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Piruvato y Nitroprusiato, Reacción

This document describes experiments to demonstrate the production of pyruvate and acetaldehyde during yeast fermentation of glucose. There are two pathways for glucose breakdown: glycolysis and gluconeogenesis. Under anaerobic conditions in yeast, glucose is broken down to pyruvate then acetaldehyde and alcohol. The experiments use inhibitors and trapping agents to show the presence of these intermediates. Yeast suspensions with glucose are incubated under different conditions, then chemical tests detect pyruvate and acetaldehyde.

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Charlie Torres
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308 views17 pages

Piruvato y Nitroprusiato, Reacción

This document describes experiments to demonstrate the production of pyruvate and acetaldehyde during yeast fermentation of glucose. There are two pathways for glucose breakdown: glycolysis and gluconeogenesis. Under anaerobic conditions in yeast, glucose is broken down to pyruvate then acetaldehyde and alcohol. The experiments use inhibitors and trapping agents to show the presence of these intermediates. Yeast suspensions with glucose are incubated under different conditions, then chemical tests detect pyruvate and acetaldehyde.

Uploaded by

Charlie Torres
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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CLS 331

Exp no. 3

The Production of Pyruvate and


acetaldehyde
during the fermentation of
glucose by Yeast

There are 2 metabolic pathways for


breakdown of glucose :

1- Glycolysis
a metabolic process that breaks down glucose through
a series of reactions and releases energy for the body in
the form of ATP.
Under aerobic conditions :
Glucose
10 steps
Pyruvate (in animal &yeast)
Under anaerobic conditions:
Animals:
Glucose
Pyruvate
Yeast:
Glucose

Pyruvate

Lactate
Acetaldehyde

Alcohol

2- Gluconeogenesis
Is a metabolic pathway that results in the generation
of glucose from non-carbohydrate carbon substrates
such as lactate, glycerol.

Principle:

In Glycolysis:
Glucose

Pyruvate (in animals &Yeast)

Under anaerobic conditions:


Animals:
Glucose
Pyruvate
Yeast:
Glucose

Pyruvate

Acetaldehyde

Lactate
Alcohol

Alcohol is formed in yeast by the oxidative decarboxylation of


pyruvate with the intermediate formation of acetaldehyde.
They normally present at low conc.

So to demonstrate their existence as intermediates on the


pathway, some means has to be found to prevent the
reaction proceeding any further.
Blocking the enzyme that catalyses the conversion of the
compound under investigation by:
-Adding an inhibitor
-Changing the physiological conditions (pH, temperature)
-Adding trapping agent which reacts with intermediate to
form a compound that is not metabolized further.
Glucose

Pyruvate

Acetaldehyde

Pyruvate decarboxylase

Alcohol

Pyruvate decarboxylase is inactive in slightly


alkaline solution so pyruvate accumulates and its
presence is demonstrated by the reaction with
sodium nitroprusside
or 2,4-dini-trophenylhydrazine.
In the 2nd experiment, sodium sulphate is added
which traps the acetaldehyde. The presence of
acetaldehyde is shown by blue colour produced
with sodium nitroprusside and piperidine.

Procedure

-Formation of pyruvate from glucose


A

10% glucose

5ml

5ml

Alkaline yeast
suspension

5ml

--------

acidic yeast
suspension

----------

5ml

Mix and incubate in water bath at 37 C for 1 hour

- Add 2ml of TCA ( trichloracetic acid) for


each tubes
- Mix and centrifuge for 10 min at 2500 g.
- Remove the supernatant
- Boil about 3ml of supernatant for 5 min.

- Sodium nitroprusside test for pyruvate


A1

solid ammonium
sulphate

inch (0.5g)

Supernatant A

2ml (boiled)

supernatant B
5% nitroprusside sol.

B1

inch (0.5g)

2ml(boiled)
2 drops

Mix

2 drops

Add conc. ammonia carefully down the side of


the tube so as to form 2 layers.
If pyruvate is present, a green or blue ring forms
at the junction of the 2 liquids.
A transient pink ring at the junction is due to the
presence of thiol group

- Test for pyruvate with 2,4-dini-trophenylhydrazine


A

2ml of supernatant A
1ml of 2,4-dinitrophenylhydrazine

inch of
solid
ammonium
sulphate

Mix

2ml of supernatant B

1ml of 2,4-dinitrophenylhydrazine

A1

2 or 3 drops
of the
mixture A

B1

1 ml NaOH
2ml water

2 or 3 drops
of the
mixture B

A red colour forms if pyruvate is present

Formation of acetaldehyde from glucose


C

D
5ml of glucose solution
5ml of yeast suspension

Mix
Incubate the two tubes at 37 C for 1h

0.5 g of sodium
sulphite
(trapping agent)

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