ATP Dehydrogenase practical

Investigation of dehydrogenase activity at different temperatures
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Skill Understood Needs work
Writing predictions
Using appropriate units
Identifying variables
Describing control experiments
Understanding reliability
Constructing tables
Drawing and plotting graphs
Describing trends
Explaining what range bars show
Identifying sources of inaccuracy and suggesting improvements
Explaining how to amend the experiment to investigate another
variable.
For my next practical I will focus on….




Introduction to the practical

, Dehydrogenase enzymes carry hydrogen atoms from substrates such as glucose and transfer them to co-
enzymes such as NAD and FAD in the mitochondria, they REDUCE the coenzymes by adding a hydrogen
atom.
The hydrogen atoms are then released from NAD and FAD (the co-enzymes are oxidised) and split into one
proton and one electron.
Methylene blue is an artificial hydrogen acceptor i.e. it can bind to hydrogen atoms therefore preventing
them from being able to reduce NAD and FAD. Methylene blue has a higher REDUCTION POTENTIAL than
NAD and FAD so when they are both present, methylene blue is reduced in preference to NAD and FAD.
When oxidised methylene blue is dark blue in colour and it loses its colour as it is reduced (gains hydrogen
atoms). The time taken for the methylene blue to lose its colour is a measure of the rate of dehydrogenase
activity. A short period of time for methylene blue to change colour indicates a high rate of dehydrogenase
activity as dehydrogenase is responsible for carrying the hydrogen atoms.


Apparatus
 Redox indicator: methylene blue (0.05 g/100cm3)
 150cm3 Yeast suspension (100 g/dm-3) 30 °C
 Water baths at 5 different temperatures
 Boiling tube x 10
 Cork/ bung for boiling tube
 10 cm3 syringe
 Stop clock
 Test tube rack
Method
1. Place 10 cm3 of the yeast suspension into a boiling tube.
2. Place the boiling tube in a water bath for 5 minutes, to equilibrate.
3. Add 3 drops of methylene blue to the boiling tube.
4. Put the bung in the top of the boiling tube and invert the test tube once, to mix.
5. Replace the boiling tube in the same water bath.
6. Time how long the indicator takes to change colour.
7. Repeat for each of the different temperatures.
8. Collect 3 sets of data for each temperature (you may have to share results with other groups)
Risk assessment




Questions