Purification and kinetics of bovine kidney cortex glutathione reductase

Article abstract of DOI:10.2174/092986610791112684

Glutathione reductase was purified 34806-fold with a final yield of 85% from the bovine kidney cortex. Some molecular and kinetic properties of purified enzyme are investigated. Product inhibition studies showed that the enzyme obeys 'branched' mechanism: KmNADPH 18 ± 3 μM and KmGSSG 65 ± 5 μM were determined.

Full text of DOI:10.2174/092986610791112684

Protein & Peptide Letters, 2010, 17, 667-674
667
Purification and Kinetics of Bovine Kidney Cortex Glutathione Reductase
Berivan Tandogan and N. Nuray Ulusu*
Hacettepe University, Faculty of Medicine, Department of Biochemistry, 06100 Ankara, Turkey
Abstract: Glutathione reductase was purified 34806-fold with a final yield of 85 % from the bovine kidney cortex. Some
molecular and kinetic properties of purified enzyme are investigated. Product inhibition studies showed that the enzyme
obeys ‘branched’ mechanism: KmNADPH 18 ± 3 μM and KmGSSG 65 ± 5 μM were determined.
Keywords: Glutathione reductase, purification, kinetics, branched mechanism.
INTRODUCTION
significance in protecting kidney cells from oxidant-induced
damage.
Glutathione reductase (GR, NADPH: oxidized glu-
tathione oxidoreductase, EC 1.8.1.7) is a ubiquitous dimeric
enzyme containing FAD in each monomer as a prosthetic
group [1]. The enzyme is a member of the flavoprotein di-
sulphide oxidoreductase family in which each subunit has
four well-delineated domains. The two active sites are at the
dimer interface, with the substrate glutathione bound by resi-
dues contributed to by both subunits. Thus only the dimeric
form of the enzyme can be catalytically active [2]. It has
been established that electrons flow from NADPH to the
substrate oxidized glutathione (GSSG) via flavin and the
redoxactive protein disulfide bridge [3]. Detailed steady-state
kinetic analysis indicated that the reaction of GR acted ac-
cording to both ping-pong and sequential mechanisms de-
pending on substrate concentrations [4, 5]. The function of
GR is to reduce the GSSG by using NADPH as the source of
reducing equivalents [6]. The reduced form of glutathione
(GSH) is the most important thiol compound, and it plays
crucial roles in nutrient metabolism, gene expression, DNA
and protein synthesis, cell proliferation and apoptosis, signal
transduction, cytokine production, immune response, protein
glutathionylation, and detoxification from free radicals,
xenobiotics, chemicals, and drugs [7]. In addition to these
numerous beneficial functions, GSH also plays a critical role
in enzymatic reactions as a co-factor and indirectly as the
major thiol-disulfide redox buffer in mammalian cells [8].
GR has been purified very rapidly in high yield by em-
ploying as affinity columns in several studies [11-14]. We
applied a heat denaturation step in addition to the affinity
and ion exchange chromatography and obtained the best re-
sult to date for GR purification. In this study we also studied
the detailed molecular properties and kinetic mechanism of
kidney cortex GR.
GR is a crucial enzyme that catalyzed the conversion of
oxidized glutathione to its reduced form. GSH is the major
low-molecular weight thiol and has an important function in
the antioxidative system. Oxidative stress, which results
from a disturbance in the GSSG/GSH balance, is the cause of
the pathogenesis of many diseases including Alzheimer’s
disease, Parkinson’s disease, liver disease, sickle cell ane-
mia, HIV, AIDS, cancer, heart attack, and diabetes. Due to
the importance of GR in many diseases, additional studies
about this enzyme have to be done in the future. The investi-
gation of the structural aspects of the enzyme function has
partly depended on the development of good purification
strategies for the enzyme.
MATERIALS AND METHODS
Materials
Bovine kidney cortex obtained from a local slaughter-
house was kept in ice and processed within 2–3 h of death.
Due to the homeostatic functions of kidneys, including
the maintenance of acid-base balance and regulation of elec-
trolyte concentrations, blood volume and pressure, it has a
very active oxidative metabolism that results in the produc-
tion of free radicals. Increased oxidative stress has been im-
plicated in various pathologies, including hypertension, athe-
rosclerosis, diabetes, and chronic kidney disease [9]. Reac-
tive oxygen species are involved in progressive renal injury,
by impairing glomerular permselective properties and induc-
ing loss of cellular phenotype and apoptosis, and finally
promoting acute and chronic inflammatory responses [10].
Therefore, in this study we investigated the molecular and
kinetic properties of kidney cortex GR because of its special
Nicotinamide adenine dinucleotide phosphate reduced
form (NADPH), oxidized glutathione (GSSG), Tris [Tris
(hydroxymethyl) aminomethane], and DEAE Sepharose Fast
Flow were obtained from Sigma Chemical Co., MO, USA.
2ꢀ, 5ꢀ-ADP-Sepharose 4B was from Pharmacia Fine Chemi-
cals, Uppsala, Sweden. BSA was from British Drug Houses
Ltd.
Assay of Glutathione Reductase
GR activity was determined according to a modified ver-
sion of Stall’s method [15]. The incubation mixture con-
tained 100 mM sodium phosphate buffer, pH 7.4; 1 mM
GSSG; and 0.2 mM NADPH. Decrease in the absorbance of
NADPH at 340 nm was monitored spectrophotometrically at
37 ºC. A unit of activity (U) was defined as the amount of
*Address correspondence to this author at the Hacettepe University, Faculty
of Medicine, Department of Biochemistry, 06100 Ankara, Turkey; Tel:
+903123245885; Fax: +903123100588; E-mail: nnulusu@hacettepe.edu.tr
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

668 Protein & Peptide Letters, 2010, Vol. 17, No. 5
Tandogan and Ulusu
enzyme that catalyzes the oxidation of 1 micromole of
NADPH in 1 min under these conditions. Specific activity is
defined as units per mg of protein.
column was washed with the same buffer (the flow rate was
10.8 mL/h) until the absorbance at 280 nm decreased to
0.004 O.D. to remove all the non-specifically bound com-
pounds. Then GR was eluted with buffer A containing 0.1
mM NADP+ (the flow rate was 10.8 mL/h). The elution pro-
file of GR from the 2', 5'-ADP-Sepharose 4B column is
given in Fig. (1).
Protein Assay
Protein concentrations in column fractions were deter-
mined by measuring the absorbance at 280 nm and protein
concentrations of the purification steps were determined as
described by Bradford [16] using bovine serum albumin as
standard.
Step 4. DEAE Fast Flow Ion Exchange Chromatography
Active GR fractions were combined and loaded onto a
DEAE Sepharose Fast Flow column (1.5 ꢀ 7.5 cm) equili-
brated with 5 mM potassium phosphate buffer, pH 6.9
(buffer B). The flow rate was maintained at 16.8 mL/h, the
column was washed with buffer B until the absorbance at
280 nm decreased to 0.004 O.D., and GR was eluted with
linear KCl (0-300 mM) gradient in buffer B Fig. (2).
Statistical Analysis of Kinetic Data
The data were analyzed and the kinetic constants were
calculated by means of a nonlinear curve-fitting program
(Statistica).
GR was purified 34806-folds from bovine kidney cortex
and had a final specific activity of 727 U/mg of protein and
an 85 % yield (Table 1). The whole purification procedure
took approximately two working days. The enzyme was sta-
ble for three weeks at 4 °C. We did not add any substance for
the stabilization of the enzyme.
Purification of Glutathione Reductase
Step 1. Homogenization and Ultracentrifugation
Bovine kidney cortex GR was purified by chromatogra-
phy consisting of two steps after ultracentrifugation by
Beckman L7-80 ultracentrifuge and heat denaturation: 2', 5'-
ADP- Sepharose 4B affinity and DEAE Sepharose Fast Flow
anion exchange chromatography. The bovine kidney cortex
was minced with scissors after washing with physiologic
saline and homogenized using an IKA ultra turrax homoge-
nizer with an S18N-10G probe at 22 000 1/min for approxi-
mately 3 minutes with 3 volumes of 10 mM Tris/HCl buffer,
pH 7.6, containing 1 mM 2-mercaptoethanol and 1 mM
EDTA (buffer A). The homogenate was centrifuged at 105
000 ꢀ g for 60 min by Beckman L7-80 ultracentrifuge. All
the procedures were carried out at +4 °C.
Polyacrylamide Gel Electrophoresis
SDS-PAGE was performed according to Laemmli [17].
We used 10% slab gels for molecular weight determination.
The gels were stained by the silver staining method de-
scribed by Merril [18].
Sephadex G-200 Gel Filtration
The molecular weight of the enzyme was estimated by
Sephadex G-200 gel filtration according to the method re-
ported by Andrews [19]. The elution volume of the enzyme
was determined by measuring the enzyme activity. The void
volume of the column was measured using Blue Dextran
2000.
Step 2. Heat Denaturation
The supernatant obtained was incubated at 50 °C for 75
min and then centrifuged at 13 000 rpm for 20 min by Ep-
pendorf 5417 R centrifuge at +4 °C.
Determination of the Isoelectric Point of GR
Step 3. 2’, 5’-ADP- Sepharose 4B Affinity Chromatography
The isoelectric point of the enzyme was determined by
chromatofocusing according to the instructions provided by
The supernatant was loaded onto a 2', 5'-ADP-Sepharose
4B column (1.5 ꢀ 6.7 cm) equilibrated with buffer A. The
Figure 1. Affinity chromatography of GR on 2’, 5’-ADP-Sepharose 4B. Column size, 1.5 x 6.7 cm; column equilibration and washing
buffer, 10 mM Tris/HCl pH 7.6 containing 1 mM 2-ME and EDTA. Enzyme elution buffer: same as the washing buffer containing 0.1 mM
NADP+; flow rate, 10.8 mL/h.

Glutathione Reductase
Protein & Peptide Letters, 2010, Vol. 17, No. 5 669
Figure 2. Gradient elution profile of GR from DEAE Sepharose Fast Flow. Column size, 1.5 x 7.5 cm. GR was eluted with 0-300 mM KCl
prepared in 5 mM potassium phosphate buffer, pH 6.9, at a flow rate of 16.8 mL/h.
Table 1. Summary of the Typical Purification Procedure of Glutathione Reductase from Bovine Kidney Cortex
Volume (mL)
Yield %
Activity
(U/mL)
Total Activity
(U)
Protein
Total Protein Specific Activ-
Purification
Fold
(mg/mL)
(mg)
ity (U/mg
protein)
Homogenate
12
1.15
1.14
13.8
8.55
55
660
0.021
0.12
100
62
1
7.5
9.5
71.25
5.74
105000xg Super-
natant
Heat denaturation
6
1.25
1.26
7.5
4.3
25.8
0.07
0.291
126
54
65
14
7.2
9.07
0.01
6026
2',5'- ADP- Sepha-
rose
9
1.31
11.79
0.002
0.02
727
85
34806
DEAE Sepharose
Fast Flow
the manufacturer (Pharmacia Chromatofocusing Instruction
Manual). Chromatofocusing in the pH range 7-4 was per-
formed on a polybuffer exchanger 94 column equilibrated
with 0.025 M imidazole-HCl, pH 7.4. Fractions of 1.2 mL
were collected and enzymatic activity, pH, and absorbance at
280 nm were measured.
NADP+ was used for the elution of the enzyme in affinity
chromatography step and DEAE Sepharose Fast Flow ion
exchange chromatography is used for the removal of NADP+
from the enzyme fractions. Gradient elution of GR from
DEAE Sepharose Fast Flow ion exchange chromatography
by KCl (0-300 mM) in buffer B is shown in Fig. (2). By lin-
ear gradient elution we get rid of unwanted proteins and ac-
complish a single band on SDS-PAGE Fig. (3). This uncom-
plicated and rapid purification method presented above
makes it possible to obtain 34806-fold purified enzyme with
high yield. A summary of the purification is presented in
Table 1. The purified enzyme gave a single band on SDS-
PAGE and the subunit molecular weight of the GR was de-
termined to be 56 ± 2 kDa Fig. (3). The subunit status of
purified GR was analyzed by size exclusion chromatography
Sephadex G–200 column. By using the Kav value of the
purified enzyme eluted as a single peak from the Sephadex
G–200 column the molecular weight of GR was calculated as
117 kDa (graph was not shown). These results (SDS-PAGE
and size exclusion chromatography) suggested that the bo-
vine kidney cortex GR purified is in its active dimeric form.
RESULTS
Purification and Some Properties of Bovine Kidney Cor-
tex Glutathione Reductase
In this study we purified GR from bovine kidney cortex
for the determination of some molecular and kinetic proper-
ties of the enzyme. The enzyme is purified by two subse-
quent chromatography steps, affinity and anion exchange
chromatography, after the ultracentrifugation and heat de-
naturation steps. By this purification protocol, adding heat
denaturation allows us to remove glucose-6-phosphate dehy-
drogenase (G6PD) and other cystosolic proteins before load-
ing to affinity chromatography. 6-Phosphogluconate dehy-
drogenase (6PGD) is not bound to the affinity column and is
eluted while washing with buffer A. The loading and elution
profile of 2', 5'-ADP-Sepharose 4B affinity chromatography
is shown in Fig. (1).
The isoelectric point of the enzyme was determined by
chromatofocusing to be 6.8 Fig. (4). This value is in agree-

670 Protein & Peptide Letters, 2010, Vol. 17, No. 5
Tandogan and Ulusu
Figure 3. SDS-PAGE of GR in which enzyme is eluted by linear gradient (0-300 mM) of KCl in buffer B. Estimation of the subunit molecu-
lar weight of GR (10% acrylamide gel was used). The gels were stained by the silver staining method of Merril [18]. Lane 1: Homogenate,
Lane 2: 105 000 x g supernatant, Lane 3: Heat denaturation, Lane 4: 2’,5’-ADP-Sepharose 4B eluant Lane 5: DEAE Sepharose Fast Flow
eluant Lane 6: The protein standards: ꢀ-Galactosidase (E. coli), 116 kDa; Hexokinase, 97 kDa; Serum Albumin (Bovine), 66 kDa; Ovalbumin
45 kDa; Carbonic anhydrase 29 kDa; cytochrome c 13, 8 kDa. The molecular weight of the enzyme was found to be 56 ± 2 kDa.
Figure 4. Chromatofocusing of bovine kidney cortex glutathione reductase. Chromatofocusing in the pH range 7-4 was performed on a poly-
buffer exchanger 94 column equilibrated with 0.025 M imidazole-HCl, pH 7.4. Fractions of 1.2 mL were collected and enzymatic activity,
pH, and absorbance at 280 nm were measured.
ment with those reported for enzyme preparations from other
sources. The isoelectric point of mouse liver [20], human
jejunal [21], and porcine erythrocytes [22] was determined to
be 6.46, 6.75, and 6.85, respectively.
(Ea), determined from the slope of the plot, was 9.62
kcal/mole.
Kinetics of Bovine Kidney Cortex Glutathione Reductase
For the optimal pH determination, the enzyme activity
was measured in different concentrations of potassium phos-
phate buffer within the pH range of 5–10. In this study, we
employed the zero buffer extrapolation method for optimal
pH determination and a value of 7.2 for bovine kidney cortex
GR was obtained. Furthermore, we observed the highest ac-
tivity in 100 mM potassium phosphate buffer.
Lineweaver-Burk double reciprocal plots obtained for
NADPH as varied substrate at different fixed GSSG concen-
trations are shown in Fig. (5). The parallel lines observed in
these double reciprocal plots are consistent with the widely
accepted idea that GR operates according to a ping-pong
mechanism. The Km values for NADPH and GSSG were
determined to be 18 ± 3 μM and 65 ± 5 μM, respectively.
The values in other kinetics studies were as follows: hu-
man jejunal GR, KmNADPH 20 μM and KmGSSG 80 μM
To obtain the optimum temperature, the activities of the
enzyme were measured at saturating substrate concentrations
in 100 mM sodium phosphate buffer, pH 7.4, between 20
and 80 °C. The optimum temperature obtained was 50 °C.
To obtain the Arrhenius plot, the activities of the enzyme
were measured between 20 and 55 °C. The activation energy
[21]; E. coli GR, Km
22 ± 2 μM and KmGSSG 97 ±
NADPH
12 μM [23]; rat liver GR, Km
7.9 ± 0.6 μM and
NADPH
KmGSSG 56.7 ± 0.4 μM [24]; anoxia tolerant turtle Tra-
chemys scripta elegans [25], KmGSSG 44.6 μM and Km
NADPH 6.82 μM, respectively.

Glutathione Reductase
Protein & Peptide Letters, 2010, Vol. 17, No. 5 671
Figure 5. Double reciprocal plots of initial velocity of the bovine kidney cortex GR against NADPH as varied substrate at different fixed
GSSG concentrations. Trendline values and standard deviations are R2 = 0.996, 0.0009; R2 = 0.946, 0.0010; R2 = 0.994, 0.0008; R2 = 0.983,
0.0006; R2=0.835, 0.0010; R2=0.984, 0.0012; respectively. The assays were carried out at 37 °C in 100 mM sodium phosphate buffer, pH
7.4, as described in the Materials and Methods.
Product Inhibition of Bovine Kidney Cortex Glutathione
Reductase by NADP+
activity of 727 U/mg by means of two subsequent chroma-
tography steps and a heat denaturation step. The molecular
weight of the enzyme was found to be 117 kDa by Sephadex
G–200 gel filtration chromatography. A protein band corre-
sponding to a subunit molecular weight of 56 ± 2 kDa was
obtained on SDS-PAGE. The isoelectric point of the enzyme
was found to be 6.8 by chromatofocusing. Optimum tem-
perature and pH were 55 °C and 7.2 respectively. The activa-
tion energy of the enzyme was 9.62 kcal/mole. Kinetic char-
acterization of bovine kidney cortex GR is also investigated.
A series of product inhibition studies were carried out to
identify the type of mechanism. Fig. (6) illustrates the inhibi-
tion pattern obtained with NADP+ as the product inhibitor
when NADPH was the variable substrate at fixed GSSG
concentrations (0.7 mM). From these steady state kinetics
data we calculated the inhibition constant (Ki) of NADP+ as
115 ± 7 μM. The inhibition by NADP+ was competitive with
respect to NADPH. In Phycomyces blakesleeanus GR, Ki
NADP+ was calculated as 61 ± 4.2 μM [26].
In this study, GR is purified from bovine kidney cortex
using two chromatographic steps and heat denaturation. This
method allows purification of the enzyme in a very short
time and high yield and purification fold. By using heat de-
naturation step, in particular, GR is separated well from
G6PD and is achieved in higher purity.
The study was repeated by varying GSSG at constant
NADPH concentrations. Initial velocities were determined at
the same concentrations of NADP+. From the statistical
analysis of the data, the inhibition type was found to be un-
competitive with respect to GSSG, and Ki was calculated as
298 ± 10 μM Fig. (7).
The kinetics of GR obeys both a ping-pong and sequen-
tial mechanism; Mannervik suggested that this type of
mechanism be named a branched mechanism [27]. A
branched mechanism is favored by the nonlinear inhibition
pattern produced by NADP+. However, at low GSSG con-
centrations the rate equation can be approximated by a sim-
ple ping-pong mechanism [24]. In this study, initial-rate
studies were performed to analyze the kinetic mechanism of
the enzyme; GSSG was used as variable substrate and the
double reciprocal plots were drawn at different fixed concen-
trations of the other substrate, NADPH. According to the
kinetic data obtained, the reciprocal plots have parallel lines
and this is consistent with the widely accepted idea that GR
operates according to a ping-pong mechanism [28]. After
that, to determine whether the real kinetic mechanism of
bovine kidney cortex GR is a ping-pong or sequential
mechanism, product inhibition studies were performed and
NADP+ was found a competitive inhibitor with respect to
NADPH. Competitive inhibition by a product against the
corresponding substrate could be expected in sequential
mechanisms (Theorell-Chance, ordered, or rapid equilibrium
random), but not in a ping-pong mechanism [29]. GSH is
expected to be noncompetitive with both substrates for either
Product Inhibition of Bovine Kidney Cortex Glutathione
Reductase by GSH
An investigation of the product inhibition by GSH was
carried out to further analyze the reaction mechanism of bo-
vine kidney cortex GR. GSH is also used in product inhibi-
tion studies. We found that GSH inhibited the reaction, but
only at high concentrations. GSH was found to be a non-
competitive inhibitor with respect to both substrates. First,
NADPH was the varied substrate Fig. (8) and GSSG was the
fixed substrate (0.7 mM), and initial velocities were meas-
ured at concentrations of GSH between 1 and 4 mM. From
the statistical analysis of the data, Ki was calculated as 8.055
± 0.33 mM. With regard to GSSG as variable substrate at
fixed NADPH concentration (0.1 mM), GSH appears to be a
noncompetitive inhibitor Fig. (9). From the double reciprocal
plots and statistical analysis, Ki was calculated as 7.203 ±
0.36 mM.
CONCLUSION
Glutathione reductase from the bovine kidney cortex was
purified 34806-fold with a final yield of 85 % and specific

672 Protein & Peptide Letters, 2010, Vol. 17, No. 5
Tandogan and Ulusu
Figure 6. Product inhibition by NADP+ of the reaction catalyzed by bovine kidney cortex GR with NADPH as the variable substrate at fixed
GSSG concentrations. Trendline values and standard deviations are R2 = 0.973, 0.0013; R2 = 0.992, 0.0021; R2 = 0.990, 0.0026; R2 = 0.997,
0.0033; R2=0.995, 0.0039; respectively. The assays were carried out at 37 °C in 100 mM sodium phosphate buffer, pH 7.4, as described in the
Materials and Methods.
Figure 7. Product inhibition by NADP+ of the reaction catalyzed by bovine kidney cortex GR with GSSG as the variable substrate at fixed
NADPH concentrations. Trendline values and standard deviations are R2 = 0.998, 0.0053; R2 = 0.999, 0.0052; R2 = 0.999, 0.0055; R2 =
0.999, 0.0060; R2=0.998, 0.0064; respectively. The assays were carried out at 37 °C in 100 mM sodium phosphate buffer, pH 7.4, as de-
scribed in the Materials and Methods.
Figure 8. Product inhibition by GSH of the reaction catalyzed by bovine kidney cortex GR with NADPH as the variable substrate at fixed
GSSG concentrations. Trendline values and standard deviations are R2 = 0.973, 0.0013; R2 = 0.993, 0.0018; R2 = 0.996, 0.0018; R2 = 0.998,
0.0018; R2=0.991, 0.0019; respectively. The assays were carried out at 37 °C in 100 mM sodium phosphate buffer, pH 7.4, as described in the
Materials and Methods.

Glutathione Reductase
Protein & Peptide Letters, 2010, Vol. 17, No. 5 673
Figure 9. Product inhibition by GSH of the reaction catalyzed by bovine kidney cortex GR with GSSG as the variable substrate at fixed
NADPH concentrations. Trendline values and standard deviations are R2 = 0.994, 0.0053; R2 = 0.999, 0.0058; R2 = 0.998, 0.0063; R2 =
0.998, 0.0068; R2=0.998, 0.0072; respectively.The assays were carried out at 37 °C in 100 mM sodium phosphate buffer, pH 7.4, as described
in the Materials and Methods.
of the two mechanisms considered [30]. In this study, we
found that GSH is a noncompetitive inhibitor of bovine kid-
ney cortex GR and these results are in agreement with the
other studies. According to our results of statistical analysis,
the ping-pong mechanism is a suitable model since the loss
function is less than the other mechanisms. However, com-
petitive inhibition by a product could be accepted in sequen-
tial mechanisms but not in a ping-pong mechanism.ꢀThere-
fore, based on the experimental data obtained in our kinetic
studies, we suggested that bovine kidney cortex GR acts ac-
cording to the branched mechanism (Scheme 1). Although
the double reciprocal plots shows bovine kidney cortex GR
follows the ping-pong mechanism, product inhibition studies
was indicated sequential ordered mechanism. This could be
explained by supposing that the GR normally functions by
branching mechanism.
6PGD
G6PD
NADP+
Ea
=
=
=
=
6-Phosphogluconate dehydrogenase
Glucose-6-phosphate dehydrogenase
Nicotinamide adenine dinucleotide phosphate
Activation energy
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ABBREVIATIONS USED
GR
=
=
Glutathione reductase
FAD
Flavin adenine dinucleotide
[12]
[13]
NADPH = Nicotinamide adenine dinucleotide phosphate
reduced form
GSSG
GSH
=
=
Glutathione oxidized form
Glutathione reduced form

674 Protein & Peptide Letters, 2010, Vol. 17, No. 5
Tandogan and Ulusu
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Received: May 19, 2009
Revised: July 23, 2009
Accepted: August 02, 2009