological Sciences, California
State University, Long Beach, CA.
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Glutathione
(GSH) is a low
molecular
weight
tripeptide
present mainly
in animals,
plants and
some bacteria
and, in these
organisms, GSH
is the most
abundant
intracellular
thiol (Nelson,
1982). In
vivo, GSH
plays a role
in maintaining
the sulfhydryl
groups of
proteins in
the reduced
state (Tietze,
1968), and it
serves as a
reducing agent
for
glutaredoxin
in
deoxyribonucleotide
synthesis.
Its redox
function is
also used to
remove toxic
peroxides
formed in the
normal course
of growth and
metabolism
under aerobic
conditions.
A deficiency
in GSH can
result in
increased
susceptibility
to cell lysis
due to lipid
peroxidation
(Nelson, Cox).
The
tri-peptide is
composed of
three amino
acids:
cysteine,
glutamic acid
and glycine.
Under
oxidizing
conditions,
two reduced
GSH molecules
can become
cross linked
via a
disulfide
bridge to form
one dimeric
oxidized
disulfide (GSSG).
Conversion
back to GSH in
the cell is
catalyzed via
a short redox
cascade
involving
NADPH and
glutathione
reductase.
In this
cascade, NADPH
serves to
reduce
glutathione
reductase,
which in turn
reduces GSSG
to GSH being
converted to
the oxidized
form of the
enzyme during
this process
(Griffith,
1980).
The following
study utilizes
this redox
chain to
establish an
assay for both
GSH and GSSG.
This assay
takes
advantage of
the reduction
of DTNB
by GSH to form
a reduced DTNB
product which
shows a high
molecular
extinction
coefficient at
412nm.
The GSSG
produced
during the
reduction of
DTNB is then
reduced by the
reductase
allowing it
re-enter the
assay.
The process
cycles
continuously
until all of
the DTNB is
reduced or
NADPH is
consumed.
The
second method
utilizes
2-vinylpyridine
to derivatize
pre-existing
GSH, thereby
preventing it
from entering
the assay
(Griffith,
1980).
Since
the rate of
this reaction
will be
dependent upon
the combined
GSSG and GSH
concentration
at a constant
glutathione
reductase and
NADPH
concentration,
the rate of
the linear
portion (R2
value = 0.99)
can be used to
generate a
standard curve.
Two methods
have been
investigated
for the
quantification
of GSSG.
The first
method
involves the
direct
addition of
glutathione to
DTNB (Ellman,
1959).
In the absence
of NADPH and
glutathione
reductase, GSH
will reduce
DTNB, but the
GSSG produced
will not enter
the enzymatic
recycling
assay. 
2-VP
has been found
to not inhibit
glutathione
reductase, and
therefore does
not interfere
with its
reductive
catalysis of
GSSG. Because
the kinetics
of the
2-VP reaction
is slow
relative to
the rate
constants for
the GSH
reductase and
the DTNB
reaction,
pre-incubation
of a GSSG and
GSH mixture
with 2-VP will
assay only for
the presence
of GSSG.
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