SKILL FOCUSPredictingPerforming and recordingAnalyzing and interpretingCommunicating resultsInvestigation 3•A
72 MHR • Unit 1 Metabolic Processes
Pre-lab Questions
What is the purpose of oxidizing succinic acid in the
Krebs cycle?
How does methylene blue indicate succinic
dehydrogenase activity?Problem
Where is succinic acid oxidized in a cell? In other words,
where does succinic dehydrogenase activity occur?Prediction
Predict how you could observe succinic dehydrogenase
activity.CAUTION: The indicator methylene blue is a dye.
Avoid any contact with skin, eyes, or clothes.
Flush spills on your skin immediately with copious
amounts of water and inform your teacher.
Exercise care when handling hot objects. Handle
thermometers with care. Follow your teacher’s
instructions on the safe use of scalpels. Dispose
of all chemicals according to your teacher’s
instructions and wash your hands before leaving
the laboratory.Materials
500 mL beaker blue coloured pencils
3 test tubes (several shades)
scalpel or sharp knife 0.5 mol/L succinic acid
3 medicine droppers methylene blue (0.01%)
hot plate distilled water
thermometer mineral oil
grease pencil two pieces of beef heart
colour chart (each about 2–3 cm^3 )Procedure
1.Set up a hot water bath in the 500 mL beaker, and
maintain it at 37°C.
2.Using the grease pencil, number each test tube.
3.Using the scalpel, cut away any fat tissue from the
beef muscle. Cut away two small pieces of tissue
and set aside.
4.In test tube 1, add a piece of beef heart, 4 drops
of succinic acid, 8 drops of methylene blue, and
8 drops of distilled water.
5.In test tube 2, add 4 drops of succinic acid, 8 drops
of methylene blue, and sufficient distilled water to
equal the volume of the mixture in test tube 1.Enzyme Activity in the Krebs Cycle
The enzyme succinic dehydrogenase catalyzes the oxidation of the four-carbon
molecule succinic acid to fumaric acid. This is an essential step in the Krebs cycle.
In this investigation, you will use a solution of methylene blue to observe the action
of succinic dehydrogenase. Fresh beef heart tissue will serve as a source of
succinic dehydrogenase.acids that are required to produce lipids. The
energy stored in these molecules can be released
later, as needed. You can find a more detailed
description of these other metabolic pathways at
the end of this section.
As the product of the transition reaction from
glycolysis, acetyl-CoA enters the Krebs cycle to
produce ATP.The Krebs Cycle
The Krebs cycle, named after scientist Hans Krebs,
is a cyclical metabolic pathway that oxidizes
acetyl-CoA to carbon dioxide and water, forming a
molecule of ATP (see Figure 3.9 on page 71). Inaddition to producing one ATP molecule, the series
of redox reactions that form the Krebs cycle involve
the transfer of electrons. This electron transfer
leads to the formation of three NADH molecules
and one FADH 2 molecule.
The Krebs cycle involves a total of nine reactions.
First, an enzyme removes the acetyl group from
acetyl-CoA and combines it with a four-carbon
oxaloacetate molecule to produce a six-carbon
citrate molecule. With the acetyl group removed,
coenzyme A is released to participate in another
reaction in the matrix.
At this point, a series of oxidation-reduction
reactions begins that will result in the formation of