Enzymes

It might be claimed that enzymes are naturally occurring catalysts that speed up chemical reactions. By offering distinct binding sites for the substrates, the enzyme catalyst enhances the likelihood that reactants will come into touch with one another in the right orientation (Madden & Shafer 2002). This experiment was specifically designed to investigate the potential influences on the pace of enzyme-catalyzed processes, or enzyme kinetics.
The mitochondria have all the necessary capacities to supply the cell and its supporting structures with energy so they can carry out various cellular operations. The matrix of the mitochondrion is where the TCA cycle occurs, in which pyruvate (oxidized from glucose in gycolysis) is converted into acetyl-CoA, then fed into the pathway to be oxidized to CO2 and its energy conserved (Michaelis and Menton 1913). Succinate dehydrogenase is the only enzyme of the TCA cycle that is also part of the electron transport system, thus, it is located in the inner membrane (Loftus, Hall, Anderson et al 1994).

Michaelis and Menton (1913) formulated a theory that describes initial events for enzyme catalyzed reaction. The theory assumes that enzyme (E) and substrate (S) combine reversibly to form an enzyme-substrate (ES) complex (Michaelis and Menton 1913). Succinate dehydrogenase (SDH) enzyme belonging to Krebs cycle is embedded in mitochondrial inner membrane. Succinate dehydrogenase (SDH) reacts as follows: Succinate +E-FAD 🡪Fumarate + E-FADH2 whereby E-FAD represents SDH with its covalently bound coenzyme, flavin adenine dinucleotide (FAD) and E-FAD complex oxidizes succinate to fumarate (Campbell 2002). The experiment was carried out to measure SDH activity occurring in isolated mitochondria during fractionation of liver cells. The expected reactions are as summarized below:

E-FADH2 + DCIP(ox) (blue) 🡪 E-FAD + DCIP(red) (colorless)

The experiment was conducted to test enzyme concentration, substrate concentration, and effects of competitive inhibitor addition in SDH. Enzyme concentration in this reaction was altered by addition of different mitochondrial fraction volumes. The substrate concentration effects was then measured through alteration of the amount of succinate that was added to the reaction mixture.



Aim:

PURPOSE

The aim of carrying out this experiment is to:

To measure succinate dehydrogenase activity in fractions isolated from liver.

Illustrate the technique of biologically isolating and organelle separation in cells.

Identify mitochondria by use of different metabolic tests.





Methods:

Methylene blue and tetrazolium chloride as metabolic test indicators were used during this experiment in identification of mitochondria. Five sterile 2ml screw-cap tubes with lids labelled WCL, SN1, SN2, SN3 and Mito. Test tubes used had labelling of according to person’s initials and practical session. The first three test tubes contained 0.001 ml of Methylene Blue indicator whereas the last two test tubes contained 0.03mL of Tetrazolium. The first two test tubes were used as control experiment for the indicators. The purpose of Methylene Blue indicator was for determining whether oxygen was used up by mitochondria in aerobic cell respiration process.

If the test tube remains blue, then there is oxygen present in the solution, therefore mitochondria are not present (Karp 2008). If the indicator turns colorless (clear) after being left overnight, then oxygen is no longer present, therefore used up by the mitochondria (Karp 2008). Salad oil was added into the solution so as to act as a prevention measure for preventing oxygen from the atmosphere from entering the solutions therefore interfering with the experiment by adding more oxygen.

The purpose of Tetrazolium Chloride indicator in this experiment is to determine if oxidation-reduction reactions occurred in mitochondria’s respiratory enzymes. When mitochondria are present, the normally colorless oxidized form of Tetrazolium that was added will turn a bright red/pink overnight, meaning reduction has taken place (Madden & Shafer 2002).

During the blending process, the cell walls and cell membranes are broken allowing the free floating organelles that are contained in the cytoplasm to be released (Karp 2008). Based upon the understanding that the mitochondria contain both an inner and outer membrane and that their relative densities are higher than most other organelles we believe we will find them in the residue.  We believe that these membranes are the primary sites for aerobic cell respiration. Therefore, we expect to receive positive test results in test tubes 3 and 4.

Procedure:

Mitochondria was first isolated then 20g of cauliflower was grounded in chilled mortar containing 40 mL of cold mannitol with grinding medium plus 5 g of cold purified sand for roughly 4 minutes. The suspension was then filtered using cheesecloth with a centrifuge - 600 g at 5° C for a period of 10 minutes. Post-mitochondrial supernatant fluid was then discarded and 7.0 mL cold mannitol assay medium was added to mitochondrial pellet.



Results:

The following tables and figures were obtained from the experiment carried out.

All the results were then recorded as raw data from the experiment alongside tube labels and absorbance readings were recorded every time from 0-3 min in the intervals of 15 sec.

Graphs of absorbance (y-axis) versus time (x-axis) was the drawn to demonstrate effects of amount of enzyme, concentration of substrate, inhibitor and enzyme activities in the experiment.

Table 1 Table of BCA and Absorbance

BCA

Absorbance

0

0

2000

1.381

1500

1.316

1000

1.193

750

1.084

500

1.052

250

0.867

125

0.519

25

0.142





Figure 1 Figure of Graph of BCA against Absorbance.



Table 2 Succinate dehydrogenase activity

Conc. Of reduced INT in sample (μmol/L)

Group

Pair initials

WCL

SN1

SN2

Mito

1

ZS

17.5

0

0

39.1

2

CP, LL

11.48

0.04

0

3.07

3

BL, MS

11.52

1.08

0

2.39

4

BW, EM

21.3

0.57

0.1

14.51

5

TB, SE

10.76

1.87

0

8.848



SA, RA

15.6

0.71

1.6

7.8



JT, BT

40.3

5.62

1.2

7.37



J, J, F

6.99

-0.93

2.33

13.2



Discussion:

Initial velocities of reactions from succinate to fumarate of different enzyme concentrations with the same amount of succinate were tested using the first two tubes. Enzyme concentration changes as per the changes in initial velocity. Forth tube was used in testing the effects that competitive inhibitor (Malonate) has on enzyme activity. The fifth tube contained Sodium azide as it was used to block the normal path of electrons in the electron transport chain, and is therefore used to compare reaction rates with those that contain sodium azide (Bregman 2002).

Succinate dehydrogenase was denatured by heating it on hot water bath and this brought it to inactivated state. The purpose of this action was to use it as control experiment for measuring absorbance changes since as it cools, there is transformation back to its native conformation, and this represents enzyme state before they undergo reactions. The absorbance changes during the 35 minutes duration should display differences in reaction rates against differences in enzyme concentration. The rate of reactions should be slowed by the competitive inhibitors presence, substrate absence and addition of enzyme blockers (sodium azide).

Succinate dehydrogenase (SDH) catalyzes a redox reaction (Mooney &Campbell 1999). Dichloroindophenol (DCIP) was used as the electron acceptor because it undergoes a color change as it becomes reduced: the oxidized form of the molecule is blue and the reduced form is colorless (Bregman 2002). The rate of color change was measured colorimetrically in determining the activities of succinate dehydrogenase.

According to the Michaelis-Menton equation, initial velocity is directly proportional to enzyme concentration (Mooney &Campbell 1999). As the number of enzymes present was increased, the substrate binding to that given enzymes became quicker due to the enzyme’s high affinity for the substrate thereby having a higher reaction of initial velocity. The rate of change in enzyme concentration increases in direct proportion to the initial reaction velocity from succinate to fumarate in the presence of increasing succinate dehydrogenase. This is as shown in Figure 1. The first tube with the least amount of mitochondrial suspension had the least succinate dehydrogenase volume thus can be seen as having the lowest initial velocity; consequently, the second tube registered the highest initial velocity due to the highest enzyme concentration of 0.9 mL mitochondrial suspension.

Malonate being a competitive inhibitor was added to the forth tube so as to use it to test how effective succinate dehydrogenase and succinate reaction can be to fumarate. Both third and fourth tubes contained the same enzyme concentration, fourth tube registered slower reaction rate due to interference that malonate had on the enzyme. This is because competitive inhibitor has similarity in molecular structure to that of succinate therefore binding can occur in the succinate dehydrogenase active site hence preventing succinate binding occurrences.



















References

Campbell, M.A. Laboratory Schedule: Molecular Biology, 2002: College Biology Department.



Loftus, T.M., Hall, L.V., Anderson, S.L. and McAlister-Henn, L, 1994: Isolation andcharacterization of the yeast gene encoding cytosolic NADP+-specific isocitratedehydrogenase. Biochemistry. 33: 9661-9667.

Madden, J. and Shafer L., 2002: Personal correspondence. Davidson College Molecular Biology Thursday Lab.

Mooney, E. and Campbell, A. M, 1999: A Project-Based Biotechnology Laboratory Course using Isocitrate Dehydrogenase. BioScene. 25 (2): 3 - 11. 1999.

Bregman, Allyn A. Laboratory Investigations in Cell and Molecular Biology. John Wiley & Sons, Inc., New York, 2002.

Karp, Gerald, 2008: Cell and Molecular Biology: Concepts and Experiments.