Carbohydrate Metabolism by Yeast
Experiment #12
Objective: To observe enzyme activities in respiring yeast by observing color changes in
methylene blue dye as a substitute for NAD+ in the yeast respiratory chain. You will observe the
oxidation of lactic acid and ethanol by yeast lactate dehydrogenase and alcohol dehydrogenase,
respectively.
Introduction
Yeast are simple organisms that can adapt to either aerobic or anaerobic conditions, and a
wide range of nutrients. In this experiment you will study the activity of two enzymes, lactic acid
dehydrogenase and alcohol dehydrogenase, which both use NAD+ as the cellular oxidizing
agent. You will use methylene blue as an artificial electron acceptor (oxidizing agent) in the
reaction, which changes from blue to colorless as a result of its reduction in the enzyme reaction.
When methylene blue is substituted for NAD+, the blue color of methylene blue will
disappear as it is reduced and the lactic acid is oxidized to pyruvic acid, or ethanol is oxidized to
acetaldehyde. NADH or reduced methylene blue can be oxidized by the mitochondrial
respiratory electron transport system when oxygen is available. This will result in the blue color
of methylene blue reappearing upon reoxidation by the respiratory chain. There are many
poisons that can block the mitochondrial electron transport chain, notably cyanide ion (CN-).
Cyanide binds to the enzyme cytochrome oxidase, which normally reduces molecular oxygen to
water in respiration. When cyanide is bound, the enzyme cannot react with oxygen, and the
electron transport process (or oxidation of NADH and methylene blue) is blocked. You will
demonstrate this blockage of the respiratory chain with cyanide.
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