Cody Ellison

Bio 107-17L

Cellular Respiration

The Effects of Succinate on the Rate of Cellular Respiration

Abstract

The main goal of this experiment was to see how increased or decreased levels of

succinate would affect the rate of cellular respiration. This experiment was focused on the sixth

step of the Krebs cycle where succinate loses electrons and hydrogen ions to FAD, forming

FADH2 to be stored for energy. To see how different levels of succinate would affect the rate of

cellular respiration an electron acceptor called DPIP had to be used instead of FAD to accept the

hydrogen ions. As DPIP is reduced it turns from blue to clear. In order to measure the rate of

color change in the DPIP we used a spectrophotometer, which measured the amount light the

DPIP was absorbing over time. Three different amounts of succinate were added to a mixture of

DPIP, buffer solution and a mitochondrial suspension. The succinate had to be added just before

placing the sample into the spectrophotometer because as soon as it is added, the redox reaction

begins. Absorption level readings were taken in five-minute increments. We hypothesized that

with increased amounts of succinate, the rate of respiration would increase. The results supported

our hypothesis; the highest amount of succinate had an absorbance of .391 after 30 minutes, and

the lowest amount had an absorbance of .738.

Introduction

Cellular respiration is a process by which a cell breaks down glucose molecules to create

and store energy, a crucial part of the life of a cell. Without energy a cell cannot operate, and

when your cells cannot operate, neither can you. This paper is focused on the Krebs cycle, the

second stage of the three major processes of cellular respiration. Before the Krebs cycle begins,

glycolysis must take place. In the glycolysis process, glucose is oxidized to form pyruvate

through ten enzyme-mediated steps. During this process, glycolysis generates two ATP and two

NADH. After pyruvate is formed it enters the mitochondria via active transport where it is

oxidized, forming acetyl CoA (acetyl coenzyme A). From there, the acetyl CoA enters the Krebs

cycle and is converted into one ATP, one FADH2 and four NADH through eight enzyme

mediated steps. The focus of the experiment is on the sixth step of the Krebs cycle process where

succinate is oxidized by transferring two hydrogen ions to FAD. During the oxidation succinate

forms fumarate while the two hydrogen ions that were transferred to FAD form FADH2, a

source of energy to be used by the cell. This paper explores how the rate of cellular respiration is

affected by using different amounts of succinate. Since cellular respiration is the process of a cell

breaking glucose molecules down to be used for energy, an increase in the rate of cellular

respiration would mean that more energy is being created. In our experiment, we focused only on

the sixth step of the Krebs cycle where the succinate transfers two hydrogen ions to FAD to

create energy in the form of FADH2. Thus, if the rate of cellular respiration increases then the

succinate molecules are transferring more hydrogen ions to FAD to form FADH2 at a faster rate

giving the cell more energy for other cellular processes required to keep the cell alive. For

example, researchers conducted an experiment exposing mungbean seedlings to doses of NaCl.

When the seedlings were exposed to the salt, activity levels of succinate dehydrogenase were

stimulated in the shoot, or stem, of the plant. As a result of being exposed to NaCl, the plant

grew better and had efficient respiration (Biswas, Kunda, Saha, 2012). Stimulated succinate

dehydrogenase activity resulted in better plant growth because the succinate dehydrogenase was

able to create more FADH2. In another experiment, a group of scientists found a toxic chemical

in a solvent used in consumer goods that inhibits succinate dehydrogenase activity in a cell,

resulting in mitochondrial dysfunction and ultimately the death of the cell (Conrad, Dunning,

Landry, Hitt, McMartin, 2013). The mitochondrial dysfunction is caused by decreased energy

production and oxygen consumption, due to the lack of succinate dehydrogenase activity

creating the necessary energy for the mitochondria to operate. Failure in the mitochondria leads

to the death of the cell. For our experiment, we hypothesized that the rate of cellular respiration

would increase with increasing amounts of succinate in the sample. We predicted that the sample

with the most amount of succinate present would reduce the DPIP at a faster rate, and result in a

lower level of absorbance, and the sample with less succinate present would not reduce the DPIP

as fast, and result in a higher level of absorbance.

Materials and Methods

To conduct this experiment we needed three test tubes labeled one though three. In test

tube one we mixed 4.4 mL buffer, 0.3 mL DPIP and 0.3 mL mitochondrial suspension. In the

second tube we mixed 4.3 mL buffer, 0.3 mL DPIP and 0.3 mL mitochondrial suspension. In the

third tube we mixed 4.2 mL buffer, 0.3 mL DPIP and 0.3 mL mitochondrial suspension. A

spectrophotometer was needed to measure the absorption levels of the DPIP. In addition, we

needed four cuvettes labeled one through three and one labeled “B” to calibrate the

spectrophotometer, in which we mixed 4.6 mL buffer, 0.3 mL mitochondrial suspension and 0.1

mL succinate. Note that before the cuvettes were placed in the spectrophotometer, they were

cleaned with a kimwipe to remove anything that might absorb the light. We then used the cuvette

labeled “B” to calibrate the spectrophotometer by clicking on the experiment menu of the

software we were using and selecting calibrate, and then spectrometer: 1. Once the

spectrophotometer was calibrated the contents from test tubes one, two and three were added to

their respective cuvettes. One at a time, the succinate was added to its respective cuvette, mixed,

and placed in the spectrophotometer. 0 mL succinate was added to the first cuvette and then

placed in the spectrophotometer for 30 minutes, absorption levels were recorded every five

minutes. This process was repeated for the second and third cuvette. 0.1 mL succinate was added

to the second cuvette and 0.2 mL succinate was added to the third. It was important not to mix

the succinate until the solution was ready to enter the spectrophotometer, because the succinate

would react with the DPIP as soon as it entered the solution.

Results

After putting the date into a line graph, you can clearly see the results of the experiment.

The data points all seem start in the same place because the succinate that had just been added to

the solutions had not had enough time to make a noticeable change. The green line represents the

third solution that contained 0.2 mL succinate, more than the other two solutions. After 30

minutes in the spectrophotometer the absorption level dropped to 0.391, the lowest absorption

level out of all the solutions. The DPIP pigment in this solution turned clear at faster rate than the

other solutions. The blue line represents the solution that contained 0.1 mL succinate. As you can

see, after being in the spectrophotometer for 30 minutes, the absorption level dropped to 0.476.

This is not as low as the solution containing 0.2 mL succinate, which means that the DPIP was

turning clear at a slower rate than the solution containing 0.2 mL succinate. The red line

represents the solution that contained no succinate at all. As you can see, after 30 minutes in the

spectrophotometer, the level of absorption is far above the final absorption levels of the solutions

containing 0.1 mL and 0.2 mL succinate, almost at the same level as it started. This means that

the pigment in the DPIP changed very little or none at all.

Discussion

Before the experiment was conducted we hypothesized that the rate of cellular respiration

would increase in solutions containing a larger amount of succinate. It was predicted that the

solution containing 0.2 mL succinate would have the highest rate of cellular respiration, the 0.1

mL succinate solution would have a higher rate of cellular respiration than the solution with no

succinate but lower than the solution containing 0.2 mL succinate and the solution containing no

succinate would have the slowest rate of cellular respiration. The results from the experiment

supported our hypothesis and predictions. The solution that contained 0.2 mL had the lowest

level of absorption, which means that the DPIP pigment was turning clear the fastest. DPIP turns

from blue to clear when it is reduced, which means that the 0.2 succinate solution reduced the

DPIP at the fastest rate. The solution containing 0.1 mL succinate reduced the DPIP faster than

the solution with no succinate but slower than the 0.2 mL succinate. In this experiment, the DPIP

is being used instead of FAD, which is what actually is present in the Krebs cycle. We chose to

use DPIP because it behaves the same as FAD does in the Krebs cycle, as an electron acceptor,

which intercepts hydrogen ions that are released from the succinate. So in this experiment the

DPIP models the FAD in the Krebs cycle. The more frequently FAD is reduced by succinate, the

more FADH2 is formed for energy. Since DPIP is acting as the FAD for our experiment, a faster

disappearance of DPIP pigment means a faster rate of reduction, which means more energy

would be produced. As you can see in results for the solution that contained no succinate, the

level of absorption barely decreased at all, if at all. Since the DPIP did not turn clear, it was not

reduced. This means that if there was no succinate available in the Krebs cycle, FAD would not

be reduced and there would be no formation of FADH2 energy for the cell to use. In conclusion,

the results from this experiment suggest that higher levels of succinate lead to a higher rate of

cellular respiration.

References

Biswas, Ak., Kunda, P. and Saha, P. (2012) Influence of Sodium Chloride on the

Regulation of Krebs Cycle Intermediates and Enzymes of Respiratory Chain in Mungbean

(Vigna radiata L. Wilczek) Seedlings, Plant Physiol Biochem, 60, 214-22.

Landry, G.M., Dunning, C.L., Conrad, T., Hitt, M.J. and McMartin, KE (2013)

Diglycolic acid inhibits succinate dehydrogenase activity in human proximal tubule cells leading

to mitochondrial dysfunction and cell death, Toxicol Lett, 221 (3), 176-84.