To study biological oxidation in liver homogenate (by assaying SDH

activity).

REQUIREMENTS:
Chemicals/reagents: 10% chicken/goat liver homogenate (made in homogenization buffer: 0.25M
sucrose, 20mM Tris HCl pH 7.5, 1mM EDTA), 0.15M Tris HCl pH 7.5, 0.2M sodium succinate (made
in 0.15M Tris HCl pH 7.5), 50mM sodium azide (made in 0.15M Tris HCl pH 7.5), 0.2M sodium
oxalate (made in 0.15M Tris HCl pH 7.5), 50mM DCPIP (made in 0.15M Tris HCl pH 7.5).
Equipment: Test tube stand, test tubes, droppers, beakers, measuring cylinder, micropipettes, microtips,
thermometer, water bath, stopwatch.

THEORY:
Biological oxidation is that oxidation which occurs in biological systems to produce energy. Oxidation
can occur by: (a) Addition of oxygen (less common); (b) Removal of hydrogen (common); (c) Removal
of electrons (most common). Electrons are not stable in the free state, so their removal from a substance
(oxidation) must be accompanied by their acceptance by another substance (reduction), hence the
reaction is called oxidation-reduction reaction or redox reaction and the involved enzymes are called
oxido-reductases.
The mitochondrion is the site of eukaryotic oxidative metabolism. It contains the enzymes that mediate
this process, including pyruvate dehydrogenase, the citric acid cycle enzymes, the enzymes catalyzing
fatty acid oxidation, and the enzymes and redox proteins involved in electron transport and oxidative
phosphorylation. In the electron-transport process, the free energy of electron transfer from NADH and
FADH2 to O2 via protein-bound redox centers is coupled to ATP synthesis (oxidative phosphorylation).
The electron-transport chain is a series of four protein complexes, embedded within the inner
mitochondrial membrane in addition to mobile electron carriers, coenzyme Q (ubiquinoneand
cytochrome c, through which electrons pass from lower to higher standard reduction potentials.
Complexes I and II catalyze electron transfer to ubiquinone from two different electron donors: NADH
(Complex I) and succinate (Complex II). Complex III carries electrons from reduced ubiquinone to
cytochrome c, and Complex IV completes the sequence by transferring electrons from cytochrome c to
O2.

Succinate dehydrogenase (SDH) is part of both the citric acid cycle and respiratory electron transfer
chain. In eukaryotes, SDH is tightly bound to the inner mitochondrial membrane; in prokaryotes, to the
plasma membrane. Hence it is the only enzyme of citric acid cycle which is membrane bound; the other
enzymes of the cycle are in mitochondrial matrix. SDH catalyzes stereospecific dehydrogenation of
succinate to trans-fumarate. The enzyme contains three different iron-sulfur clusters and one molecule
of covalently bound FAD. Electrons pass from succinate through the FAD and iron-sulfur centers before
entering the chain of electron carriers in the mitochondrial inner membrane (or the plasma membrane in
bacteria). Electron flow from succinate through these carriers to the final electron acceptor, O 2, is
coupled to the synthesis of about 1.5 ATP molecules per pair of electrons (respiration-linked
phosphorylation).

Malonate, oxalate, malate & oxaloacetate, all resembling succinate due to the presence of two
carboxylic groups, are competitive inhibitors of SDH and their addition to mitochondria blocks the
activity of the citric acid cycle.
Being a stable enzyme, which is present only in the mitochondria, SDH is frequently used as a
mitochondrial marker in eukaryotic organelle preparations.

PRINCIPLE:
Enzymatic activity or the velocity of an enzymatic reaction can be measured either by the rate of
formation of its products or by the rate of disappearance of substrate. In this experiment, the latter
criterion is used. The reaction of succinate oxidation to trans-fumarate is measured by observing the
reduction of 2,6-dichlorophenolindophenol (DCIPP), an artificial electron acceptor. (Methylene blue is
another frequently used electron acceptor.) Adding sodium azide blocks the electron transport chain so
electrons cannot be transferred to molecular O 2 in the electron transport chain. Instead, the electrons are
transferred from E-FADH2 to DCPIP. (The standard redox potential of DCPIP/DCPIP-H 2 is +0.217V
being higher than that of FAD/FADH 2, DCPIP can take up electrons from FADH 2.) The reduction of
DCPIP can be identified by a color change; the oxidized form of the electron acceptor is blue and its
reduced form is colorless:

E-FADH2 + DCPIPox (blue) → E-FAD + DCPIPre (colorless)

The time taken for DCPIP to decolorize is noted as the end point of the enzymatic activity. Appropriate
control (containing buffer instead of enzyme) is processed simultaneously. Also, the effect of a
competitive inhibitor of SDH, oxalate, on the activity of SDH, is studied. The enzyme substrate reaction
is carried out at optimum conditions of temperature (37oC) & pH (7.5).

PROCEDURE:
1. Enzyme preparation: 10% (w/v) crude chicken/goat liver homogenate was made in cold
homogenization buffer and was kept on ice throughout the experiment.
2. Set up three test tubes and labeled them as control, E+S, E+S+I. In each test tube, various
reagents/chemicals were added as mentioned in the following table:
 Test Tube Control Expt. 1 (Enzyme Expt. 2 (Enzyme
+ Substrate) + Substrate +
Inhibitor)
0.15M Tris HCl 2.4 ml 1.4 ml 0.4 ml
(pH 7.5)
Sodium succinate 1.0 ml 1.0 ml 1.0 ml
(final: 40mM)
Sodium azide 1.0 ml 1.0 ml 1.0 ml
(final: 10mM)
Sodium oxalate -- -- 1.0 ml
(final: 40mM)
DCPIP (final: 100 μl 100 μl 100 μl
0.5mM)

3. In the test tubes labeled E+S and E+S+I, 0.5 ml of 10% liver homogenate was added, mixed
well, a picture of all three tubes was taken, and the tubes were kept in the water bath at 37-40°C.
The time of addition of homogenate was noted.
4. Kept close watch on the tubes with intermittent shaking of the test tubes. As soon as the solution
in tube labeled E+S became colorless, all the test tubes were removed immediately from the
water bath & a picture of all three tubes was taken. The time for decolorization was noted.

RESULT:

The control tube showed no change in color. The reaction was complete in ______ minutes in E+S tube,
as indicated by the decolorization of DCPIP. As could be seen by comparing the E+S tube with E+S+I
tube, the reaction was slower in the latter, & was far from completion at the time of decolorisation in
E+S tube.

DISCUSSION:
Effect of competitive inhibition on enzyme kinetics:
A competitive inhibitor competes with the substrate for the active site of an enzyme. While the inhibitor
(I) occupies the active site it prevents binding of the substrate to the enzyme. Many competitive
inhibitors are compounds that resemble the substrate and combine with the enzyme to form an EI
complex, but without leading to catalysis. Even fleeting combinations of this type will reduce the
efficiency of the enzyme. By taking into account the molecular geometry of inhibitors that resemble the
substrate, we can reach conclusions about which parts of the normal substrate bind to the enzyme.
Competitive inhibition can be analyzed quantitatively by steady-state kinetics. In the presence of a
competitive inhibitor, the Michaelis-Menten equation becomes

Because the inhibitor binds reversibly to the enzyme, the competition can be biased to favor the
substrate simply by adding more substrate. When [S] far exceeds [I], the probability that an inhibitor
molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax. However, the [S]
at which V0 =1/2 Vmax, the apparent Km, increases in the presence of inhibitor by the factor α. This effect
on apparent Km, combined with the absence of an effect on Vmax, is diagnostic of competitive inhibition.
SDH can be inhibited by a number of compounds that resemble succinate in the presence of two
carboxylic groups like malonate, malate, oxalate, oxaloacetate, and glutarate. In the present experiment,
the addition of sodium oxalate affected SDH by slowing down the reaction, as monitored by oxidation
of succinate to fumarate with concomitant reduction of DCPIP. When monitored spectrophotometrically
at ~600nm, the decolorization/reduction of DCPIP can be used to calculate the degree of increase in K m
(αKm) in the presence of sodium oxalate.

PRECAUTIONS:
1. The liver tissue should be homogenized properly with no clumps left. It can be filtered
through 4 layers of cheesecloth if necessary.
2. The homogenate must be kept on ice throughout the experiment to prevent degradation of
enzymes.
3. All tubes in water bath must be shaken at regular intervals and the temperature of water bath
should be maintained constant between 37-40°C.
4. The tubes should all be immediately taken out of the water bath as soon as the reaction is
complete (decolorization of DCPIP) in E+S tube, and a picture should be taken, as eventually
reaction would get completed in E+S+I tube as well (but in a longer time).