Difficulty: Intermediate | Prerequisites: Enzyme Structure and Function (Doc 1), Enzyme Kinetics and Inhibition (Doc 2). Familiarity with pipetting and basic lab safety.
This is the applied lab component where you use a real enzyme, polyphenoloxidase (PPO), extracted from potato, to test the kinetics and inhibition concepts from the previous notes. You will measure reaction rates by tracking colour change with a spectrophotometer, manipulate enzyme and substrate concentrations via serial dilution, and test the effects of temperature, inhibitors, cofactor removal, and salt on enzyme activity. The lab also teaches experimental design: every experiment includes proper controls so you can attribute changes in rate to the variable you are testing.
Polyphenoloxidase (PPO) from potato catalyses the oxidation of catechol to coloured quinone products, measured at 525 nm. The lab tests how enzyme concentration, substrate concentration, temperature, inhibitors (potassium arsenite, PHBA), cofactor chelators (PTU, potassium cyanide), and salt affect reaction rate. Controls (no enzyme, no substrate) establish that the reaction requires both components. PPO uses copper as a cofactor and is part of the plant's defence system against microbial and insect attack.
Polyphenoloxidase (PPO) / tyrosinase / catechol oxidase
The enzyme studied in this lab. Found widely in plants and animals. It catalyses the oxidation of catechol (and similar phenolic compounds) using molecular oxygen. All three names refer to the same enzyme or closely related enzymes.
Catechol
The substrate used in the lab. A phenolic compound with two hydroxyl groups on a benzene ring. PPO oxidises it to orthoquinone (benzoquinone).
Benzoquinone / orthoquinone
The immediate product of the PPO-catalysed oxidation of catechol. Reddish-brown in colour. It then undergoes further condensation reactions to produce darker coloured products.
Quinones
The broader class of oxidised phenolic compounds produced by PPO. These are toxic to many microbes and insects, which is why the plant makes them.
Homogenate
A suspension of broken cells prepared by blending the potato in water. Contains the enzyme along with other cellular contents.
Supernatant
The liquid portion above the pellet after centrifugation. In this lab, the supernatant contains the dissolved PPO enzyme and is used as the enzyme source.
Pellet
The solid material at the bottom of the centrifuge tube after spinning. Contains cell walls, unbroken cells, and large organelles. Discarded in this experiment.
Centrifugation
Spinning samples at high speed to separate components by density. Heavy particles (cell debris) sink to form the pellet; lighter dissolved proteins (including PPO) stay in the supernatant.
Cuvette
A small, transparent container used to hold samples in the spectrophotometer. Must be kept clean, unscratched, and consistently oriented.
Control tube
A tube that omits one key component to establish a baseline. Control 1 (no enzyme) shows that catechol alone does not produce colour. Control 2 (no substrate) shows the enzyme's own absorbance.
Treatment tube
The experimental tube containing both enzyme and substrate under standard conditions, or under the condition being tested.
Chelating agent
A compound that binds tightly to metal ions, removing them from the enzyme. Used here to investigate whether PPO requires a metal cofactor.
Phenolthiourea (PTU)
A chelating agent specific for copper. If PTU inhibits PPO, it indicates copper is a necessary cofactor.
Potassium cyanide
A chelating agent that binds both copper and iron. Comparing its effect with PTU's helps determine which metal ion PPO requires.
Para-hydroxybenzoic acid (PHBA)
A compound structurally similar to catechol, tested as a potential competitive inhibitor of PPO.
Potassium arsenite
A compound that disrupts disulfide bonds in proteins, tested as a potential noncompetitive inhibitor of PPO.
PPO is part of the potato's defence system. When cells are damaged (by cutting, bruising, or microbial/insect attack), PPO catalyses the oxidation of phenolic compounds to quinones.
Quinones are broadly toxic to microbes and insects. They also polymerise into the dark brown/black pigments you see on a cut potato or apple.
This browning reaction is an adaptive defence: plants that produce more quinones in response to attack have better survival (natural selection).
PPO is also responsible for the tanning of human skin (the related enzyme tyrosinase produces melanin from tyrosine).
PPO catalyses: catechol + 1/2 O2 --> orthoquinone + H2O.
The orthoquinone is reddish-brown. It undergoes further non-enzymatic condensation reactions to produce darker products.
We measure the coloured products at 525 nm (green light) because that wavelength gives the strongest absorbance signal for these products.
Homogenisation: Blend potato in distilled water to rupture cells and release PPO into solution.
Filtration: Strain through cheesecloth to remove large debris.
Centrifugation: Spin at 5000 rpm for 5 minutes. The supernatant (liquid on top) contains dissolved PPO. The pellet (solid at bottom) is discarded.
Spectrophotometry: Measure absorbance at 525 nm over time. Each reading is taken at one-minute intervals for 10 minutes.
Serial dilution: Two-fold dilutions (1 ml sample + 1 ml water) to create concentrations of 1/2, 1/4, 1/8, etc.
Every experiment includes a treatment tube (enzyme + substrate under the test condition) and control tubes.
Control 1 (no enzyme): Buffered catechol + water instead of enzyme. Shows whether colour change occurs without the enzyme.
Control 2 (no substrate): Distilled water + enzyme. Shows the enzyme's own baseline absorbance.
The control results confirm that both enzyme and substrate are required for the reaction to proceed.
The enzyme-only control typically has higher absorbance than the catechol-only control because the potato extract itself contains coloured compounds.
Part IV tests reaction at 0, 20, 45, and 65 degrees C.
Low temperatures slow molecular motion, reducing the rate of enzyme-substrate collisions. The reaction is slower but the enzyme is not damaged.
Moderate warming (up to an optimum) increases rate because molecules move faster and collide more frequently.
High temperatures (65 degrees C) denature the enzyme, destroying its three-dimensional structure and abolishing activity.
Part V tests whether temperature damage is permanent.
Frozen-then-thawed enzyme: freezing may or may not permanently damage PPO. Activity should be partially or fully recoverable.
Boiled-then-cooled enzyme: boiling denatures the protein irreversibly. Activity should not recover after cooling.
Potassium arsenite (Part VI): Disrupts disulfide bonds (sulfhydryl groups) in the protein. This is a structural effect, not an active-site competition. Predicted to be a noncompetitive inhibitor. Percent inhibition should remain constant across substrate concentrations.
PHBA (Part VII): Structurally similar to catechol. Predicted to be a competitive inhibitor because it can fit into the active site. Percent inhibition should decrease at higher substrate concentrations as catechol outcompetes PHBA.
PPO contains a copper cofactor at its active site.
PTU (phenolthiourea) chelates copper specifically. If PTU inhibits PPO, it confirms that copper is required.
Potassium cyanide chelates both copper and iron. Comparing its effect with PTU's determines whether iron is also involved.
If both inhibit PPO, and PTU alone is sufficient, the cofactor is copper (not iron).
Increasing NaCl concentration destabilises ionic bonds that help maintain the enzyme's conformation.
At some salt concentration, the enzyme begins to lose its shape and activity declines.
This experiment demonstrates that the ionic environment matters for enzyme function, not just temperature and pH.
Catechol oxidation (the reaction PPO catalyses):
\text{catechol} + \frac{1}{2}\text{O}_2 \xrightarrow{\text{PPO}} \text{orthoquinone} + \text{H}_2\text{O}The orthoquinone then undergoes further non-enzymatic condensation reactions to produce darker coloured products measured by the spectrophotometer.
Enzymatic browning is a significant problem in the food industry. Sliced apples, potatoes, bananas, and avocados all darken because of PPO activity. Food producers use various strategies to slow browning: acidification (lemon juice lowers pH), exclusion of oxygen (vacuum packaging), blanching (brief heat treatment to denature PPO), or chemical inhibitors such as ascorbic acid. Understanding PPO's mechanism and cofactor requirements directly informs these preservation techniques.
Students often assume the brown colour on a cut potato is caused by oxidation of starch. It is caused by PPO oxidising phenolic compounds to quinones, which then polymerise.
A common error is thinking the spectrophotometer measures the enzyme directly. It measures the coloured products of the reaction. More product means higher absorbance.
Students sometimes think the enzyme-only control should show zero absorbance. The potato extract itself is coloured, so the enzyme-only control typically has a non-zero (but stable) absorbance reading.
Some students confuse "the reaction slowed down" with "the enzyme was inhibited." In a normal run, the reaction slows because substrate is consumed, not because the enzyme is damaged.
⚠️ Be able to explain the purpose of each control tube and what its results tell you.
⚠️ Know why the enzyme-only control has higher absorbance than the catechol-only control (the potato extract is itself coloured).
⚠️ Understand the logic for predicting whether PHBA is competitive (structural similarity to catechol) and whether potassium arsenite is noncompetitive (disrupts protein structure, not active-site binding).
⚠️ Know that PPO has a copper cofactor, and be able to explain how PTU and potassium cyanide experiments distinguish copper from iron involvement.
⚠️ Be ready to explain why boiled enzyme does not recover activity after cooling (irreversible denaturation), while frozen enzyme may partially recover.
True or False: The spectrophotometer directly measures the amount of enzyme in the cuvette.
Fill in the blank: PPO catalyses the oxidation of catechol to ______.
True or False: PTU chelates iron specifically.
Fill in the blank: After centrifugation, the enzyme is found in the ______, not the pellet.
True or False: A boiled enzyme that is cooled back to room temperature should recover its catalytic activity.
Answers: 1. False (it measures coloured products). 2. Orthoquinone (benzoquinone). 3. False (PTU chelates copper). 4. Supernatant. 5. False (boiling causes irreversible denaturation).
Q: What do the results from the two control tubes (catechol-only and enzyme-only) tell you?
A: Control 1 (catechol-only) shows that catechol does not spontaneously produce coloured products without the enzyme. Control 2 (enzyme-only) shows the baseline absorbance of the potato extract itself, confirming that the enzyme alone does not generate additional colour without substrate. Together, they confirm that both enzyme and substrate are required for the reaction.
Q: Why does the enzyme-only control have higher absorbance than the catechol-only control?
A: The potato extract contains naturally coloured compounds (pigments from the potato cells). These absorb light at 525 nm even though no enzymatic reaction is occurring. Catechol solution, by contrast, is colourless or nearly so.
Q: If you used 250 microlitres of potato extract instead of 100 microlitres, how would you expect the absorbance-vs-time graph to change?
A: The initial rate (slope of the linear phase) would be steeper because more enzyme is present, so more substrate is converted per unit time. The reaction would also reach its plateau sooner because substrate would be consumed faster.
Q: PPO is incubated with PTU and its activity drops to near zero. It is then incubated with potassium cyanide and its activity also drops to near zero. What can you conclude about PPO's metal cofactor?
A: Since PTU (copper-specific) alone is sufficient to abolish activity, PPO requires copper as a cofactor. The potassium cyanide result (which removes both copper and iron) is consistent but does not add new information beyond confirming that a metal ion is essential. The copper-specific result from PTU is the key finding.
Q: You test PHBA as an inhibitor at three substrate concentrations. Percent inhibition is 55% at low substrate, 35% at medium, and 15% at high substrate. What type of inhibitor is PHBA?
A: Competitive. Percent inhibition decreases as substrate concentration increases, indicating that the substrate is outcompeting PHBA for the active site. This is consistent with PHBA's structural similarity to catechol.
Q: Why must you pre-warm or pre-cool your reagents before starting a temperature experiment?
A: If you mix room-temperature reagents and then place them in a hot or cold bath, the reaction starts at room temperature and only gradually reaches the target temperature. Pre-equilibrating the reagents ensures the reaction runs at the intended temperature from the start, giving accurate rate measurements for that condition.
The PPO lab ties together enzyme fundamentals (Doc 1) and kinetics/inhibition theory (Doc 2) in a hands-on setting. The concept of cofactors and chelating agents connects to broader inorganic biochemistry and metalloproteins. The evolutionary angle (PPO as an adaptive defence mechanism, quinone toxicity as a result of natural selection) links to ecology and evolutionary biology. The spectrophotometry technique is used across biology and chemistry courses.
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