Analytical Biochemistry 442 (2013) 1923

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

A spectrophotometric coupled enzyme assay to measure the activity

of succinate dehydrogenase
Andrew J.Y. Jones, Judy Hirst
Medical Research Council Mitochondrial Biology Unit, Cambridge CB2 0XY, UK

a r t i c l e i n f o a b s t r a c t

Article history: Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the
Received 4 June 2013 electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked
Received in revised form 9 July 2013 to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichi-
Accepted 12 July 2013
ometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time
Available online 22 July 2013
method to detect the production of fumarate from succinate by complex II that is easy to implement
and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses
Keywords:
fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydro-
Coupled assay
Fumarate hydratase
genase to convert malate to pyruvate and to convert NADP+ to NADPH; the NADPH is detected spectro-
Oxaloacetate decarboxylating malic enzyme metrically. Simple protocols for the high-yield production of the two enzymes required are described;
Succinate:ubiquinone oxidoreductase oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the
activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on
articial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specic and stoi-
chiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses
of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and
complex compositions.
2013 The Authors. Published by Elsevier Inc. Open access under CC BY license.

In mammalian mitochondria, succinate:ubiquinone oxidore-
ductase (succinate dehydrogenase, complex II, EC 1.3.5.1) catalyzes
the oxidation of succinate to fumarate in the matrix and the reduc-
tion of ubiquinone-10 in the inner membrane [1,2]. Thus, it links
the tricarboxylic acid cycle to the respiratory electron transport
chain. Complex II comprises four subunits [13]. In the hydrophilic
domain, the largest subunit houses a covalently bound FAD (avin
adenine dinucleotide) cofactor and the succinate-binding site, and
a smaller subunit contains three ironsulfur clusters that transfer
electrons from the avin to ubiquinone. The binding site for the
ubiquinone substrate is located close to a heme cofactor in the
membrane-associated domain that is composed of two hydropho-
bic subunits.
Homologues of mammalian complex II are widely conserved
in aerobic organisms, in both eukaryotes and prokaryotes.
Some prokaryotes use fumarate as a terminal electron acceptor
in anaerobic respiration by using enzymes that are closely re-
lated to complex II to catalyze the reverse reaction, reduction
Corresponding author.
E-mail address: jh@mrc-mbu.cam.ac.uk (J. Hirst).
of fumarate to succinate [4]. Some parasites, such as Ascaris
suum, switch between succinate oxidation and fumarate reduc-
tion during different life stages, and a similar switch has been
proposed to occur in mammalian cells during hypoxia [5].
Mutations in complex II have been identied as a cause of
Leigh syndrome, a neurodegenerative disease [6], and complex
II was recently identied as a signicant source of mitochon-
drial reactive oxygen species production [7]. Complex II muta-
tions have also been linked to hereditary paragangliomas,
tumors in the head and neck [8], and there is currently signif-
icant interest in how mitochondrial succinate and fumarate
levels are linked to the stabilization of HIF-1a and, thus, to cell
proliferation [9].
Due to its central role in metabolism and medicine, measure-
ments of complex II activity have long been used to assess the activ-
ity of both the enzyme itself and the mitochondrial electron
transport chain as a whole. However, there is no convenient and
reliable method for testing complex II activity. Clark oxygen elec-
trodes are often used [10] (see, e.g., Refs. [7,11]), but they are expen-
sive in their sample requirements, and because they measure
succinate:O2 oxidoreduction they can assess only the combined
activity of respiratory complexes II, III, and IV. Alternative real-time
assays of complex II rely on articial electron acceptors that change
color on reduction and, therefore, can be monitored spectrophoto-

0003-2697 2013 The Authors. Published by Elsevier Inc. Open access under CC BY license.
http://dx.doi.org/10.1016/j.ab.2013.07.018
20 Coupled enzyme assay for succinate dehydrogenase / A.J.Y. Jones, J. Hirst / Anal. Biochem. 442 (2013) 1923
oxaloacetate
succinate fumarate decarboxylating
dehydrogenase hydratase malic enzyme
NADP+ NADPH
Q H2O
succinate fumarate malate pyruvate
-O
QH2 -O -O
O O O
OH
O O O O
O- O- O-
Scheme 1. Reaction scheme for the coupled assay, illustrating the use of FumC and
MaeB to couple the oxidation of succinate to the reduction of NADP+ in a 1:1
stoichiometry.
metrically. 2,6-Dichlorophenolindophenol (DCPIP)1 and 2-(4-iodo-
phenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) are
reduced directly by complex II and also by the quinol product of com-
plex II catalysis (see, e.g., Refs. [1214]). DCPIP and other tetrazolium
salts (which do not react efciently with complex II directly) have
also been used as secondary electron acceptors, with phenazine
methosulfate (PMS) to mediate electron transfer from complex II
(see, e.g., Refs. [12,14,15]). However, all of these assays lack specic-
ity. When either the electron acceptor or PMS reacts with the enzyme
directly it fails to focus on the complete catalytic cycle of succi-
nate:ubiquinone oxidoreduction, the reactivity is not specic to com-
plex II, and O2 interferes with the results [16]. On the other hand, the
reaction with ubiquinol is inefcient, electrons are not scavenged
stoichiometrically when the respiratory chain is turning over, and
the measured rate is always a combination of all possible pathways.
Here, we present a convenient, stoichiometric, and precise
coupled enzyme assay for succinate oxidation (see Scheme 1).
Our assay uses two enzymes: fumarate hydratase (FumC, EC
4.2.1.2) converts the fumarate product of succinate oxidation to
malate [17], and then oxaloacetate decarboxylating malic dehydro-
genase (MaeB, EC 1.1.1.40) couples the conversion of malate to
pyruvate to the reduction of NADP+ to NADPH [18]; the NADPH
is detected spectrophotometrically. Because our assay quanties
succinate oxidation directly, it is suitable for measuring succi-
nate:ubiquinone oxidoreduction by isolated complex II as well as
succinate consumption in membrane and tissue preparations.
Materials and methods
Preparation of FumC and MaeB
FumC and MaeB from Escherichia coli were overexpressed in E.
coli. Plasmid constructs encoding the fumC and maeB genes were
supplied by Todd Weaver (University of WisconsinLa Crosse)
[17] and Mara Drincovich (National University of Rosario, Argen-
tina) [18], respectively. The same protocol, adapted from Refs. [17]
and [18], was used for expression and purication of both proteins
unless otherwise stated. E. coli BL21(DE3) cells (New England Bio-
labs) were transformed with the maeB or fumC plasmid. Cells were
grown at 32 C in LB medium supplemented with 50 lg ml1 ampi-
cillin until A600 reached approximately 0.6, and then protein expres-
sion was induced using isopropyl b-D-1-thiogalactopyranoside
(IPTG, 1 mM for FumC and 0.1 mM for MaeB) for 18 h at 20 C. Cells
were harvested by centrifugation (3500g, 10 min), resuspended in
buffer A (50 mM potassium phosphate and 300 mM NaCl, pH 7.8,
1
Abbreviations used: DCPIP, 2,6-dichlorophenolindophenol; INT, 2-(4-iodo-phe-
nyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride; PMS, phenazine methosulfate;
FumC, fumarate hydratase; MaeB, oxaloacetate decarboxylating malic dehydroge-
nase; EDTA, ethylenediaminetetraacetic acid; SMP, submitochondrial particle.
at 4 C) for FumC or buffer B (20 mM TrisCl, 100 mM NaCl,
25 mM imidazole, and 10% [w/v] glycerol, pH 7.4, at 4 C) for MaeB,
and then lysed in one passage through a Constant Systems model B
2.2-kW Z cell disruptor at 30 psi. The lysates were centrifuged
(150,000g, 45 min),the pellets were discarded, and the supernatants
were ltered (0.45 lm) before being loaded onto a preequilibrated
+ CO2 25 ml NiSepharose 6 Fast Flow column (GE Healthcare). The col-
umn was washed using buffer A + 60 mM imidazole for FumC or
buffer B for MaeB, and then the required proteins were eluted in buf-
O
fer A + 400 mM imidazole for FumC or buffer B + 300 mM imidazole
for MaeB. Relevant fractions were identied by activity assays,
O-
pooled, and concentrated (Amicon Ultra-15 50-kDa centrifugal lter
units) and then were dialyzed overnight in buffer C (10 mM Tris
SO4, 5 mM ethylenediaminetetraacetic acid [EDTA], and 1 mM
dithiothreitol, pH 7.0, at 4 C) for FumC or buffer D (60 mMTris
SO4, 20 mM b-mercaptoethanol, 25 mM imidazole, and 10% [w/v]
glycerol, pH 7.4, at 4 C) for MaeB. Typical yields were 36 and
60 mg per liter of culture for FumC and MaeB, respectively.
Preparation of complex II-containing samples
Submitochondrial particles (SMPs) were prepared from bovine
heart mitochondria as described previously [11]. Complex II
was solubilized from bovine heart mitochondrial membranes
( 10 mg protein ml1 in 10 mM TrisSO4 [pH 7.4], 250 mM
sucrose, 1 mM EDTA, and 0.005% phenylmethanesulfonyl uoride
[PMSF]) by the addition of 0.5% n-dodecyl-b-D-maltopyranoside
(DDM); the sample was stirred on ice for 30 min and then
centrifuged (48,000g, 30 min), and the supernatant was collected.
Complex I was prepared from bovine heart mitochondria as
described previously [19]. Tissue samples were prepared from rat
skeletal muscle using a procedure modied fromRef. [20]. Briey,
the leg muscle was stripped of fat and connective tissue and
chopped nely (<1 mm pieces), and then it was homogenized in
15 volumes of buffer (320 mM sucrose and 10 mM TrisHCl, pH
7.0, at 4 C) using a tight-tting glass Teon homogenizer. The
sample was centrifuged at 1000g for 5 min at 4 C, and the super-
natant was collected.
Kinetic activity measurements
All assays were carried out in 10 mM TrisSO4 (pH 7.4 at 32 C),
250 mM sucrose, 2 mM MgSO4, and 1 mM K2SO4 at 32 C unless
otherwise stated. Gramicidin (20 lg ml1) was included to dissi-
pate the proton motive force in SMPs. Standard concentrations
were 5 mM succinate, 2 mM NADP+, 60 lg ml1 FumC, and
300 lg ml1 MaeB. The NADPH concentration was followed at
340 to 380 nm (e = 4.81 mM1 cm1) using a Molecular Devices
SpectraMax Plus 384plate reader. The reductions of DCPIP
(100 lM, 600 nm, e = 21 mM1 cm1, blue to colorless) [14] and
INT (100 lM, 500 nm, e = 19 mM1 cm1, colorless to red) [13]
were measured in the presence and absence of 100 lM decylubiq-
uinone, 5 mM succinate, or 100 lM NADH. When required, ubiqui-
nol oxidation by the respiratory chain was inhibited by 400 lM
NaCN, and atpenin A5 (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) was used to inhibit complex II and added from a concentrated
stocksolution in dimethyl sulfoxide (DMSO). O2 consumption by
SMPs was measured using a Clark electrode in a stirred 1 ml
Perspex cell held at 32 C (Rank Brothers, Cambridge, UK).
Results and discussion
Kinetic characterization of FumC and MaeB
Fig. 1 shows how the rates of catalysis by the FumC and MaeB
enzymes used here depend on the concentrations of fumarate
 Coupled enzyme assay for succinate dehydrogenase / A.J.Y. Jones, J. Hirst / Anal. Biochem. 442 (2013) 1923 21

but they catalyze reversibly to reach an equilibrium position and,

300

(mol min-1 mg-1)

thus, can provide only a comparative evaluation of enzyme activi-
200 ties. The MaeB preparation described here compares favorably on
Rate both criteria; it catalyzes irreversibly and can be prepared easily
100 and cheaply in large amounts.

0
A
0 5 10 15 20 Measurement of rate of succinate oxidation by submitochondrial
[Fumarate] (mM) particles

8 8 Fig. 2A shows a typical assay trace recorded to follow succi-

(mol min-1 mg-1)

6 6 nate:O2 oxidoreduction by respiratory complexes II, III, and IV in

SMPs. The rate of NADPH formation is not constant throughout
Rate

4 4
2 2
0
B 0
0 10 20 30 40 50 60
[Malate] (mM)
Fig.1. Kinetic characterization of the FumC and MaeB enzymes. (A) Rate of
conversion of fumarate to malate by FumC, detected by the production of NADPH
that is driven by the conversion of malate to pyruvate by MaeB. The concentrations
were 1.7 lg ml1 FumC, 300 lg ml1 MaeB, and 2 mM NADP+. (B,C) Rates of
conversion of malate to pyruvate by MaeB, detected by the production of NADPH.
The concentrations were 44.2 lg ml1 MaeB and 4 mM NADP+ (B) and 40 mM
malate (C). In all cases, assays were carried out in triplicate with standard error bars
reported, at 32 C, in buffers containing 10 mM TrisSO4 (pH 7.4), 250 mM sucrose,
2 mM MgSO4, and 1 mM K2SO4.
and of malate and NADP+, respectively. First, the assay buffer used
for all experiments contained 2 mM Mg2+ because MaeB requires
Mg2+ for catalysis [18]; as expected, no catalysis was observed in
the absence of Mg2+, and concentrations above 4 mM have been
shown previously to inhibit [18]. K+ (2 mM) was also included in
the assay buffer because it was shown previously to activate MaeB
by approximately 30% [18]. The rate of MaeB catalysis is easily
measured by the accumulation of NADPH at 340 to 380 nm;
Fig. 1B shows that the apparent KM for malate is relatively high
the assay; it begins slowly and then speeds up to reach a constant
value after approximately 100 s. During the assay lag phase, the
C intermediate fumarate and malate concentrations accumulate to
0 1 2 3 4 their steady-state values. Thus, the lag phase can be eliminated
[NADP+] (mM) by increasing the concentrations of FumC and MaeB. However,
the nal rate measurement is unchanged, and because of the in-
creased enzyme requirements we consider this to be unnecessary.
To conrm the specicity of the coupled assay for the conver-
sion of succinate to fumarate, rates of NADPH formation were mea-
sured in the absence of various assay components. In the absence
of SMPs, succinate, MaeB, or NADP+ the observed rates were negli-
gible, but in the absence of FumC a signicant rate, approximately
10% of the control, was observed. Based on the other control exper-
iments, we conclude that this results from a low level of endoge-
nous fumarate hydratase in the SMP preparationwhich does not
affect the assay results because it merely reduces the load on the
exogenous FumC.
Fig. 2B shows that our standard working concentrations of
MaeB (300 lg ml1) and FumC (60 lg ml1) are adequate for the
quantitative detection of succinate oxidation by SMPs (provided
that the lag phases are discarded), and Fig. 2C shows that the rate
0.5
Abs. 340-380 nm

(7.3 0.9 mM) and the Vmax is poor (8.37 0.08 lmol min1 mg1
or 92 0.93 s1), and Fig. 1C shows that for NADP+ the KM is lower 0.4
(52 2.6 lM). Both KM values were measured with the second sub- 0.3
strate at a high enough concentration for Vmax. Our values are 0.2
comparable to those reported previously (KM(malate) = 3.4 mM, 0.1
KM(NADP+) = 41.5 lM, Vmax = 67 s1) [18]. Fig. 1A shows how the rate 0
A
of conversion of fumarate to pyruvate, in the presence of FumC and 0 100 200 300
MaeB, depends on the concentration of fumarate present; the KM Time (s)
for fumarate is 64 7.8 lM and Vmax is 293 7 lmol min1 mg1
or 975 21.7 s1. The values are similar to those reported previ- mol min-1 mg-1 Abs min-1
ously (207 lM and 1149 s1) [21]. For these measurements, the 0.8 0.3
concentration of MaeB was set to 300 to 600 lg ml1 (0.45 0.6
Rate

0.90 lM) to overcome its poor Vmax value; increasing the MaeB 0.2
0.4 MaeB = 300 g ml -1
concentration further did not increase the observed rate of cataly- FumC = 60 g ml -1
0.1
sis (see below also). The concentration of NADP+ was set to 2 mM, 0.2
approximately 20 times higher than KM. Along with the release of B C
0 0
CO2, the high NADP+/NADPH ratio precludes the reverse reaction, 0 1 2 3 4 0 100 200
conversion of pyruvate to malate, which has been demonstrated Relative enzyme [SMP] (g ml -1)
to occur slowly under some conditions [18]. The lower KM and
concentration
higher Vmax values observed for FumC allowed it to be used at Fig.2. Quantifying the rate of succinate oxidation by the respiratory chain in
lower concentrations, 60 to 120 lg ml1 (0.30.6 lM), than MaeB submitochondrial particles. (A) Assay trace measuring succinate oxidation by SMPs
in subsequent experiments (see below also). (40 lg ml1), detected by the coupled FumCMaeB assay system as the formation of
NADPH (300 lg ml1 MaeB and 60 lg ml1 FumC). The trace becomes linear after
Previously, the decarboxylating malic enzyme from pigeon liver
approximately 100 s. (B) Observed rates of succinate oxidation by SMPs
(equivalent to MaeB) was used in an assay for fumarate hydratase (40 lg ml1), detected by the coupled FumCMaeB assay system as the formation
activity [22]. Similar assays have been used recently in studies of of NADPH, over a range of FumC and MaeB concentrations. (C) Rates of succinate
fumarate hydratase deciencies (see, e.g., Ref. [23]). However, oxidation by SMPs, detected by the coupled FumCMaeB assay system as the
commercially available decarboxylating malic enzymes (EC formation of NADPH, over a range of SMP concentrations (300 lg ml1 MaeB and
60 lg ml1 FumC). In panels B and C, assays were carried out in triplicate with
1.1.1.38, -.39, or -.40) are prohibitively expensive, precluding them
standard error bars reported. In all cases, assays were at 32 C in buffers containing
from being applied extensively in kinetic studies. Alternatively, 10 mM TrisSO4 (pH 7.4), 250 mM sucrose, 5 mM succinate, 2 mM NADP+, 2 mM
malate dehydrogenases (EC 1.1.1.37 or -.82) are much cheaper, MgSO4, and 1 mM K2SO4.
22 Coupled enzyme assay for succinate dehydrogenase / A.J.Y. Jones, J. Hirst / Anal. Biochem. 442 (2013) 1923
of NADPH reduction by the detection system depends directly on
the SMP concentration over the whole range tested. Therefore,
the detection system is not rate limiting in any of these experi-
ments, and the observed rates accurately dene the rates of succi-
nate oxidation.
Comparison of coupled assay system with alternative methods
Succinate oxidation by complex II in SMPs is coupled to the
reduction of O2 by complex IV, via the electron transport chain,
in a 2:1 succinate:O2 stoichiometry. Therefore, measurements of
O2 consumption, using a Clark electrode, are the gold standard
for conrming the accuracy of any new assay. Fig. 3A shows that
measurements of the rate of succinate oxidation by SMPs using
our coupled assay system agree very well with Clark electrode
measurements. Note that our assay system can be applied to mea-
surements on isolated complex II, whereas the Clark electrode can
be used only when complex II catalysis is linked to O2 reduction by
the respiratory chain. Our assay has the additional advantages of
being a spectrophotometric method, with signicantly lower sam-
ple requirements, improved convenience, and quicker and easier
data accumulation.
To investigate alternative methods that are applicable to iso-
lated complex II as well as to membrane and tissue preparations,
we compared our coupled assay for succinate oxidation with an as-
say using DCPIP [12,14] (see Table 1). The rate of succinate oxidation
by detergent-solubilized bovine heart mitochondrial membranes
was measured in the presence of a short-chain ubiquinone, decyl-
ubiquinone (the solubilization isolates complex II from the other
enzymes and precludes signicant turnover from endogenous ubi-
quinone-10). Using 30 lg ml1 solubilized membranes, 5 mM suc-
cinate, and 100 lM decylubiquinone, the observed coupled assay
rate was 0.41 0.023 lmol min1 mg1 (the rate was negligible in
the absence of any of the components). With 100 lM DCPIP, a sub-
stantial rate of DCPIP reduction (0.062 0.003 lmol min1 mg1)
was observed even in the absence of decylubiquinone. A similar re-
sult (0.088 0.005 lmol min1 mg1) was obtained when 100 lM
1.2
(mol min-1 mg-1)
0.8
Rate
0.4
0
0 5 10
[Succinate] (mM)
100 100
Rate (%)
80 80
60 60
40 40
20 20
B C
0 0
0 20 40 60 80 100
[Atpenin] (nM)
Fig.3. Kinetic parameters describing succinate oxidation and its inhibition by the
respiratory chain in SMPs. (A) Rates of succinate oxidation by SMPs detected by the
coupled assay system (solid symbols) or by the Clark O2 electrode (open symbols).
(B,C) Rates of succinate oxidation by SMPs detected by the coupled assay in the
presence of varying concentrations of atpenin A5 (B) and malonate (C). Inhibition
curves were t using the standard doseeffect relationship with Hill slope = 1. The
assays contained 120 lg ml1 FumC, 600 lg ml1 MaeB, 5 mM succinate (B,C),
2 mM NADP+, 2 mM MgSO4, and 1 mM K2SO4 in 10 mM TrisSO4 (pH 7.4) and
250 mM sucrose. The measurements were carried out in triplicate at 32 C and are
reported with standard error bars.
NADH was added instead of succinate. Therefore, DCPIP is reduced
directly by the respiratory chain enzymes without the participation
of ubiquinone, so the assay does not address the complete complex
II turnover cycle. When 100 lM decylubiquinone and 100 lM
DCPIP were present (the DCPIP should be reduced by the decyl-
ubiquinol product), the rate of DCPIP reduction increased to
0.20 0.007 lmol min1 mg1. This value is signicantly less than
the value from our coupled assay even if the ubiquinone-indepen-
dent rate is not subtracted. Furthermore, when the full respiratory
chain is present, DCPIP is unable to compete with complex III for
ubiquinol, so complex IV must be inhibited to allow ubiquinone
reduction to be measured. For example, when complex I NADH:ubi-
quinone oxidoreduction was measured with 30 lg ml1 SMPs and
100 lM NADH, in the absence of a complex IV inhibitor, the rate
of NADH oxidation measured directly was 0.38 0.005 lmol
min1 mg1, but the rate of DCPIP reduction was much lower,
0.18 0.009 lmol min1 mg1. Rates of succinate oxidation by
solubilized complex II determined using 100 lM INT [13] instead
of DCPIP were similarly lower than the control value (see Table 1).
Measurement of complex II activity in tissue samples
To detect biochemical defects in complex II in patients with
mitochondrial diseases, rates of complex II catalysis are typically
measured in tissue homogenates from biopsy samples. Specically,
the rate of DCPIP reduction is monitored in the presence of succi-
nate and in the presence and absence of ubiquinone-1 [24]. There-
fore, measurements from our coupled assay were compared with
DCPIP measurements on rat skeletal muscle homogenates (see Ta-
ble 1). Our coupled assay, with 5 mM succinate, 50 lM
ubiquinone-1, and 1.5 mM NaCN, gave a rate of 0.12 0.006
lmol min1 mg1 that was fully sensitive to 2 lM atpenin A5, a
complex II inhibitor [14]. Using the same substrate concentrations,
the rate measured using 100 lM DCPIP was only 0.06 0.001
lmol min1 mg1. In the absence of both ubiquinone-1 and NaCN,
the rates were 0.054 0.002 lmol min1 mg1 (coupled assay)
and 0.019 0.001 lmol min1 mg1 (DCPIP). As expected, in the
absence of ubiquinone-1, NaCN inhibited succinate oxidation in
the coupled assay fully, but the DCPIP assay gave a rate of
0.018 0.001 lmol min1 mg1. Typical rates reported using succi-
nate, ubiquinone-1, NaCN, and DCPIP in human tissue samples are
0.061 to 0.079 lmol min1 mg1 [24], comparable to the rate de-
tected here in rat tissue by the same method. Thus, the same picture
emerges for tissues samples as for solubilized membranesthat
rates of succinate oxidation measured using DCPIP are a signicant
A underestimate of the true rates. However, we are aware of the
15 20 importance of standardized measurements for the comparison of
values from tissue biopsy samples [24] and, thus, that continuing
with established assay protocols may be preferable in this case.
Determination of kinetic parameters for succinate dehydrogenase
catalysis
The coupled assay was used to determine the KM and Vmax val-
ues for complex II in SMPs (see Fig. 3A) and the IC50 values for two
0 1 2 3 4 5 complex II inhibitors (Figs. 3B and C). The KM value obtained,
[Malonate] (mM) 410 55 lM, is within the range of values reported previously
(using DCPIP) of 130 lM [25], 303 lM [26], 1.3 mM [27], and
1.5 mM [28]. By using the published value of 0.19 nmol of complex
II per milligram of protein in a mitochondrial inner membrane
preparation [29], our Vmax of 1.2 0.03 lmol min1 mg1
(although depending strongly on the assay conditions and compo-
sition), equates to a turnover number of 100 2.7 s1. Note that
our value is similar to that reported previously from a Clark elec-
trode measurement in 10 mM succinate (0.94 lmol min1 mg1
[30]); both values are higher than a previous value from this labo-
 Coupled enzyme assay for succinate dehydrogenase / A.J.Y. Jones, J. Hirst / Anal. Biochem. 442 (2013) 1923
Table 1
Comparison of values measured using the different assay methods.
Preparation Substrate(s)
a
Complex II Succinate + DQ
Complex IIa Succinate only
Tissueb + NaCNc Succinate + Q1
Tissueb + NaCNc Succinate
Tissueb Succinate
Note. All values are reported in lmol min1 mg1 and measured with 5 mM succinate and 100 lM decylubiquinone (DQ) or 50 lM ubiquinone-1 (Q1) as stated.
a
Detergent-solubilized bovine heart mitochondrial membranes in which complex II is functionally isolated.
b
Rat skeletal muscle homogenate.
c
NaCN inhibits cytochrome c oxidase; it prevents reoxidation of endogenous ubiquinol-10 so that no ubiquinone-10 is present.
ratory (0.264 lmol min1 mg1 [11]) due to improvements in mito-
chondria preparation. Fig. 3B shows that atpenin A5, an inhibitor
that binds in the complex II ubiquinone-binding site, has an IC50
value of 2.4 1.2 nM, in good agreement with the published value
of 3.6 nM [14] measured using DCPIP in mitochondria. Fig. 3C
shows that malonate, an inhibitor that binds in the complex II a-
vin site, has an IC50 value of 96 1.3 lM, in line with the values of
42 lM in rat mitochondria [31] and 180 to 210 lM in human cell
lines [32] measured using DCPIP. Inhibition of complex II by the
avin site inhibitor oxaloacetate could not be quantied because
it is an intermediate of the MaeB reaction and so interferes with
our detection system.
Acknowledgments
This research was funded by the Medical Research Council. We
thank Todd Weaver (University of WisconsinLa Crosse) and Mara
Drincovich (National University of Rosario, Argentina) for the fumC
and maeB plasmids, respectively.
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