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

Mode of Action of Enzymes

Enzymes are biological catalysts that accelerate chemical reactions without being consumed. They work by lowering the activation energy (\$E_a\$) required for a reaction to proceed, allowing the reaction to occur at the temperature and pH found in living cells.

Key Features of Enzyme Action

• Specificity – each enzyme binds to a particular substrate or group of related substrates.
• Active site – a region of the enzyme where substrate molecules bind and undergo a chemical transformation.
• Enzyme–substrate complex – a temporary association that brings reactants into the correct orientation.
• Induced fit – binding of the substrate induces a conformational change that enhances catalysis.
• Regeneration – after the reaction, the enzyme returns to its original state, ready for another catalytic cycle.

Factors Influencing Enzyme Activity

1. Temperature – increases reaction rate up to an optimum; beyond that, denaturation occurs.
2. pH – each enzyme has an optimum pH; deviation can affect ionisation of active‑site residues.
3. Substrate concentration – follows Michaelis–Menten kinetics; rate plateaus at \$V_{\max}\$.
4. Inhibitors – competitive, non‑competitive or uncompetitive, altering \$Km\$ or \$V{\max}\$.
5. Presence of cofactors or co‑enzymes – required for activity of many enzymes.

Using a Colourimeter to Measure Enzyme‑Catalysed Reactions

A colourimeter measures the intensity of colour (absorbance) of a solution at a specific wavelength. When an enzyme reaction produces or consumes a coloured product, the change in absorbance can be used to monitor the progress of the reaction in real time.

Principle of Colourimetric Measurement

The Beer‑Lambert law relates absorbance (\$A\$) to concentration (\$c\$) of the coloured species:

\$\$

A = \varepsilon \, b \, c

\$\$

where \$\varepsilon\$ is the molar extinction coefficient (L mol⁻¹ cm⁻¹) and \$b\$ is the path length of the cuvette (cm). By measuring \$A\$ at regular intervals, the concentration of product formed (or substrate consumed) can be calculated.

Typical Set‑up

• Colourimeter with selectable wavelength (usually 400–700 nm).
• Standard cuvettes (1 cm path length) or disposable cuvettes.
• Blank solution (reaction mixture without enzyme) to zero the instrument.
• Reaction mixture containing substrate, buffer, and enzyme.
• Timer or data‑logging function to record absorbance at set intervals.

Step‑by‑Step Procedure

1. Prepare a series of identical reaction tubes containing buffer and substrate.
2. Place a blank tube (buffer + substrate, no enzyme) in the colourimeter and set the wavelength to the absorbance maximum of the coloured product.
3. Zero the instrument using the blank.
4. Add a measured volume of enzyme solution to the first reaction tube and immediately mix.
5. Transfer an aliquot (e.g., 1 mL) to a cuvette and record the initial absorbance (\$A_0\$).
6. At predetermined time points (e.g., every 30 s), withdraw an aliquot, place it in a cuvette, and record the absorbance (\$A_t\$).
7. Repeat steps 4–6 for different enzyme concentrations or temperatures if required.
8. Plot \$A_t\$ versus time to obtain a reaction progress curve.

Data Presentation

Concentration is calculated from the absorbance using the rearranged Beer‑Lambert equation:

\$\$

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

\$\$

Interpreting the Results

• A linear increase in absorbance indicates a constant rate of product formation (initial rate region).
• The slope of the \$A\$ vs. time plot gives the initial rate (\$\Delta A/\Delta t\$), which can be converted to \$V_0\$ (µmol L⁻¹ s⁻¹) using the extinction coefficient.
• Comparing slopes under different conditions (e.g., temperature, pH, inhibitor presence) reveals how those factors affect enzyme activity.
• When the curve plateaus, the reaction has reached equilibrium or substrate depletion.

Safety and Practical Tips

• Wear gloves and goggles when handling enzymes and chemicals.
• Keep cuvettes clean and free of fingerprints; scratches affect absorbance readings.
• Use a consistent cuvette orientation to avoid path‑length variation.
• Prepare a fresh blank for each set of measurements if the buffer composition changes.
• Record temperature of the reaction mixture; colourimeter chambers can be temperature‑controlled.

Summary

Colourimetric assays provide a rapid, quantitative method for monitoring enzyme‑catalysed reactions that involve a colour change. By measuring absorbance at the product’s λ_max, students can calculate reaction rates, explore the effects of experimental variables, and reinforce the concept that enzymes accelerate reactions by lowering activation energy.