outline the use of a colorimeter for measuring the progress of enzyme-catalysed reactions that involve colour changes

Enzyme Action (Syllabus 3.1)

Definition: Enzymes are biological catalysts that increase the rate of a chemical reaction without being consumed. After each catalytic cycle the enzyme returns to its original (unchanged) form, ready to bind another substrate molecule.

• Active site – a specialised pocket on the enzyme where the substrate binds.
• Enzyme–substrate (ES) complex – a temporary association that correctly orients the reactants and lowers the activation energy (E_a).
• Lock‑and‑key model – the substrate fits the active site like a key in a lock.
• Induced‑fit model – binding of the substrate induces a conformational change that enhances catalysis.
• Overall effect – the enzyme provides an alternative reaction pathway with a lower E_a, allowing the reaction to proceed rapidly under the mild temperature and pH conditions found in living cells.

Factors Affecting Enzyme Activity (Syllabus 3.2)

Definitions of Kinetic Parameters (AO1)

• V_max – the maximum reaction rate when every enzyme molecule is saturated with substrate.
• K_m – the substrate concentration at which the reaction rate is half of V_max. It is an inverse measure of enzyme–substrate affinity (low K_m = high affinity).

Michaelis–Menten Kinetics

The Michaelis–Menten equation relates initial rate (v) to substrate concentration ([S]):

\[

v = \frac{V{\max}[S]}{Km + [S]}

\]

• At low [S] the curve is steep (rate proportional to [S]).
• As [S] increases the curve approaches a horizontal asymptote at V_max.
• Plotting v against [S] gives a hyperbolic curve; a Lineweaver‑Burk double‑reciprocal plot (1/v vs 1/[S]) yields a straight line from which V_max (y‑intercept) and K_m (‑1 × x‑intercept) can be calculated.

Inhibition (AO2)

• Competitive inhibition: Inhibitor resembles the substrate and binds to the active site. Increases K_m (more substrate needed) but V_max is unchanged.
• Non‑competitive inhibition: Inhibitor binds elsewhere, altering enzyme shape. Decreases V_max (fewer active enzymes) while K_m remains unchanged.
• Both types can be demonstrated with a colourimetric assay, because the change in absorbance directly reflects the rate of product formation.

Colourimetric Measurement of Enzyme‑Catalysed Reactions

Principle (AO1)

A colourimeter measures the intensity of colour (absorbance, A) of a solution at a selected wavelength. When an enzyme reaction produces or consumes a coloured species, the change in A over time is proportional to the change in concentration of that species.

Beer‑Lambert law:

\[

A = \varepsilon \, b \, c

\]

• ε = molar extinction coefficient (L mol⁻¹ cm⁻¹)
• b = path length of the cuvette (usually 1 cm)
• c = concentration of the coloured component (mol L⁻¹)

Re‑arranged to calculate concentration:

\[

c = \frac{A}{\varepsilon \, b}

\]

Typical Set‑up (AO2)

• Colourimeter with selectable wavelength (400–700 nm) and data‑logging function.
• Standard 1 cm cuvettes (glass or disposable).
• Blank solution (reaction mixture without enzyme) to zero the instrument.
• Reaction mixture containing buffer, substrate (coloured or producing a coloured product), and enzyme.
• Thermostatically controlled water bath or incubator if temperature is a variable.

Step‑by‑Step Procedure (Example: Catalase + H₂O₂, coloured product o‑dianisidine) (AO3)

1. Prepare a series of identical reaction tubes containing a fixed concentration of buffer and substrate (e.g., 10 mM H₂O₂ + 0.2 mM o‑dianisidine).
2. Place a blank tube (buffer + substrate, no enzyme) in the colourimeter. Select the wavelength corresponding to the λ_max of the coloured product (≈ 460 nm for o‑dianisidine).
3. Zero the instrument with the blank.
4. Add a measured volume of enzyme solution to the first reaction tube, mix quickly, and start a stopwatch.
5. Immediately withdraw an aliquot (e.g., 1 mL) and record the initial absorbance (A₀).
6. At regular intervals (e.g., every 30 s) withdraw a fresh aliquot, place it in a cuvette, and record the absorbance (A_t).
7. Repeat the experiment with different enzyme concentrations, temperatures, pH values, or inhibitor concentrations as required.
8. Plot A versus time; the gradient of the initial linear region (ΔA/Δt) is converted to an initial rate (V₀) using the extinction coefficient.

Data Recording (example table)

Concentration is obtained from c = A / (ε b). For o‑dianisidine, ε ≈ 12 000 L mol⁻¹ cm⁻¹.

Data Analysis (AO3)

• Initial‑rate region: A straight‑line increase in absorbance indicates a constant rate of product formation. The slope (ΔA/Δt) is proportional to V₀.
• Effect of variables: Compare slopes when temperature, pH, enzyme concentration, substrate concentration, or inhibitor is altered. Explain the observed changes using the factors table.
• Plateau: When the curve levels off, substrate is exhausted or the reaction has reached equilibrium.
• Kinetic parameters: Repeat the assay with a range of substrate concentrations. Plot V₀ against [S] to obtain V_max and K_m. A Lineweaver‑Burk plot (1/V₀ vs 1/[S]) provides a straight line for easier determination.

Evaluation Checklist (AO3)

• Was the blank prepared with exactly the same buffer and substrate concentrations as the test tubes?
• Were cuvettes clean, scratch‑free and oriented consistently?
• Did temperature remain constant throughout each trial (use a thermostatted cuvette holder if possible)?
• Was the timing accurate when withdrawing aliquots?
• Potential sources of error: stray light, pipetting inaccuracies, enzyme denaturation during handling, and substrate depletion before the final reading.
• How could reliability be improved? (e.g., repeat each condition three times and take the mean, use a calibrated spectrophotometer, ensure thorough mixing).

Safety and Practical Tips

• Wear gloves, lab coat, and safety goggles when handling enzymes and chemicals (e.g., H₂O₂, o‑dianisidine).
• Use clean, scratch‑free cuvettes; fingerprints can affect absorbance readings.
• Always insert cuvettes with the same orientation to avoid path‑length errors.
• Prepare a fresh blank each time the buffer composition or substrate concentration changes.
• Record the temperature of the reaction mixture; many colourimeters have a temperature‑controlled cuvette holder.
• If using immobilised enzymes, verify that the support material does not leach coloured substances.

Connections to Other Syllabus Topics

Understanding enzyme kinetics is essential for:

• Respiration (Topic 12) – e.g., the role of catalase in protecting cells from H₂O₂ generated during aerobic metabolism.
• Photosynthesis (Topic 13) – e.g., the kinetic properties of Rubisco and how temperature/pH affect carbon fixation.
• Biotechnology (Topic 15) – immobilised enzymes in industrial processes such as starch conversion by amylase.

Summary (AO1/AO2)

• Colourimetric assays give a rapid, quantitative way to monitor enzyme reactions that involve a colour change.
• By measuring absorbance at the product’s λ_max and applying the Beer‑Lambert law, students can calculate product concentration and initial reaction rates.
• Repeating the assay with varying substrate concentrations allows determination of V_max and K_m (Michaelis–Menten analysis).
• Changing temperature, pH, enzyme/substrate concentration or adding inhibitors demonstrates how each factor influences activity, directly addressing syllabus requirements.
• The practical also develops AO3 skills: planning, data collection, analysis, and critical evaluation.