BIO 220 Laboratory Exercises
Laboratory II: Kinetic Characterization
of Succinate Dehydrogenase
The goal of this lab is to estimate the kinetic parameters (Km and Vmax) of succinate dehydrogenase, a mitochondrial marker enzyme.
Students will examine the effects of four variables on the rate of an enzymatic reaction:
1) enzyme concentration
2) substrate concentration
3) presence of a competitive inhibitor
4) a variable of the student's choice, ex. temperature, different concentrations of enzyme,
substrate, or inhibitor, or other similar perturbation
Introduction
Enzymes are naturally occurring catalysts that increase the rate of a chemical reaction. The enzyme catalyst increases the chances that reactants will contact each other in the proper orientation by providing specific binding sites for the substrates. In this experiment, we are interested in factors that affect the rate of enzyme-catalyzed reactions, a field known as enzyme kinetics. The pioneering work in this field was done by Michaelis and Menton (1913), who formulated a theory that describes the initial events in an enzyme catalyzed reaction. The theory assumes that enzyme (E) and substrate (S) combine reversibly to form an enzyme-substrate (ES) complex. The ES complex then breaks down to form free enzyme (E) and product (P). (See Chapter 6 of Becker et al.1 for more information.)
Succinate dehydrogenase (SDH) is an enzyme of the Krebs cycle and is embedded within the mitochondrial inner membrane. It catalyzes the following reaction:
Figure 1. The reaction2 of succinate dehydrogenase (SDH), which may also be expressed as: Succinate +E-FAD àFumarate + E-FADH2. E-FAD represents SDH with its covalently bound coenzyme, flavin adenine dinucleotide (FAD). The E-FAD complex oxidizes succinate to fumarate. Hydrogens removed from succinate are passed to the enzyme-bound FAD, which is converted to FADH2. SDH is also part of respiratory complex II of the electron transport chain. In this project, you will measure SDH activity in the mitochondria isolated during fractionation of rat liver cells. The rate of conversion of succinate to fumarate will be measured by monitoring the reduction of an artificial electron acceptor by the E-FADH2 complex. Since SDH is also part of the electron transport chain, hydrogens from the reduced SDH complex (E-FADH2) are naturally transferred to the next component of the electron transport chain, and so on, ultimately leading to the production of ATP. (See Chapter 10 of Becker et al.1 for details.) To force these hydrogens onto an artificial electron acceptor, their normal path into the electron transport chain must be blocked. The respiratory chain poison sodium azide will be used to block the electron transport chain, forcing the hydrogens (and electrons) from E-FADH2 onto the artificial electron acceptor dichlorophenolindophenol (DCIP). The reduction of DCIP by E-FADH2 can be measured spectrophotometrically, since the oxidized form of the dye is blue and the reduced form is colorless. This reaction can be summarized:
E-FADH2 + DCIP(ox) (blue) à E-FAD + DCIP(red) (colorless)
Thus, the rate of decrease in absorbance at 600 nm (D A600 nm / min) can be used to measure the velocity of the SDH reaction.
This experiment is designed to test the effects of three factors on the velocity of SDH: enzyme concentration, substrate concentration, and addition of a competitive inhibitor. The concentration of the enzyme in the reaction will be altered by adding different volumes of mitochondrial fraction. The effects of substrate concentration will be measured by altering the amount of succinate added to the reaction mixture. Malonate will be used to demonstrate the effects of a competitive inhibitor on SDH activity.
Procedure
Safety Notice:
Azide is a respiratory chain poison. Ingestion of even a small amount (5-10 mg) of sodium azide has been shown to cause toxic symptoms3. Gloves must be worn when handling azide.
1) Allow the spectrophotometer to warm up for at least 5 min. Set the wavelength to 600 nm.
2) Label 11 disposable cuvettes according to Table 1.
3) Determine the volume of the homogenization buffer that should be added to each cuvette to
achieve a final reaction volume of 3 mL.
4) Prepare each cuvette by adding the solutions as outlined in Table 1.
****DO NOT ADD MITOCHONDRIAL SUSPENSION YET.**** Except for the ice-
cold mitochondrial suspension, all solutions should be at room temperature.
5) When you are ready to begin the reaction and record absorbance values, add the
mitochondrial suspension to each tube.
Important notes: 1) Before the removal of each aliquot, resuspend the material of the
mitochondrial pellet solution by gentle swirling. 2) You may need to dilute the
mitochondrial suspension if the reaction rate is too rapid. 3) A total of 3-4 mL of
mitochondrial suspension will be needed for the entire series of reactions. If your reaction
rate is too rapid, or if you have less than 2-4 mL of material, notify your instructor.
6) After mitochondria are added, begin recording A600 nm values every 15 s for 3 min. The
absorbance should decrease as DCIP is reduced, i.e. converted from blue to colorless.
7) Design tube 11 to contain your own creation of reactants to answer a question not addressed
already. Some ideas include: the effects of deleting azide or succinate from the reaction
mixture, the addition of varying concentrations of azide or malonate, the presence of SDH
activity in another subcellular fraction (nuclear pellet or post-mitochondrial supernatant), or
in the mitochondria from another organ (this assumes you prepared mitochondria from a
different tissue in the last lab). You may also examine other factors, such as temperature.
Table 1. Recipe for preparation of the enzymatic assays.
Tube / Buffer(mL) / Azide
(mL) / DCIP
(mL) / Malonate
(mL) / Succinate
(mL) / Mitochondrial suspension
(mL) / Comment
0.04 M
stock / 0.5 mM
stock / 0.2 M
stock / 0.02 M
stock / DO NOT
ADD YET
1 / 0.5 / - / - / 0.5 / 0.1 / Blank for tube 2
2 / 0.5 / 0.5 / - / 0.5 / 0.1 / [enzyme]
3 / 0.5 / - / - / 0.5 / 0.2 / Blank for tube 4
4 / 0.5 / 0.5 / - / 0.5 / 0.2 / [enzyme]
5 / 0.5 / - / - / 0.5 / 0.3 / Blank for tubes 6-11
6 / 0.5 / 0.5 / - / 0.5 / 0.3 / Optimized condition
for comparison of [enzyme], [S] and [I]
7 / 0.5 / 0.5 / 0.2 / 0.5 / 0.3 / [I]
8 / 0.5 / 0.5 / - / 0.1 / 0.3 / [S]
9 / 0.5 / 0.5 / - / 0.05 / 0.3 / [S]
10 / 0.5 / 0.5 / - / 0.01 / 0.3 / [S]
11 / YOUR OWN DESIGN
Lab Report Write-Up Guide
Results Section
1) Table 1: Record all the raw data from your experiment with tube numbers 2, 4, 6, 7, 8, 9, 10, 11 along the top of the table and absorbance readings taken at each time from 0-3 min in 15 sec intervals.
2) Prepare 4 graphs of absorbance (y-axis) versus time (x-axis) to show the effects of enzyme amount, substrate concentration, inhibitor and your own experiment on enzyme activity. Also use these graphs to measure Vo (Dabs/min) of each reaction. For the effects of amount of enzyme, use tubes 2, 4, 6; for substrate concentration use tubes 6, 8, 9, and 10; for presence of inhibitor, use tubes 6 and 7. For your own experiment tubes 11 and the appropriate control. Label your axes with titles and units, and provide a legend indicating the different reaction conditions (mM succinate, mM malonate, or mg protein) for each kinetic trace.
3) Table 2: Prepare a Master Summary Table to organize not only the data, but also your thoughts regarding the experiments. Suggested column headings for this table are: Tube number, volume of succinate (mL), concentration of succinate (mM), volume of mitochondrial suspension (mL), total protein in the assay (mg), volume of malonate, concentration of malonate, Vo (initial velocity, D A600 nm/min), and SA (specific activity, D A600 nm/min/mg protein).
Calculation Hints:
To calculate the amount of protein in each assay, refer to data from the previous lab, i.e. concentration of protein (mg/mL) of the mitochondrial pellet suspension. Initial velocity is measured from graphs of A600 nm versus time in the initial linear portion of the graph. To calculate the substrate concentration, use the following formula: (stock conc.)(volume) = (assay conc.)(total assay volume).
4) Using data from reactions 2, 4, and 6, graph enzyme amount (mg protein, x-axis) versus initial velocity (D A600 /min, y-axis).
5) Using data from 6, 7 make a bar graph to show the effects of inhibitor on the enzyme specific activity (SA, y-axis, D A600 /min/mg protein).
6) Using data from reactions 6, 8, 9, and 10, graph substrate concentration (x-axis, mM) versus specific activity (SA, y-axis, D A600 nm/min/mg protein) of the enzyme.
7) Prepare a Lineweaver-Burk plot of 1/[S] (x-axis) versus 1/SA (y-axis). Perform a linear fit of the data to determine the Km and Vmax for the enzyme. Provide proper units for these parameters.
***Make sure to include appropriate graphs and tables for the data collected in tube 11 - your individual experiment. It may be helpful to compare tube 11 to tube 6. Tube 6 was the "optimized" condition for the reaction.***
Discussion Section
1) In the discussion section, summarize all of the results in the first paragraph. In subsequent paragraphs, address the effects of enzyme concentration, substrate concentration and inhibitor on enzyme activity. Include references to specific graphs and tables presented in the results section to support your conclusions. Also, explain the results of the individual experiment, "tube 11".
2) Compare your calculated values for Km, Vmax, and/or SA to published values. Provide possible reasons for any differences. (Note: If you are extra ambitious, rates expressed as "D A600 nm/min" can be converted to "mM accumulation of product/min" using the extinction coefficient of DCIP.)
3) Don't forget to include a reference section (2 references minimum). Most credit will be given for this section when you find sources from the primary literature. Use these references in the text of your report. It would be appropriate to reference a paper which explains the findings of your "tube 11" experiment.
References
1) Becker, W.M., et al., eds. (2009) The World of the Cell, 7th ed. Pearson Benjamin Cummings.
2) Kanehisa, M. and Goto, S. (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28:
27-30. Figure R00408.gif from database Release 49.0, January 1, 2009.
3) Richardson, S.G.N., et al. (1975) Two cases of sodium azide poisoning by accidental ingestion of Isoton.
J. Clin. Path. 28:350-1.