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Analytical Biochemistry
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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
210
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
B
3000
a
a
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).
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.
References
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Mitochondrial complex I deciency in Parkinsons disease, J. Neurochem. 54
(1990) 823827.
[2] N.A. Riob, E. Clementi, M. Melani, A. Boveris, E. Cadenas, S. Moncada, J.J.
Poderoso, Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase
activity through peroxynitrite formation, Biochem. J. 359 (2001) 139145.
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