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Date: Wednesday 20th October 2010

Course Code & Title of Lab: BIOL 2363 – Properties of an enzyme: Succinate Dehydrogenase

Aim: To determine the absorbance produced by the reduction of one mol of Neotetrazolium by μplotting a calibration curve; to explore the aspects of preparing the enzyme succinate dehydrogenase from rat liver; to determine the amount of reduced neotetrazolium in the presence and absence of succinate with time via absorbance readings; to investigate the effect of increasing substrate/succinate concentration on the oxidation reaction of succinate to fumarate and the effect of the competitive inhibitor malonate and increasing concentrations of succinate on the oxidation reaction of succinate to fumarate via spectrophotometric methods.

Theory:

Enzymes are catalysts that change the rate of a reaction without being changed themselves. They are highly specific and their activity can be regulated (Hames and Hooper 2005).

The citric acid cycle is the central metabolic hub of the cell. The citric acid cycle, in conjunction with oxidative phosphorylation, provides the vast majority of energy used by aerobic cells (Berg, Tymoczko and Stryer 2002).

A diagram of the citric acid cycle is shown in Fig. 1.

(Nelson and Cox 2008)

Fig.1: Reactions of the Citric Acid Cycle

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Succinate Dehydrogenase is the enzyme involved in the oxidation of succinate to fumarate in the citric acid cycle.

The structure of succinate dehydrogenase is show in Fig. 3 and the oxidative reaction in Fig. 4.

Fig. 3: Structure of Succinate Dehydrogenase (Nelson and Cox 2008)

In this experiment, the compound neotetrazolium replaces FAD as the hydrogen acceptor to allow the oxidation of succinate. Its activity can be followed by observing the colour change from yellow/brown to violet.

Neotetrazolium + 4H+ Neotetrazolium diformazan(Yellow/brown) (Violet)

The catalytic rate of an enzyme can be lowered by inhibitor molecules. A competitive inhibitor typically has close structural similarities to the normal substrate for the enzyme. Thus it competes with substrate molecules to bind to the active site as shown in Fig. 5.

(Hames and Hooper 2005)Fig. 5: The characteristics of competitive inhibition

Competitive inhibitors bind reversibly to the active site therefore at high substrate concentrations the action of a competitive inhibitor is overcome (Hames and Hooper 2005).

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Fig. 4: Oxidation reaction of Succinate to fumarate. FAD removes two hydrogens from succinate to form fumarate (Alberts, et al. 2003).

The enzyme succinate dehydrogenase can be inhibited by the competitive inhibitor malonate. Malonate differs from succinate in having one methylene group compared to succinate which has two methylene groups. Fig 6 shows the inhibition of succinate dehydrogenase by malonate.

Fig. 6: Inhibition of succinate dehydrogenase by malonate (Hames and Hooper 2005)

Competitive inhibition can be quantitatively analyzed by steady-state kinetics. In the presence of a competitive inhibitor, the Michaelis-Menten equation becomes:

Where [I] – concentration of inhibitor[E] - concentration of enzyme[S]- Concentration of substrateKm - substrate concentration at which V0 is one-half VmaxVO – Initial velocity

When substrate concentration far exceeds the concentration of the inhibitor, the probability that an inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax (velocity at maximum substrate concentration). However, the substrate concentration at

which V0 =12

Vmax, the apparent Km, increases in the presence of inhibitor by the factor . This α

effect on apparent Km, combined with the absence of an effect on Vmax, is indicative of competitive inhibition (Nelson and Cox 2008).

This can be seen in the double reciprocal plot shown on Fig. 7.

(Nelson and Cox 2008)

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Fig.7: Double-reciprocal plots. One obtained in the absence of inhibitor and two at different concentrations of a competitive inhibitor. Procedure:

REAGENTS:

1. 0.35% Na2S2O4 – (very labile) freshly prepared in ice cold distilled water. 2. 0.2% NT in water – freshly prepared.3. 0.02M sodium succinate.4. 0.01M sodium malonate.5. 0.25M sucrose.6. 0.5M phosphate buffer, pH7.5.7. Acetone.

Preparation of Calibration Curve:

Eight test tubes were obtained and labelled 1-8. 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of 0.06% NT was added to each tube respectively. The volumes of the tubes were then made up to 1.0mL using distilled water. 2mL of cold Na2S2O4 was added to each tube. The tubes were then kept in the dark and stored on ice. 7mL of acetone was added to dissolve the coloured formazan and after 10minutes, the absorbance of the solutions of each tube was measured at 540nm. A calibration curve was then plotted.

Preparation of the Enzyme:

A rate was sacrificed and its liver removed. Approximately 5g of liver was weighed out and its weight recorded. The liver was cut into small pieces and placed in a homogenization tube and immersed in a beaker of chipped ice and water. 10mL of 0.25M sucrose solution was added to the tube and homogenized. The homogenate was poured into a graduated cylinder through a double layer of gauze and sucrose was added to make the volume up to 10 times the weight of the tissue used. The homogenate was poured into centrifuge tubes and centrifuged at 600g for 10minutes at 4°C. The supernatant was transferred to an Erlenmeyer flask in an ice bath and mixed.

Time Course:

Eight tubes were obtained and labelled 1-8. Tube 1 contained 0.0mL succinate, 0.5mL buffer, 0.5mL NT and 1.2mL water. Tube 2 contained 0.9mL succinate, 0.5mL buffer, 0.5mL NT and 0.3mL water. Tube 3 contained 0.9mL succinate, 0.5mL buffer, 0.5mL NT and 0.3mL water. Tube 4 contained 0.9mL succinate, 0.5mL buffer, 0.5mL NT and 0.3mL water. Tube 5 contained 0.9mL succinate, 0.5mL buffer, 0.5mL NT and 0.3mL water. Tube 6 contained 0.9mL succinate, 0.5mL buffer, 0.5mL NT and 0.3mL water. Tube 7 contained 0.9mL succinate, 0.5mL buffer, 0.5mL NT and 0.3mL water. Tube 8 contained 0.0mL succinate, 0.5mL buffer, 0.5mL NT and 1.2mL water.

The eight tubes were placed in a water bath at 37°C for 3 minutes and 7mL of acetone was added to tubes 1 and 2. 1mL of homogenate was then added to each tube and the time noted. The tubes were then incubated at 37°C for the following times: tube 1 – zero minutes, tube 2- zero minutes, tube 3- 5 minutes, tube 4 – 10 minutes, tube 5 – 15 minutes, tube 6 - 20 minutes, tube 7 – 30 minutes and tube 8- 30 minutes. 7.0mL of acetone was then added to tubes 3 – 8. The contents of

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each tube were then transferred to a numbered centrifuge tube and a small amount of ammonium sulphate added. The tubes were spun at 3000rpm for 5 minutes. The absorbance of the clear supernatant was measured at 540nm using acetone as the blank.

Effect of Substrate Concentration and the effect of Malonate:

Tubes were obtained and labelled 9 – 24. Tube 9 contained 0.0mL succinate, 0.5mL buffer, 0.5mL NT and 1.2mL water. Tube 10 contained 0.05mL succinate, 0.5mL buffer, 0.5mL NT and 1.15mL water. Tube 11 contained 0.1mL succinate, 0.5mL buffer, 0.5mL NT and 1.1mL water. Tube 12 contained 0.25mL succinate, 0.5mL buffer, 0.5mL NT and 0.95mL water. Tube 13 contained 0.4mL succinate, 0.5mL buffer, 0.5mL NT and 0.8mL water. Tube 14 contained 0.5mL succinate, 0.5mL buffer, 0.5mL NT and 0.7mL water. Tube 15 contained 1.0mL succinate, 0.5mL buffer, 0.5mL NT and 0.2mL water. Tube 16 contained 1.1mL succinate, 0.5mL buffer, 0.5mL NT and 0.1mL water.

Tube 17 contained 0.0mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 1.1mL water. Tube 18 contained 0.05mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 1.05mL water. Tube 19 contained 0.1mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 1.0mL water. Tube 20 contained 0.25mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 0.85mL water. Tube 21 contained 0.4mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 0.7mL water. Tube 22 contained 0.5mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 0.6mL water. Tube 23 contained 1.0mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 0.1mL water. Tube 24 contained 1.1mL succinate, 0.1mL malonate, 0.5mL buffer, 0.5mL NT and 0.0mL water.

The tubes were placed in a water bath at 37°C for 3 minutes. 1mL of homogenate was then added to each tube and the time noted. The tubes were then incubated at 37°C. The contents of each tube were then transferred to a numbered centrifuge tube and a small amount of ammonium sulphate added. The tubes were spun at 3000rpm for 5 minutes. The absorbance of the clear supernatant was measured at 540nm using acetone as the blank.

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References

Alberts, Bruce, et al. Essential Cell Biology. London: Garland Science, 2003.

Berg, Jeremy M, John L Tymoczko, and Lubert Stryer. Biochemistry. New York: W. H. Freeman and Company, 2002.

Hames, David, and Nigel Hooper. Instant Notes Biochemistry 3/e. New York: Taylor & Francis Group, 2005.

Nelson, David L, and Michael M Cox. Lehninger Principles of Biochemistry. United States of America: W.H. Freeman and Company, 2008.

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