BIOCHEMISTRY - Laboratory Practical - NAU 2015

MODULE IV

METABOLISM OF MAJOR CLASSES OF BIOMOLECULES

Laboratory Work 11

EXPERIMENTAL SIMULATION OF GLYCOLYTIC REACTIONS

Objective: to experimentally confirm The conversion of glucose via The Glycolytic Pathway.

Basic Theoretical Background

Glycolysis is the metabolic pathway that converts glucose into Pyruvate (under aerobic conditions) or lactate (under anaerobic conditions). Anaerobic glycolysis is the primary pathway of METABOLISM/26.html">Energy Metabolism in the absence of oxygen. The net energy yield of glycolysis is two ATP molecules per glucose molecule.

The Enzymes of glycolysis are located in the Cytoplasm. Glycolysis consists of two stages. As a result of The First stage—phosphorylation—glucose, which is a hexose, is converted into two phosphotrioses. One of them, glyceraldehyde 3-phosphate, serves as the substrate for the Second Stage of glycolysis, known as the glycolytic oxido-reduction stage. Glyceraldehyde 3-phosphate is oxidized; reducing equivalents from it and from inorganic phosphate are accepted by the coenzyme NAD⁺, which transfers two hydrogen atoms to pyruvate, converting it into lactate.

The process of glycolysis can be experimentally confirmed by The formation of lactic acid and a decrease in inorganic phosphate content in the incubation mixture in the presence of glycolytic enzymes and the substrate, glucose.

Equipment: test tubes, pipettes, porcelain mortar and pestle, quartz sand, cotton wool, funnel, pH meter, filter paper, thermostat, balance, dropping pipette.

11.1. Study of Glycolysis in Yeast Suspension

Materials and Reagents: brewer's yeast (5 g) ground in a porcelain mortar with 2–3 ml of distilled Water and a small amount of quartz sand, mixed with 8–10 ml of water, and filtered through cotton wool; phosphate buffer 1/15 M, pH = 7.4 (9.46 g/L Na₂HPO₄; 10.40 g/L NaH₂PO₄ · 2H₂O, adjusted to pH 7.4 by mixing the solutions); 1% glucose solution; liquid paraffin (vaseline oil); 2% trichloroacetic acid (TCA) solution; 1% FeCl₃ solution; 1% phenol solution; ammonium molybdate solution (0.5 g of pure ammonium molybdate dissolved in
100 ml of water, filtered, and mixed with 100 ml of 10 M sulfuric acid solution); 0.1% ascorbic acid solution.

Procedure

Two test tubes are filled with reagents as indicated in Table 11.1.

Class="right">Table 11.1

Contents of Control and Test Tubes

Add 2 ml of trichloroacetic acid solution immediately to one of the test tubes (control sample) to inactivate the enzymes, and mix.

Overlay both test tubes with liquid paraffin to establish anaerobic conditions. Incubate in a thermostat at 37 °C. After 1 hour, add 2 ml of trichloroacetic acid solution to the test sample and mix. Filter the contents of both test tubes to remove Proteins.

Following incubation, determine The amount of lactic acid formed (the end product of glycolysis) and the decrease in inorganic phosphorus content (utilized in phosphorylation reactions).

Determination of Lactic Acid in the Incubation Medium

Take 1.0 ml of solution from each test tube, add
5 drops of 1% FeCl₃ and 1.0 ml of 1% phenol solution to each. The interaction between the purple iron phenolate and lactic acid yields yellow-green iron lactate.

Observe the appearance of a yellow-green coloration.

Determination of Inorganic Phosphorus in the Incubation Medium

Take 1.0 mL of liquid from each tube, add 1.0 mL of ammonium molybdate, and observe the color change. The interaction of inorganic phosphorus with ammonium molybdate in an acidic medium leads to the formation of an ammonium salt of phosphomolybdic acid—(NH₄)₃РО₄ · 12МоО₃ · 6Н₂О—which has a characteristic yellow color.

Add 1 mL of ascorbic acid solution. Mix the contents of the tubes thoroughly. Upon reduction by ascorbic acid, ammonium molybdate turns into phosphomolybdic blue, the color intensity of which is proportional to the phosphorus content in the test sample.

After 10 min, measure the optical density of the samples using a red light filter.

Processing of Experimental Data

Write out sequentially all the reactions of anaerobic glycolysis, highlight the individual stages, and indicate the phosphorylation, substrate-level phosphorylation, and redox reactions. Point out the reaction that utilizes inorganic phosphate and the one that yields lactate.

Record the experimental results in Table 11.2.

Table 11.2

Determination of lactic acid (formed during glycolysis) and inorganic phosphate (utilized during glycolysis)

Selection/41.html">Review Questions and Tasks

1. Write the reaction for the formation of iron phenolate and iron lactate.

2. Explain the principle behind the determination of inorganic phosphate.

3. Explain why the yeast needs to be ground with Glass sand.

4. Explain the difference between aerobic and anaerobic glycolysis. Which specific reaction sets them apart?

5. What are the end products and energy yield of complete AEROBIC GLUCOSE OXIDATION? Outline The sequence of processes involved.

References: [1; 4—7].