Vol. 266,No.
OF BIOLOGICAL
CHEMISTRY
THEJOURNAL
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Issue of November 15,PP. 21409-21415.1991
Printed in U.S.A.
Redox Formsof Human Placenta Glutathione Transferase*
(Received for publication, April 16, 1991)
Giorgio RicciSg, Gilbert0 Del BoccioS, Alfonso PennelliS, Mario
Lo Belloll, Raffaele Petruzzellill,
Anna Maria Caccurill,Donatella Barrall ,and Giorgio Federicin
From the $Instituteof Biochemical Sciences, University of Chieti “G. D’Annunzio,” Chieti, the VDepartment of Biology,
University of Rome “Tor Vergata” and the I/Department of Biochemical Sciences, University of Rome “La Sapienza,” Rome, Italy
Human placentaglutathione
transferase (EC inhibitor specificity and immunological properties (4).All the
2.5.1.18) T undergoes anoxidative inactivation which cytosolic enzymes have a dimeric structure due to a noncoleads to the formation of an inactive enzymatic form valent association of identical or different subunits with a
which is homogeneous inseveral chromatographic and molecular mass of 23-28 kDa (3). Moreover, each monomer
electrophoretic conditions. This process
is pH depend- contains one binding site for glutathione (GSH) (G-subsite)
ent, and it occurs at appreciable rate in alkaline con- and another for the hydrophobic substrate (H-subsite) (3).
ditions and in the presence of metal ions. Dithiothreitol
treatmentcompletelyrestoresthe
active form. -SH Several studies have been performed to define the catalytic
mechanism of this enzyme, but this remains obscure at the
titration data and electrophoretic studies performed
present;
similarly, the topography of the subsites is not clarboth on the oxidized and reduced forms indicate that
one intrachain disulfide is formed, probably between ified despite the primary structure of a number of isoenzymes
the two faster reacting cysteinyl groups of each sub- established so far (3). At this regard one important question
unit. By the use of a specific fluorescent thiol reagent is the role, if any, of the sulfhydryls present in each subunit
the disulfide forming
cysteines have been identifiedas of the Piclass isoenzymes. It has been recently observed that
the 47th and 101th residues. The disulfide formation the covalent modification of a single cysteine residue/moncauses changes in thetertiary structure ofthis trans- omer causes a dramaticloss of activity of the homodimeric Pi
ferase as appears by CD, UV, and fluorometric anal- isoenzymes such as the rat GST 7-7 (5), the human placenta
yses; evidences are provided that one orboth trypto- GST-T (5, 6), the mouse GST M I1 (5), and thehorse erythphanyl residues of each subunit together
with a number
rocyte GST (7). This sulfhydryl is the most reactive among
of tyrosyl residues are exposed to a
more hydrophilic
has been
environment in the oxidized form. Moreover,electro- the 4 cysteine residues of each subunit,andit
phoretic data indicate that the subunit of the oxidizedidentified as the 47th amino acid both in the rat 7-7 and
human placentaT isoenzymes (5,6). Whether this thiol group
enzyme has an apparent molecular mass lower than
is important for the maintenance of a catalytically active
that of the reduced transferase, thereby confirming
structure or whether it is implicated in the catalytic mechastructural differences between these forms.
nism remains to be clarified. Probably related to theintegrity
of this residue are also the observations that oxidizing agents
or disulfides cause a loss of the GSTactivity (4,
Glutathione transferase (EC 2.5.1.18) (GST)’ is a family of such as Hz02
enzymes found in numerous species and in many tissues of 5, 7-10) and that a number of GST purifications were permammals (1, 2). They represent one of the most efficient formed in the presence of reducing agents to prevent enzyme
biological systems for the detoxification of electrophilic al- inactivation. These data suggest a correlation between the
redox state of the Pi class GST and its activity; this was
kylating agents (3). They may also be implicated in other
cellular metabolisms such as binding of hydrophobic com- recently suggested by Shaffer et al. (8)who observed that the
pounds, i.e. drugs and bilirubin, steroid isomerization, and bovine placenta GST exists in an active reduced form and a
less active oxidized form. These forms behave differently in
reduction of hydroperoxides (3).
sedimentation analysis, gel chromatography, and gel electroThe mammalian enzymes have been grouped into three
distinct classes named Alpha, Mu, and Pi on the basis of phoresis (8). Unfortunately,they did not perform further
several criteria including amino acid sequence, substrate, and characterizations of the oxidized form. Therefore, up to now,
the term “oxidized GST” is ambiguous since the number of
* The costs of publication of this article were defrayed in part by involved sulfhydryls and their identification in the primary
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734 structure are notyet defined, the homogeneity of the oxidized
form(s) as well as the factors affecting this process are unsolely to indicate this fact.
V This paper is dedicated to Prof. Doriano Cavallini oh the occasion known. The aim of the present paper is a quantitative apof his 75th birthday
proach for the characterization of the redox states of the
To whom correspondence should be addressed Istituto di Scienze human placenta GST.
Biochimiche, Universita “G. D’Annunzio,” Via dei Vestini 6, Chieti,
We present evidence that one intrachain disulfide bond
Italy.
The abbreviations used GST,Glutathionetransferase;
DTT, may be formed in each subunit of this transferase between
DTNB, 5,5’-di- cysteines 47 and 101. This occurs in metal-catalyzed and pHdithiothreitol; CDNB, l-chloro-2,4-dinitrobenzene;
thiobis-(2-nitrobenzoic acid); TNB-, thionitrobenzoate; DTDP, 4,4’- dependent processes. This oxidative reaction yields one single
dithiodipyridine; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide
inactive enzyme population which appears homogeneous ungel electrophoresis; Et-SH, mercaptoethanol; FPLC, fast protein liquid chromatography; HPLC, high pressure liquid chromatography; der several chromatographic and electrophoretic analyses.
ANM, N-(4-anilino-l-naphthyl)maleimide;
TPCK, L-l-tosyl-amido- Moreover, these techniques together with UV, circular di2-phenylethyl chloromethyl ketone.
chroism, and fluorescence data let us hypothesize remarkable
21409
Redox Forms of GST
21410
conformational differences between
the reduced and oxidized
forms.
EXPERIMENTAL PROCEDURES
Enzyme Preparation-Human placenta GST T was prepared as
previously described (11).About 20 mg of this enzyme were further
purified by CM52 column (2 X 10 cm) equilibrated with 10 mM Kphosphate buffer, pH 6.8 containing 1 mM EDTA. The acidic isoenzyme was not retained whereas several protein contaminants were
bound to the resin. Aliquots of GST were then treated with 10 mM
dithiothreitol (DTT) atpH 8.0 (0.1 M K-phosphate buffer and 1 mM
EDTA) for 60 min a t 37 “C toachieve the maximal specific activity.
DTT was removed by a G-25 Sephadex column (1 X 40 cm) equilibrated with 0.1 M K-phosphate buffer, pH 7.0 (orother buffers
depending by the subsequent experimental conditions) and 1 mM
EDTA. This activity value remains unchanged for a t least 3 h a t
37 “C (at pH 7.0) after the Sephadex chromatography. The active
isoenzyme appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophyoresis (SDS-PAGE), disc-gel electrophoresis under nondenaturating conditions and onfastprotein
liquid
chromatography (FPLC). This GSTis named in this paper as “fully
reduced GST” (220 units/mg). By incubating such enzyme in 0.1 M
K-phosphate buffer, pH 8.0, for 48 h at 25 “C,we obtained an inactive
form (<I unit/mg) which is defined as “fully oxidized GST” or “fully
inactivated GST.” The inactive form obtained after reaction with
stoichiometric or catalytic amounts of cupric ions was dialyzed overnight before any experiment.
Enzyme Activity-GST activity was assayed spectrophotometrically at 340 nm (12). Standard incubation mixture contained 1 mM
l-chloro-2,4-dinitrobenzene(CDNB) and 2 mM GSH in 2 ml (final
volume) of 0.1M K-phosphate buffer, pH 6.5, containing 1mM EDTA.
One enzyme unit is the amount of GST thatcatalyzes the conjugation
of 1 pmol of GSH/min at 37 “C. Spectrophotometric measurements
were corrected for the spontaneous reaction of GSH with CDNB.
Protein concentration was determined as described by Lowry (13).
Molarity of GST was calculated on the basis of a molecular mass of
46 kDa.
Inactivation Experiments-About 0.7 mg of fully reduced GST (15
WM) were incubated at 25 “C in the presence of 0.1 M K-phosphate
buffers, pH 7.0-8.0, and 0.1 M Tris-HC1 buffers, pH 8.5-9.0, in the
presence or absence of 1 mM EDTA (1ml final volume). At various
times 10-pl aliquots were assayed for GST activity as above described.
Thiol Group Titration-Protein sulfhydryls were titrated essentially by following the Ellman and Grassetti and Murray procedures
(14, 15); about 230 pg of oxidized or reduced GST were reacted with
0.1 mM 5,5’-dithiobis-(2-nitrobenzoicacid) (DTNB) in 1 ml final
volume of 0.1 M K-phosphate buffer, pH 8.0, and 1 mM EDTA. The
same amount of reduced and oxidized forms were reacted with 0.15
mM 4,4’-dithiodipyridine (DTDP) in 1 ml final volume of 0.07 M Kphosphate buffer, pH 7.0, and 1 mM EDTA. The reactions were
followed spectrophotometrically in continuous a t 412 nm (14) and at
324 nm (15), respectively. Titration data with DTNB at pH 7.0 (0.1
M K-phosphate buffer) and at pH5.0 (0.1 M Na-acetate buffer) were
obtained by taking into account the extinction coefficients of thionitrobenzoate (TNB-) at these pH values.
ElectrophoreticAnalyses-SDS-PAGE was carried out in adiscontinuous slab gel as described by Laemmli (16) by omitting mercaptoethanol (Et-SH) in the run buffer and in the sample. Discgel
electrophoresis under nondenaturating conditions were performed
essentially as described by Ornstein and Davis (17) with the exception
that only one buffer (12 mM Tris-glycine, pH 8.5) was used. Gels at
10% acrylamide were prerun at 3 mA/tube for 60 min to remove
persulfate. About 12pg of GST were run for 6 ha t 2.5 mA/tube. Gels
were stained with Coomassie Blue R-250.
Chromutofocusing Analysis-Fully oxidized GST (about 0.5 mg in
0.01 M K-phosphate buffer, pH 8.0, were analyzed by a FPLC system
(LKB, Sweden) equipped with a Mono PHR 5/20 chromatofocusing
column (Pharmacia, Sweden) before and after incubation with 5 mM
DTT for 30 min at 37 “C. The column was equilibrated with 25 mM
methylpiperazine/HCl buffer, pH 5.7, and eluted with Polybuffer 74/
HCl, pH 4.0, at a flow rate of 0.5 ml/min.
CD Analysis-CD spectra were performed with a Jasco J-500A
spectropolarimeter equipped with Jasco DP-500N data processor. The
reduction of the fully oxidized GST (25 p ~ was
) performed in the
presence of 100 p~ DTT in 3ml (final volume) of 0.01 M K-phosphate
buffer, pH 8.0 (20 “C).At fixed times 10-p1aliquots were assayed for
GST activity.
UV and Fluorescence Analyses-The fully oxidized GST (1.3 mg/
ml) was incubated with 1 mM Et-SH in 2.5 ml (final volume) of 0.1
M K-phosphate buffer, pH 8.0, a t 25 “C. The reduction process was
followed spectrophotometrically with a double beamUvikon 860
spectrophotometer Kontron equipped with a thermostatted cuvette
compartment. Blank cuvette contained the same amount of enzyme
without Et-SH. At fixed times, 1 0 - ~aliquots
1
were assayed for GST
activity. The same conditions were employed for fluorometric analyses that have been performed with a Jobin Yvon 3D fluorometer
equipped with a thermostatted sample holder. Samples were excited
at 280 2 5 nm and 295 k 5 nm, and the emission spectra were
recorded between 300 and 400 nm. Experiments were done at 22 ‘C.
Identification of the Sulfhydryls Involved in the Disulfide-About
3 mg of the fully oxidizedGST were dialyzed overnight against 0.1 M
Tris-HC1, pH 8.0, and 1mM EDTA. The enzyme was then denatured
with 6 M guanidinium chloride and carboxymethylated with monoiodoacetate according to the procedure described elsewhere (18).No
reducing agents were added during the denaturation. S-Carboxymethylated GST was digested with TPCK-trypsin in 0.1 M ammonium bicarbonate, pH 8.0, a t 37 “C for 24 h and dried under vacuum.
The mixture of peptides thus obtained was incubated with 1 mM
DTT in 1 ml of 0.1 M K-phosphate buffer, pH 7.0 (30 min, 37 “C)
and then reacted with 50 mM N-(4-anilino-1-naphthy1)maleimide
(ANM) for 30 min in the same conditions. High pressure liquid
chromatography (HPLC) purification of peptides was carried out
with a Beckman System Gold equipped with a Beckman programmable detector Module 166 and a Shimadsu (Kyoto, Japan) RF-530
fluorescence monitor, connected in tandem. Automated Edman degradation of the fluorescent peptides was performed by using an
Applied Biosystems model 470 A gas-phase sequencer, equipped with
an Applied Biosystems model 120 A PTH-analyzer.
RESULTS AND DISCUSSION
Oxidative Inactivation of Placenta GST-7F”ST spontaneously inactivates in a pH-dependent process (Fig. 1, Table
I). The inactivation is faster by increasing the pH values and
it follows pseudo-firstorder kinetics (Fig. 1B).In our experimental conditions the pH-independent inactivation due to a
A
0-.-e-.-
3
1
5
7
hours
0
51
‘0
\
1
3
5
?
hours
FIG. 1. pH-dependent inactivation of GST.Experimental conditions were as described under “Experimental Procedures.” A, time
course of the inactivation of the fully reduced GST (1.5 p M). 0,pH
8.0 + 1 mM EDTA; 0,pH 9.0 + 1 mM EDTA; A,pH 8.0; .,pH 8.5;
A, pH 9.0; 0, pH 8.5 + 1.5 PM CuSO,. B, semilog plots of the
inactivation data from A.
Redox Forms of GST
TABLEI
Standard inactivation procedures a t various pH values and activity
determinations were performed as described under “Experimental
Procedures”
Pseudo-first
PH
EDTA Temp.
order constant
min”
25
25
9.0
9.0
“C
“C
-
0.0076
0.0007
25 8.5
25 8.5
37 8.5
8.5
“C
“C
“C
0 “C
-
0.0029
0.000
0.0057
0.000
25 8.0
25 8.0
‘C
-
“C
+
0.0013
0.000
25 7.0
25 7.0
“C
“C
+
-
0.000
0.000
+
+
-
TABLE
I1
Rate constants for dithiothreitol and glutathione reactivation of the
fully inactivated GST
The reactivations were carried out in 0.07 M potassium phosphate
buffers, pH 7.0 and 8.0, and Tris-HC1 buffer, pH 9.0,with 4 mM DTT
or 4 mM GSH at 37 “C. Fully inactivated GST was 4.6 p ~ At. fixed
times 5-pl aliquots were assayed for activity as described under
“ExDerimental Procedures.”
Pseudo-first
PH
Reagent
order constants
min”
DTT
DTT
DTT
GSH
GSH
0.077 GSH
7.0
8.0
9.0
7.0
8.0
9.0
0.31
>1
>>1
0.018
0.053
teine residues/subunit. These data arise from the complete
primary structure as deduced from the correspondent cDNA
and the gene structure (20, 21). Direct titration of sulfhydryl
groups in the placenta GST can be performed with thiolspecific reagents such as DTNB or DTDP although it has
been observed that theclass Pi isoenzymes possess a number
of masked -SH groups that do not react with these reagents
even under denaturating conditions (7). In our experimental
conditions the fully active enzyme has two fast reacting
sulfhydryls/subunit both titrable with DTNB and DTDP and
a third slow reactive thiol group (Fig. 2, Table 111).The fourth
cysteine residue is not titrable in 8 M urea, 6 M guanidine, or
after NaBH4 treatment and urea denaturation (Table
111).On
the contrary, the fully inactivated GST (both spontaneously
and copper catalyzed) has only one slow reactive -SH group/
subunit (Fig. 2, Table 111).From these data it appears that
the fully inactive GST differs from the active form of two
disulfides/dimer, but do not clarify whether these are intrachain or interchain bridges. On the otherhand thesedisulfides
seem to involve only the four faster reacting sulfhydryls.
Other interesting data arise from the -SH group titration
with DTNB performed on the native enzyme at low pH values.
As shown in Fig. 2 at pH 7.0 and 5.0 only four -SH groups
are detectable. At pH 5.0 two fast and two slow reacting
6-
A
e-S-0-A-
A-B-0’
*$-o~O
...-.-.-.-.-.-.-.-.-.-
*’o’o’
.I-
A 0
_””
””
W
.+ 2
solvation of this transferase and occurring at low enzyme
concentration (19) is negligible as appears from the experiment performed at pH7.0. The inactivated enzyme at pH9.0
after 8 h of incubation and that obtainedafter 24 h of
incubation at pH8.0 have approximately the same activity of
less of 1%. The time course of this process is affected by the
temperature(Table I) and does not occur under nitrogen
(data not shown). The presence of metal-chelating agents
such as EDTA dramatically lowers the inactivation rate (Fig.
1,Table I) thereby indicating a probable involvement of metal
ionsascatalysts.
As shown in Fig. 1 the presence of a
stoichiometric amount of cupric ions enhances about 10-fold
the inactivation rate of GST. Catalytic amountsof cupric ions
also yield a higher rate of inactivation, butin that case a more
complex kinetic behavior has been observed. Both the spontaneous and copper-catalyzed inactivatedGSTs recovered
completely the original activity by treatment with 4 mM DTT
within 10 min of incubation a t pH 8.0 (37 “C). Thereactivation process is pH dependent, and it follows a pseudo-first
order kinetic (Table 11).GSH yields much slower reactivation
rates than DTT at all pHs tested; this could reflect both a
difference in the redox potential values of the oxidized GST
and glutathione or a steric hindrancefor GSH in itsreaction
with the oxidized protein groups.
All these data point out the probable involvement of a
number of protein cysteine residues in a reversible redox
process as also hypothesized for the bovine placenta GST (8)
which undergoes a similar reversible inactivation in the presence of H202.
Thiol Group Titration-Human placenta GST has 4 cys-
21411
/.””“
I
;
j
p A - d - A - ~ d - ~ -
./)
./.A
60
120
min
FIG. 2. Thiol group titration. Experimental conditions are as
reported under “Experimental Procedures.” Fully reduced GST (5
p ~ reacted
)
with 0.1 mM DTNB a t pH 8.0 (0);0.15 mM DTDP at
pH 7.0 (A); 0.1 mM DTNB at pH 7.0 (0);0.1 mM DTNB at pH 5.0
(dotted line). Fully oxidized GST (4 p M ) reacted with 0.1 mM DTNB
at pH 8.0 (B); 0.15 mM DTDP at pH 7.0 (A).In the ordinate are
reported the number of titrated sulfhydryls/mol of GST (46 kDa).
TABLE
I11
-SH group titration data
Titration procedures were performed as described under “Experimental Procedures.”
pH
Titrated -SH
Reagent
GST
groups/dimer
DTDP
Active form
7.0
4.0 fast +slow
1.9
Active form +
DTDP
7.0
6.2
fast
8 M urea
Active form +
DTDP
7.0
6.1
fast
8 M urea + NaBH4
DTDP
7.0
1.8 slow
Inactive form
DTNB
Active form
8.04.2
fast + 2.0slow
DTNB
Active form
4.1
7.0
fast
DTNB
Active form
5.0
2.2 fast + 1.9slow
DTNB
Active form +
6.0 fast
8.0
8 M urea
Active form +
DTNB
8.0
6.2
fast
8 M urea + NaBH4
DTNB
slowform 2.0
8.0
Inactive
~~~
Redox Forms of GST
21412
sulfhydryls can be observed.A more detailed kinetic analysis
performed accordingly to Frost and Pearson (22) (data not
shown) allows the calculation of apparent pseudo-first order
constants of0.34 and 0.031 rnin”, respectively. During the
experiment performed at pH 5.0, we also evaluated the loss
of activity asa function of thetitrated sulfhydryls. The
enzymatic activity was reduced to 10% when two sulfhydryls
are reacted with DTNB thereby confirming the importance
of only 1 reactive cysteine residue/subunit as previously observed with other thiol reagents (6, 7). The observed spontaneous disulfide formation in GST let us explore the possibility
that this may also be formed during the reaction with DTNB,
for example, by a thiol-disulfide exchange between a protein
-SH group and theprotein-DTNB mixed disulfide.When the
DTNB-reacted enzyme at pH 7.0 (four titrated -SH groups)
was purified by a G-25 chromatography and treated with 1
mM DTT, the retrotitration data reported in Fig. 3 clearly
indicate that four TNB- are bound to theenzyme, and therefore no protein disulfide bridgehas been formed.
Electrophoresis under Denuturating and Nondenuturating
Conditions-SDS-PAGE was performed both on the spontaneous and copper-inactivated GSTs and compared with the
fully active enzyme. As reported under “Experimental Procedures,’’ the standardized procedure by Laemmli (16) was
modified by omitting Et-SH in the sample and in the run
buffer. Underthese conditionsthe active enzyme givesa single
band with an apparent molecular mass of 23 kDa identical to
that found in the standardized conditions with Et-SH (11)
(Fig. 4).On the other hand both the inactivated forms appear
as homogeneous components with the smaller apparent molecular mass of20.5 kDa. Pretreatment of the inactivated
GST with 1mM DTT prior electrophoresis restores the single
band at 23 kDa.The lack of bands with molecular mass higher
than 23 kDa points out that no intersubunit disulfide exists
in these inactivated forms. Moreover, these results suggest
that the spontaneous and copper-catalyzed oxidations lead to
the formation of similar homogeneous populations of inactive
transferase with subunits of smaller apparent molecular mass;
this may be dueto a conformational change of the molecular
shape as also hypothesized for
the oxidized formsof the bovine
min
FIG. 3. Retrotitration of the DTNB-treated GST. Fully re) reacted with DTNB (1mM) in 2 ml (final
duced GST (1.5 p ~ were
volume) of 0.1 M K-phosphate buffer, pH 7.0. The absorbance a t 412
nm reached a plateau in about 40 min corresponding to four -SH
groups titrated/mol of GST. The enzyme was almost inactivated. The
excess of DTNB was removed by a Sephadex G-25 column (1 X 40
cm) equilibrated with 0.1 M K-phosphate buffer, pH 8.0. The enzyme
was concentrated to 2.5 ml and reacted with 1mM DTT. Theincrease
of absorbance a t 412 nm was followed in continuous and at fixed
times 10-pl aliquots were tested for GST activity. 0,TNB- released/
mol of GST; 0,percentage of GST activity.
I
#
I
/
4
1
FIG. 4. Electrophoresis under denaturating and nondenaturating conditions. Electrophoretic conditions areas reported
under “Experimental Procedures.” SDS-PAGE lines a and f, molecular mass markers; Lines b and 1, fully reduced GST, lines c, d, and e,
fully oxidized GST obtained by incubation a t pH 8.0, 9.0, and 8.0
stoichiometric CuS04, respectively. Lines g-i, samples of lines c-e
treated with 50 mM DTT for 20 min. Disc gel electrophoresis: line 1;
fully reduced GST; lines 2-4, fully oxidized GST as in lines c-e,
respectively.
+
isoenzyme (8).We also observed a different chromatic intensities when identical amounts of oxidized and reduced GSTs
were stained with Coomassie Brillant BlueR-250ongel.
These differences werealso detected in solution by following
the Bradford procedure (23); the oxidized form gives an absorption at 595 nm about 20% lower than that of the active
form. This is a further indication of structural differences
between these forms. The homogeneity of the GST-oxidized
forms and their likeness were also supported by the disc gel
electrophoresis under nondenaturating conditions (Fig. 4).
Additionally in this case, the oxidized forms migrate as a
single band with a higher mobility than the reduced GST.
Under these conditions, however, we cannot establish whether
the higher mobilityof the inactive GSTs is due to a structural
modification or to anenhancement of the netnegative charge
on the protein. Moreover, both the oxidized GSTs are indistinguishable on chromatofocusing analysis giving a single
peak witha retention time (24 min) different from that of the
active enzyme (30 min). This result is again an indication
that both the spontaneously and copper-inactivatedGSTs are
identical and that thedisulfide formation causes a change of
the chemicophysical properties of this transferase.
Identifitation of the 2 Cysteinyls Involved in the Disulfide
Bridge-The spontaneously oxidized transferase was denatured in 6 M guanidinium chloride and then reacted with
monoiodoacetate to alkylate other sulfhydrylsnot involved in
the disulfide bond as described under “Experimental Procedures.” The modified enzyme wasthen digested withTPCKtrypsin for 24 h. After this procedure the peptide mixture was
reduced with 1 mM DTT and reacted with 50 mM ANM, a
specific fluorescent probe for -SH-containing compounds,
recently used for the detection of the high reactive cysteine
47 of this isoenzyme (6). The HPLC pattern of this peptide
mixture revealed only two majorfluorescentpeaks eluted after
29 and 33 min, both exhibiting approximately the same fluorescent intensity (Fig. 5). HPLC of the peptide mixture
reacted with ANM without any DTT treatment lacks of these
major fluorescent peaks, thereby indicating the absence of
any free sulfhydryls in the peptides. The automated Edman
degradation gave the following amino acid sequence for the
first faster fluorescent peak: Asp-Gln-Gln-Glu-Ala-Ala-Leu-
Val-Asp-Met-Val-Asn-Asp-Gly-Val-Glu-Asp-Leu-Arg-XxxLys; the same analysis performed on the second fluorescent
Redox Forms of GST
21413
I
0
TIMElmnl
FIG. 6. Near U V circular dicroism spectra. Fullyoxidized
GST (25 PM) was reactivated with 100 PM DTT in 2.5 ml of 0.01 M
K-phosphate buffer, pH8.0 (20 "C). At fixed times 10-pl aliquots
were assayed for GST activity.A, CD spectrum of the fully oxidized
form plus DTT (t")(solid line) and CD spectrum of the fully reduced
form (210 of min incubation, 220 units/mg) (dotted line). E, differential CD spectrum between oxidized and reduced forms. Inset, kinetics of CD perturbations at 270 nm (El) and 283 nm (0)compared
with the reactivation kinetic (0);CD data are reported as percentage
of maximal CD perturbations (210 min of reactivation).
FIG.5. HPLC pattern of the ANM-labeled TPCK-trypsin
digest. Before HPLC analysis the fully oxidized GST wastreated as
reported under "Experimental Procedures." HPLC was carried out
by using a reverse-phase Aquapore RP-300 column (Applied Biosystem) (250 X 4.6-mm inner diameter). Gradientsystem was composed
of 0.2% trifluoroacetic acid (solventA) and 70% acetonitrile (solvent protein disulfides are broad with no clear-cut fine structure
B). Flow rate was 1 ml/min. Peptides were detected by monitoring (31). It is interesting to note that the peak centered at 270
the absorbance at 220 nm (A) and the fluorescence at 448 nm (LX
= nm resembles that caused by the optically active disulfides of
355 nm) ( E ) .
the oxidized forms of human somatotropin (24) and human
apolipoprotein B (32). Tentatively we may assign the major
peak gave the following sequence; Ala-Ser-Xxx-Leu-Tyr-Gly-band centered at 270 nm to thedisulfide of the oxidized GST
Gln-Leu-Pro-Lys. By comparing these sequences with the and thesecond band at 283 nm to aromatic residues involved
complete primary structure of this isoenzyme (20, 21) the 2 in some structural change. On the contrary, the far UV-CD
modified cysteines can be identified as the101 and 47 residues, spectra of the oxidized and reduced forms of GST are very
respectively.
similar (data notshown) thereby indicating that no consistent
CD Spectra-All the spectroscopic analyses were performed differences in the secondary structure occur between these
on the spontaneously inactivated GST toavoid any interfer- forms.
ence due to cupric ions. Moreover, since the spontaneous
UV Spectra-For UV differential spectra between the reoxidation occurs at a very low rate, we always obtained duced and oxidized forms of GST, several parameters have
spectroscopic data by starting with the fully oxidized GST been optimized to obtain reliable spectrophotometric data.
and by following the reactivation process by DTT or Et-SH. We followed the reduction process of oxidizedGST at pH8.0
In particular, circular dichroism experiments have been per- and in the presence of 1 mM Et-SH; under these conditions
formed with DTT as reducing agent since neither the reduced Et-SH which has a pKsH = 9.6 (33) is largely undissociated.
DTT nor the disulfide bond in the oxidized DTT give signif- The small amount of the ionized form (about 25 p ~ is)
icant dichroism over the concentrations and thespectral range stoichiometric in respect to the oxidized protein (28 p ~ ) ,
studied (24). From Fig. 6A, it is evident that CD spectrum of however, the presence of an high amount of undissociated Etthe fully active enzyme markedly differs from that of the SH as "buffer" makes constantthe concentration of the
oxidized form in the 250-300-nm region. The interpretation dissociated form during the reduction process. These condiof such spectral changes cannot be made without an accurate tions are importantsince the thiolate ion absorbs at 230-240
knowledge of the contributions of tryptophanyls, tyrosyls, nm and any change of its concentration yields interferences
and other chromophores to the totalspectrum of the reduced in the UV region even at higher wavelengths. Similarly the
form of placenta GST. These data are unknown up to now use of DTT as reducing agent was avoided since its oxidized
although the CD spectra of the Pi isoenzymes have been form absorbs at 283 nm. We also tested that thespontaneous
reported and compared with those of the Mu and Alpha classes autoxidation of Et-SH, which leads to the formation of a
isoenzymes (25). In spite of this lack several considerations disulfide absorbing at 250 nm, was negligible until the reaccan be made. First, the simple subtraction of the spectrum of tivation went to completion. As shown in Fig. 7, the reduction
the reactivated GST from that of the oxidized form generates of GST is accompanied by UV perturbations in the 270-310a broad negative band with a maximum at 270 nm and a
nm region. It is well known that the 270-310-nm perturbasecond smaller peak at about 283 nm (Fig. 6B),thereby tions are mainly due to changes in the environment of trypindicating at least two groups of chromophores to be involved tophyl and tyrosyl residues (34). The reduced form of GST
in this spectral change. A second observation is that change shows an increased absorption in this region with maxima
of the ellipticity values at 270 nm parallels the reactivation centered a t 291, 287, 285, 280, and 274 nm. A maximum
kinetic (Fig. 6B, inset). It has been claimed that low energy perturbation with
= 710 M" cm" was obtained after
disulfide bond transitions may occur at any wavelength be- 90 min of incubation. Moreover, a broad peak can be observed
tween 250-340 nm, depending on the environment of the near 300 nm. On the basis of the reported absorption differdisulfide and on the dihedral angle and thatthey can generate ence for tyrosyls and tryptophanyls due to several perturbants
a conspicuous negative contribution to the ellipticity band (34), the peaks at 291, 285, and 274 nm may be due to an
(26-30). Except for a few proteins, generally the CD peaks of enhanced hydrophobicity near one or both tryptophanyls of
Redox Forms of GST
21414
Ii\
1
I
toward the blue with respect to the oxidized form (Fig. 8).
These differences are mainly due to tryptophanyl(s) since
proportionally identical perturbations have been observed by
exciting at 295 nm (data notshown). A kinetic study of these
fluorescence spectra changes (Fig. 8, inset) reveals at least
two distinct phenomena to occur during the reduction process.
The faster blue shift of the fluorescence emission which may
correspond to a movement of the tryptophanyl(s) toward a
perturbation
more hydrophobic environment (36); this
reaches a maximum in about 60 min of incubation with 1 mM
I
I
270
290
310
Et-SH andparallels the reactivation kinetic. A slower quenchnm
ing may bedue to a charge approach nearthe tryptophanyl(s)
FIG. 7. Differential UV spectra. Fully oxidized GST (28 p ~ ) (37) and it reaches to a maximum in about 100 min reactivawereincubated with 1 mM Et-SH in 2.5 ml of 0.1 M K-phosphate tion.
buffer, pH 8.0 (25 “ C ) .BK cuvette contained all reagents except EtSH. At fixed times, 10-pl aliquots were assayed for GST activity.
Dotted line, differential UV spectrum at zero time; solid line, differential UV spectrum after 90 min of reactivation. Inset, kinetics of the
UV perturbations at 291 nm (0)and 300 nm (A) compared with the
reactivation kinetic (0).UV data are reported as percentage of the
maximal UV perturbations (100 min of reactivation).
1oc
A
3
W
0
:: 5c
W
B
5
Y
1
310
,
330
,
.
350
1
,
370
1
1
a
390
nm
FIG. 8. Fluorescence analysis. Fully oxidized GST (28 p ~were
)
incubated with 1 mM Et-SH in 2.5 ml of 0.1 M K-phosphate buffer,
pH 8.0 (22 “ C ) .Sample was excited at 280 nm. At fixed times 10-pl
aliquots were assayed for GST activity. A, fluorescence emission
spectra (LX
= 280 nm) at zero time ( a ) , after 40 min of incubation
( b ) ,and after 100 min of incubation (c). B, kinetics of the fluorescence
quencing (0)and blue shift (0) compared with the reactivation
kinetic (0).Data are expressed as percentage of the maximal perturbations (110 min of reactivation).
the GST subunit, and the peaks at 280 and 285 nm to a
similar change of polarity involving a number of tyrosyls. It
is interesting to note that while these major absorption peaks
increased paralleling the reactivation process, the minor peak
near 300 nm follows a different kinetic pattern (Fig. 7, inset).
The presence of an atypical extremum near 300 nm in solventperturbated or denaturated proteins has been explained as a
signal that thetotal net charge around a tryptophyl chromophore is made less positive or more negative (35). Our data
are therefore consistent with two kinetically different movements of tryptophanyls during the reduction process: a first
approach of the tryptophanyl(s) to a more hydrophobic environment which causes a shift of the major absorption bands
and a slower removal from a positive charge or, alternatively,
a slower approach to a negative charge. Obviously, since GST
has two tryptophanyls in each subunit, these two distinct
modifications may reflect changes of the environment of both
these aromatics as well as of a single residue.
Fluorescence Spectra-Reactivation of the oxidized form of
GST is accompanied by a quenching of the intrinsic protein
fluorescence when the enzyme is excited at 280 nm (Fig. 8).
The reduced form exhibits an emission spectrum with a
maximum at 336 nm, which corresponds to a4-nmshift
CONCLUDING REMARKS
Our experimental data indicate that the human placenta
GST ?r may undergo an oxidative inactivation due to an
intrachain disulfide formation. This process occurs at appreciable rate only in alkaline conditions and probably requires
traces of metal ions as demonstrated by the inhibiting effect
of EDTA (Fig. 1).This is also confirmed by the enhancement
of the oxidation rate produced by catalytic amounts of cupric
ions. Both the “spontaneous” and thecopper-catalyzed processes lead to the formation of an inactive transferase which
is homogeneous on SDS-PAGE, FPLC,and PAGE under
nondenaturating conditions (Fig. 4). The analytical data on
SDS-PAGE, performed without reducing agents, clearly indicate that no interchain disulfide bonds have been formed.
Moreover, the subunit of the oxidized form of GST shows an
apparent molecular mass of 20.5 kDa which is smaller than
the corresponding reduced form. Since a complete recovery of
the original band of 23 kDa has been observed after DTT
treatment (Fig. 4)) a conformational modification maybe
supposed due to a disulfide formation. This was then confirmed by the spectroscopic data whichwillbe
discussed
below. Our data are indicative that the oxidation involves 2
cysteines among the 4 present in each subunit. These data
arise from titration experiments performed with DTNB and
DTDP (Table I11 and Fig. 2). In particular, with both these
reagents it appears that among the threetitrable sulfhydryls/
monomer, the 2 faster reacting cysteines are probably involved
in the disulfide formation. The occurrence of a disulfide was
confirmed by tryptic digestion of the oxidized transferase
after blockage of the remaining free sulfhydryls with monoiodoacetate. When the peptides were reduced by DTT and
labeled with ANM, only two major fluorescent peaks were
detected on HPLC (Fig. 5). This is an indication that only
two sulfhydryls have been oxidized in each subunit and that
the disulfide is identical in both subunits.
The AA sequence of these peptides allowed the identification of the cysteine 47 and the cysteine 101 as involved in
this disulfide. This may be informative about the tridimensional structure of the subunit of this isoenzyme: these cysteinyls being far in the primary sequence, it seems to be
reasonable that in the coiled native structure they must be
sufficiently close to interact and then to form the disulfide
bridge. The redox process is accompanied by changes of the
tertiarystructure of this protein as it appears by several
spectroscopic evidences obtained during the reduction of the
fully oxidized GST. Forexample, the differential CD spectrum
between the reduced and oxidized forms (Fig. 6) shows a
spectral modification centered at 283 nm probably caused by
a structural change near some aromatic group. The second
perturbation at about 270 nm has been tentatively explained
as the contribution of the disulfide to the total spectrum of
Redox Forms of GST
the oxidized form. No differences have been observed in the
far UV CD spectrum therebyindicating that no change in the
secondary structure occurs. The perturbation of aromatic
groups in the redox process of this transferase was confirmed
by UV and fluorometric analyses. During the disulfide breakdown both the UV differential spectra in the 270-310 nm
region (Fig. 7) and theblue shift of the intrinsic fluorescence
(Fig. 8) areconsistent with a movement of one or both
tryptophanyls into a more hydrophobic environment. These
experiments also indicate that a second kinetically different
process occurs, probably due to theapproaching of a negative
charge near the tryptophan(s).
All these spectroscopic data allow hypothesis of a role for
the crucial cysteine 47. It has been demonstrated that the
modification of this residue is incompatible with the catalytic
activity of this transferase (5,6). As recently observed for the
isoenzyme K from horse erythrocyte, the possibility that the
inactivation is due to conformational modification by the
steric hindrance of the -SH reagents is improbable as complete inactivation has been observed with many reagents of
different sizes and charges (7). The involvement of this group
in thecatalytic mechanism of the GST Pi
cannot be excluded,
but there is no evidence for that at present. On the other
hand the hypothesis for a structural role of this sulfhydryl is
in agreement with the data in this paper: during the redox
process involving the critical cysteine 47 both inactivation
and structural change have been observed simultaneously.
Therefore it should be important for the maintenance of a
proper tertiary structure.
From another point of view these data put forth an interesting question about the possible regulation of the activity
of this transferase in uiuo, based on a redox process. It has
been well known that many enzymatic activities maybe
modulated by redox reactions such as SH/SS exchange with
biological disulfides (38-41). This has also been recently suggested for this isoenzyme (10). The present data open the new
possibility that the activity of this isoenzyme may also be
regulated by an intramolecular disulfide. We are now studying
the possible existence of this inactive oxidized GST in several
tissues and the possibility that this form is more easily attacked by proteolytic enzymes.
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