Rate of an Enzyme Controlled Reaction Revision Notes for AQA A-Level Biology

Rate of an Enzyme Controlled Reaction

Purpose and principle

This practical investigates how a named variable affects the rate of an enzyme-controlled reaction. The rate of reaction can be influenced by several factors including temperature, pH, substrate concentration, and enzyme concentration. By changing only one variable whilst keeping all others constant, we can determine its specific effect on enzyme activity.

The practical uses trypsin, a protease enzyme, to break down casein protein found in milk. When casein is hydrolysed, the milk changes from opaque to colourless, providing a clear visual endpoint to measure reaction time.

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The opacity change in milk provides an excellent visual indicator for enzyme activity. As the casein protein is broken down, the milk becomes increasingly transparent, allowing for easy observation of the reaction endpoint.

Apparatus and materials

The following equipment is required for this investigation:

Powdered milk suspension
Trypsin solution (0.5%)
Distilled water
Hydrochloric acid (0.1M)
5cm³ syringes
Flat-bottomed tubes
Water bath
Timer

Method

This procedure investigates temperature as the independent variable:

Prepare control samples: Use two flat-bottomed tubes. Add 5cm³ of milk suspension to both tubes. To one tube, add 5cm³ of distilled water (negative control showing no enzyme activity). To the other tube, add 5cm³ of hydrochloric acid (positive control showing complete hydrolysis).
Set up test samples: Place three test tubes each containing 5cm³ of milk suspension in a water bath at 10°C. Allow 5 minutes for equilibration to ensure all samples reach the target temperature.
Start the reaction: Add 5cm³ of trypsin solution to each test tube simultaneously and immediately start timing. The enzyme will begin breaking down the casein protein.
Record observations: Measure how long it takes for the milk samples to become completely colourless, indicating full protein hydrolysis.
Repeat at different temperatures: Repeat the procedure at 20°C, 30°C, 40°C, and 50°C to investigate the temperature effect.
Calculate rate: Find the mean time for complete hydrolysis at each temperature. Calculate the rate of reaction using:

$\text{Rate of reaction} = \frac{1}{\text{mean time}}$

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Worked Example: Calculating Rate of Reaction

If the mean time for complete hydrolysis at 30°C is 4.5 minutes:

$\text{Rate of reaction} = \frac{1}{4.5 \text{ minutes}} = 0.22 \text{ min}^{-1}$

This means the reaction rate is 0.22 per minute at 30°C.

Risk assessment and safety

Several hazards must be considered during this practical:

Broken glass presents a risk of cuts. Handle glassware carefully and keep equipment away from bench edges. If cuts occur, elevate the wound and apply pressure.
Hydrochloric acid may cause irritation to eyes and skin. Wear eye protection and avoid skin contact. If contact occurs, wash immediately with cold water and flood eyes if necessary.
Hot liquids in the water bath may cause scalding. Use tongs to remove tubes and wear eye protection. Keep equipment away from bench edges.
Enzymes may trigger allergic reactions in sensitive individuals. Avoid skin and eye contact and wear protective equipment.

Data collection and processing

Record results in a table showing temperature, individual times for each repeat, mean time, and calculated rate. Ensure measurements include appropriate units and precision. Plot a graph with rate of reaction on the y-axis against temperature on the x-axis to visualise the relationship.

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When plotting your graph, you should observe a typical enzyme activity curve - initially increasing with temperature, reaching a peak at the optimum temperature, then declining sharply as the enzyme denatures.

Analysis and interpretation

Expected results: As temperature increases from 10°C, kinetic energy increases, leading to more frequent collisions between enzyme and substrate molecules. More enzyme-substrate complexes form, increasing the reaction rate up to the optimum temperature.

Beyond the optimum temperature, bonds within the enzyme's tertiary structure begin to break, changing the shape of the active site. The enzyme and substrate are no longer complementary, reducing the rate of reaction significantly.

The milk contains casein protein which, when hydrolysed by trypsin, causes the milk to become colourless. Trypsin is a protease enzyme that specifically breaks down proteins through hydrolysis reactions.

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The concept of enzyme-substrate complementarity is crucial here. Think of it like a lock and key mechanism - when the enzyme's active site changes shape due to high temperature, the 'key' (substrate) no longer fits the 'lock' (active site) properly.

Sources of error may include temperature fluctuations in the water bath, timing inaccuracies, or incomplete mixing of reactants. To improve reliability, conduct multiple repeats and calculate mean values. Validity is ensured by controlling all variables except temperature.

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Key Points to Remember:

Rate of reaction is calculated as $\frac{1}{\text{mean time}}$ - the faster the reaction, the higher the rate
Trypsin is a protease enzyme that hydrolyses casein protein in milk, causing it to become colourless
Increasing temperature initially increases reaction rate due to higher kinetic energy and more enzyme-substrate complex formation
Beyond the optimum temperature, enzyme tertiary structure is damaged and the active site shape changes
Always use control variables to ensure only the independent variable (temperature) affects the results

Rate of an Enzyme Controlled Reaction (AQA A-Level Biology): Revision Notes