6. Methods of measuring enzyme activity

This section is about ideas (e.g. 'fair test') and possible experimental procedures for investigating the factors that control the rates of enzyme catalysed chemical reactions.

I do not give detailed recipes for the experiments - just lots of ideas, there is much 'stuff' to think about!

See also  'decay' investigation using milk and lipase  gcse biology revision notes

Its part of the web page Carbon cycle, nitrogen cycle, water cycle, decomposition - decay investigation

How might the factors controlling enzyme activity be investigated in the laboratory?

Introduction

In principle you can measure the rate of an enzyme reaction in two ways:

Method 1. Measuring the rate of formation of a product (product/time)

e.g. how much product is formed in a give time.

In method 1. the decomposition of hydrogen peroxide by catalase is followed by measuring how much oxygen is formed.

Method 2. Measuring the rate at which the substrate is used up

e.g. the time taken for a given amount of substrate to react and be used up.

In method 2. the hydrolysis of starch to maltose by amylase is followed by measuring the time a given amount of starch is used up.

The experiments described below can be adapted to look at the four main factors (variables) which can affect and control the speed of an enzyme catalysed reaction.

Theoretically there are four main variables you can investigate for any enzyme reaction.

 

All of the following can all affect the speed of an enzyme catalysed reaction.

The concentration of the substrate molecules.

The concentration of the enzyme.

The temperature of the reaction mixture.

The pH (how acidic/neutral/alkaline) of the reaction medium

 

This means three of the variables involved in an experimental investigation must be kept constant except for the variable you are investigating e.g.

(a) Varying the concentration of the substrate or enzyme at a constant temperature and constant pH.

Room temperature is convenient, but reaction might be too slow - trial and error!

You must keep either the substrate concentration, or the enzyme concentration constant, and monitor the rate effect of changing the concentration of the other.

The laboratory temperature can vary even in a single lesson so using a thermostated water bath is advisable - if available. A temperature of 25^oC to 35^oC is likely to be suitable, but must be kept constant.

The pH should be automatically fairly constant and no buffer should be needed unless any acidic or alkaline substances are formed or removed as a consequence of the enzyme catalysed reaction.

If in doubt use a buffer of pH close to the optimum pH of the enzyme.

A buffer is a special solution of chemicals that resists pH changes if small amounts of acid or alkali are added to, or removed from, a solution.

If in doubt, use a buffer solution that matches the optimum pH of the enzyme.

Your body automatically does this by various chemical means, to keep most of your cell and body fluids around ~pH 7.3, safe!

 

(b) Varying the temperature of the reaction mixture keeping the concentrations of both substrate and enzyme constant and at a constant pH.

If in doubt about the constancy of the pH, use a buffer matching the enzyme's optimum pH.

Again, using a thermostated water bath is advisable and the best way to vary the temperature e.g. conducting experimental runs at 20^oC, 25^oC, 30^oC, 35^oC, 40^oC etc.

 

(c) Varying the pH of the reaction medium using different buffer solutions. Ideally using a range of at least five buffers ranging from pH 2 to pH 11. The temperature and concentrations of substrate and enzyme must ALL be kept constant.

Again, using a thermostated water bath is advisable to keep the temperature constant e.g. somewhere in the range 25^oC to 35^oC.

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Two methods for following an enzyme catalysed reaction are illustrated below, full details on other pages in Parts 7. and 8.

Above and below: Part 7. Method 1. involving collecting a gas given off for all the details.

 

Above and below: Part 8. Method 2 Measuring a colour change for all the details.

To get good results takes a lot of hard careful work and sometimes 'trial and error', but the experiments can be divided out amongst a class ad the pooled data analysed by all the class.

To get good data in one lesson the work may be split between groups of 2-3 students.

See also GCSE chemistry notes: How we measure the speed or rate of a chemical reaction

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Key points Based on the syllabus-specifications for students taking the AQA, Edexcel and OCR GCSE level biology examinations (~US grades 9-10).

Some key ideas

Measuring the Rate of Enzyme Reactions

Common Methods:

1. Measuring Gas Production

   • Used when enzymes catalyse reactions that release gases (e.g. catalase breaking down hydrogen peroxide to release oxygen).

   • Collect gas in a syringe or over water and measure the volume produced over time.

2. Observing Colour Change

   • If the reaction involves a colour change, use a colorimeter or compare against a standard.

   • E.g. amylase breaking down starch – test with iodine: starch turns blue-black, and fades as it’s broken down.

3. Measuring Mass Loss

   • If a gas is released, use a balance to detect a loss in mass over time.

4. Using a pH Indicator

   • If the reaction produces or uses up acidic/basic substances, monitor pH changes using a pH probe or indicator.

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Key Variables in Enzyme Experiments

• Independent Variable – the one you change:
  E.g. temperature, pH, enzyme concentration, substrate concentration.

• Dependent Variable – what you measure:
  E.g. volume of gas, colour intensity, time taken for a reaction to complete.

• Control Variables – what you keep constant:
  E.g. same enzyme source, reaction volume, same type of substrate.

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Summary of learning objectives and key words or phrases

Understand and describe the principles and methods of measuring the rate of an enzyme catalysed reaction, including fair test criteria and how to control variables.

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