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Analytical Biochemistry 415 (2011) 209211

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Analytical Biochemistry
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Notes & Tips

Microplate-based kinetic method for assay of mitochondrial NADH and


succinatecytochrome c reductase activities
Apurva Kumar R. Joshi, N. Raju, P.S. Rajini
Food Protectants and Infestation Control Department, Central Food Technological Research Institute (CSIR Laboratory), Mysore 570020, India

a r t i c l e

i n f o

Article history:
Received 2 February 2011
Received in revised form 8 April 2011
Accepted 11 April 2011
Available online 20 April 2011

a b s t r a c t
This article describes a microplate-based kinetic assay for mitochondrial NADH and succinatecytochrome c reductase activities in rat brain mitochondria. The assay reported here is based on the conventional spectrophotometric method and involves substrate-driven reduction of exogenous cytochrome c.
Conditions regarding linearity with respect to time and protein concentration have been standardized.
Furthermore, the methods were tested for inhibition of respective activities by specic inhibitors. The
microplate format described here can be employed for rapid and simultaneous measurements of mitochondrial NADH and succinatecytochrome c reductase activities in a large number of samples.
2011 Elsevier Inc. All rights reserved.

Defects in mitochondrial electron transport chain are an important cause of several diseases. Hence, NADHcytochrome c reductase (NCCR)1 and succinatecytochrome c reductase (SCCR)
activities are the most routinely studied activities to detect functionality of electron transport chain. Alternately, these activities have
been assessed for identifying defects at either the complex I or complex II level [1,2]. Hence, it is of practical importance to develop a rapid assay method that will facilitate reliable quantitative assessment
of NCCR and SCCR activities. Traditionally, NCCR and SCCR activities
have been assayed by measuring reduction of exogenous cytochrome c driven by NADH and succinate, respectively [3,4], using a
spectrophotometer. However, spectrophotometric methods are
time-consuming and may require calculating activities from initial
linear portions of the graph.
We have adopted the conventional spectrophotometric method
of substrate-driven reduction of exogenous cytochrome c to develop a microplate-based kinetic method for assay of mitochondrial
NCCR and SCCR activities. Furthermore, we investigated whether
the microplate method detects inhibition of NCCR and SCCR activities by rotenone, a complex I inhibitor [5], and 2-thenoyltriuoroacetone (TTFA), a complex II inhibitor [6], respectively. We isolated
mitochondria from whole brain of male rats (CFTWistar,
200 10 g, maintained as per the institutional animal ethics committee guidelines) for the study. The mitochondria were isolated as
described by Mattiazzi and co-workers [7] and were subjected to
three freezethaw cycles [8] to make mitochondrial membranes
permeable to substrates. The downsized assay was conducted in
Corresponding author. Fax: +91 821 2517233.
E-mail address: rajini29@yahoo.com (P.S. Rajini).
Abbreviations used: NCCR, NADHcytochrome c reductase; SCCR, succinate
cytochrome c reductase; TTFA, 2-thenoyltriuoroacetone; BSA, bovine serum albumin; OD, optical density.
1

0003-2697/$ - see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2011.04.014

a 96-well plate for monitoring reduction of exogenous cytochrome


c (100 lM) at 30 C in the presence of NaCN (2.0 mM), a complex IV
inhibitor, and consisted of varying amounts of freezethawed
mitochondrial preparations in a nal volume of 300 ll. The NCCR
assay consisted of NADH (300 lM) and glycylglycine buffer
(50 mM, pH 8.5), whereas the SCCR assay consisted of succinate
(10 mM), bovine serum albumin (BSA, 0.25%), rotenone (5.0 lM),
and sodium phosphate buffer (25 mM, pH 7.4). NCCR and SCCR activities were initiated by the addition of NADH and cytochrome c,
respectively, using a multichannel pipette. In the case of SCCR, a mixture containing succinate, mitochondria, BSA, and rotenone was preincubated for 1 min at 30 C before the addition of cytochrome c. The
reduction of cytochrome c was recorded by monitoring the increase
in absorbance per minute at 1, 2, 3, and 4 min at 550 nm in a microplate reader as change in OD per minute at the end of time intervals
mentioned above. The amount of enzyme resulting in a change in
absorbance of 0.001/min was considered as 1 unit of enzyme. Protein
content of mitochondrial preparation was estimated by a previously
described method [9]. Inhibition of NCCR by rotenone and of SCCR by
TTFA was studied after 5 min of preincubation with the respective
inhibitor followed by the conduct of assay.
NCCR activity was found to be linear with respect to protein
concentrations up to change in optical density (OD)/min of 0.39
absorbance units only during the rst minute. Linearity in the case
of the well containing 30.4 lg of protein was slightly compromised
during the second minute of assay, whereas that of 60.8 lg of protein was greatly compromised (Fig.1A and B). For practical purposes, accurate results may be obtained by using protein
concentrations giving a change in absorbance of 0.10.15/min
and strictly setting the run time for only 1 min. SCCR activity
was found to be linear during the rst minute up to change in
OD/min of 0.18 absorbance units given by the maximum protein
concentrations (76 lg) used in the assay. During the second

210

Notes & Tips / Anal. Biochem. 415 (2011) 209211

1min

2min

3min

4min

0.5

NCCR

NCCR

0.4

OD / min

0.4

OD / min

0.5

0.3
0.2

0.1

(60.8)

0.3

(30.4)

0.2

(15.2)

0.1

(7.6)
0

0
0

10

20

30

40

50

60

70

Protein (g/well)

1min

2min

3min

4min

0.2

0.2

SCCR

(76)

SCCR
0.15

OD / min

0.15

OD / min

Time (min)

0.1

0.05

(57)
0.1

(38)
0.05

(19)

10

20

30

40

50

60

70

80

Protein (g/well)

Time (min)

Fig.1. Rates of substrate-driven reduction of exogenous cytochrome c in the presence of brain mitochondria. Each data point is the mean standard deviation of four
determinations. Panels A and B demonstrate NCCR (NADHcytochrome c reductase) activity driven by NADH, and panels C and D demonstrate SCCR (succinatecytochrome c
reductase) activity driven by succinate. Numbers presented in parentheses in panels B and D indicate amounts of protein (lg) added to assay mixture.

minute of the reaction, a slight compromise in linearity was observed in the case of the highest protein concentration, whereas
good linearity was observed up to 57 lg of protein (change in
OD/min of 0.13 absorbance units) (Fig.1C and D). For practical purposes, assay can be conducted using protein concentrations giving
a change in OD/min of approximately 0.1 absorbance units and setting the run time for 1 min because it offers linearity over a broad
range of protein concentrations. Reliability of the microplate methods for assay of NCCR and SCCR activity is further strengthened by
detection of strong inhibition of both activities by their specic

inhibitors (Fig.2). Rotenone (1.0 nM10 lM) was found to affect


3681% inhibition of NCCR activity, whereas TTFA (0.011.0 mM)
caused 5185% inhibition of SCCR activity.
In conclusion, this article has described microplate methods for
kinetic assay of mitochondrial NCCR and SCCR activities. We stress
the importance of the use of multichannel pipettes to add assay
initiator and recording rates without delay. The microplate format
described here offers advantages of simple yet reliable measurement of NCCR and SCCR activities, particularly in situations where
large numbers of samples need to be analyzed.

B
3000
a
a

SCCR (units/mg protein)

NCCR (units/mg protein)

8000

6000
b

4000
c
d

2000

2000
b

1000
c

-9

10

-8

10

-7

10

Rotenone (M)

-6

10

-5

10

-7

10

-6

10

-5

10

-4

10

-3

10

TTFA (M)

Fig.2. Inhibition of NCCR and SCCR activities by specic inhibitors. Each data point is the mean standard deviation of four determinations. Points with different letters are
statistically different (P < 0.001 by analysis of variance and Tukeys test). Panel A depicts inhibition of NCCR activity by rotenone, and panel B depicts inhibition of SCCR
activity by TTFA (2-thenoyltriuoroacetone).

Notes & Tips / Anal. Biochem. 415 (2011) 209211

Acknowledgments
The authors thank the Director of the Central Food Technological Research Institute (CFTRI, Mysore, India) for his support for the
study. The rst author (A.K.R.J.) acknowledges nancial support
from Council of Scientic and Industrial Research (CSIR, India) in
the form of a research fellowship.
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