BASIC BIOCHEMISTRY/MOLECULES OF LIFE
THE ENZYMOLOGY OF β-GALACTOSIDASE I
β-galactosidase is an enzyme that normally catalyses the breakdown of lactose in the lactose
metabolic pathway. Lactose is the substrate in this reaction and it fits into the active site of the
enzyme, which then mediates the hydrolysis of the disaccharide lactose into two
monosaccharides: glucose and galactose. Please see the top reaction in the diagram below.
However it is quite complicated to monitor this reaction using chemistry so scientists have
devised a visual method. They have chemically synthesised an artificial substrate in which a
galactose molecule has been covalently joined to a colourless molecule called orthonitrophenol
making a molecule called o-nitrophenylgalactoside (ONPG). This molecule can also fit into the
active site of the enzyme but the products of the reaction are now galactose and orthonitrophenol.
The latter is yellow when it is no longer covalently bound to Galactose. The reaction can
therefore be monitored by the accumulation of this yellow product in the reaction tube. The
precise concentration can be accurately measured by using a spectrophotometer set at 420
nanometers. (This is written as: λmax = 420 nm, where λ is wavelength.) We shall be using this
substrate to explore the enzymology of β-galactosidase in practicals 2, 3 and 4.
A protein’s function is inextricably linked to its 3-D structure. This in turn arises from the
interaction of amino-acids in its primary structure with one another, and with the surrounding
environment. In the case of enzymes their catalytic activity arises from:
 the shape of their active site, which is governed by the way the protein folds and
 the chemistry of the catalytic site, which depends upon the chemical nature of the ‘R’
groups of the amino acids involved in catalysis.
If we change the chemistry of the protein molecule, the enzyme’s catalytic effect may also
change. Can you suggest how? Record this in your practical book. This can provide a key insight
into how the enzyme carries out its catalysis. In practical 2 we shall focus our attention on the
effect of pH on the activity of this enzyme.
THE EFFECT OF PROTEIN STRUCTURE ON ENZYME ACTIVITY.
1A. ONE HOUR PREPARATORY SESSION.
Objectives:
 To re-enforce the use of a spectrophotometer and the accurate use of micropipettes.
 To appreciate the specificity of substrate-enzyme interaction.
 To appreciate the possible effect of pH on protein structure and function.
 To plan a series of reaction conditions to test the enzyme activity of β-galactosidase.
Resources:
 A spectrophotometer.
 P20, P200 and P1000 micropipettes and tips.
 Information on a number of solutions.
 Your group members and a facilitator.
PROCEDURE:
1. Ensure that you are familiar with the correct use of the spectrophotometer
allocated to your bench.
2. Also ensure that you are familiar with the accurate use of the P20, P200 and
P1000 micropipettes.
3. Use the information presented in the diagrams above to explore the shape and
orientation of the substrate that binds to the enzyme. Record this in your lab
book.
4. Explore how changing pH might alter the structure of the protein, and the
chemistry of the active site. Record this in your lab book.
(Although outside the first year syllabus please also be aware that pH may also
affect the chemistry of the substrate and its products.)
5. Use the information on the next page to draw up a table for the preparation of
reaction solutions with different pH values.
You will be supplied with:
0.48M Tris Acetate
0.48M phosphate buffer pH 7.5.
1.8mM ONPG
2.4 units/ml of β-galactosidase
Use these solutions and water as necessary to prepare 3ml reaction volumes in cuvettes. The final
concentration of each constituent should be as follows:
0.08M Tris Acetate
0.08M phosphate buffer pH 7.5.
0.3mM ONPG
0.4 units/ml of β-galactosidase
Draw up this table in your book:
Sample 1
At pH 4.0
Sample 2
At pH 6.0
Sample 3
At pH 7.5
Sample 4
At pH 9.0
Sample 5
At pH 12.0
Volume of 0.08M Tris
Acetate
Volume of 0.08M
phosphate buffer
1.8mM ONPG
2.4 units/ml of βgalactosidase
H2O
Total Volume
You will also need to set up two controls to check that the substrate and enzyme are BOTH
required for the reaction to occur. What will they be? Add these to your book.
Sample 6
Control (substrate)
Volume of 0.08M Tris Acetate
Volume and pH of 0.08M phosphate buffer
ONPG
2.4 units/ml of β-galactosidase
Total Volume
Sample 7
Control (enzyme)
1B. 90 MINUTE INDIVIDUAL SESSION
Objectives:
 To gain experience in the use of a spectrophotometer.
 To gain experience in the accurate use of micropipettes.
 To plot enzyme activity against time using a range of substrate concentrations.
 To use this information to calculate the Km and Vmax of β-galactosidase.
Resources:
 Your laboratory notebook – updated with information from the one hour group
preparation session.
 A spectrophotometer set at 420nM and 3ml cuvettes.
 P20, P200 and P1000 micropipettes and tips.
 A solution of 2.4units/ml of β-galactosidase.
 A solution of 10mM ONPG.
 Tubes and buffer for ONPG dilutions.
 A facilitator.
PLEASE NOTE THAT TIME ON THE SPECTROPHOTOMETERS IS LIMITED BY
THE CLASS SIZE. WITH RESPECT TO YOUR SEVEN SAMPLES:
 FOUR CAN BE LEFT TO STAND ON THE BENCH WHILE YOU
MONITOR THE OTHER THREE FOR FOUR MINUTES AT 30 SECOND
INTERVALS.
 THE FOUR ‘BENCH’ SAMPLES CAN THEN BE READ AS SINGLE
OBSERVATIONS.
PROJECT:

Use the tables in your notebook to set up samples 1, 5, 6 and 7. Label these and
leave these to one side on your bench. Note the exact time in your book.

Zero your spectrophotometer by inserting either tubes 6 or 7 and pressing the
DARK BLUE button.
NB. ALWAYS ENSURE THAT THE CUVETTES HAVE BEEN DRIED AND ARE
FACING THE CORRECT WAY SO THAT THE CLEAR FACES ARE IN THE LIGHT
PATH WHEN USING SPECTROPHOTOMETERS.

Now you are ready to proceed with samples 2, 3 and 4.

Use the tables in your notebook to set up sample 2 and immediately replace the blank
with sample 2 and press the GREEN button.

Round the readings to the nearest 0.1 and record them in a table in your book every 30
seconds for four minutes.

Zero the machine again and then set up and record sample 3.

Zero the machine again and repeat for sample 4.

Zero again and read the ‘bench’ samples (1, 5, 6, and 7). Note the time in seconds since
these were set up.

Use the data from samples 2, 3, and 4 to plot a graph of activity as increase in absorbance
at 420nM (O/D 420) against time (secs). Use this graph to work out the rate of ONPG
hydrolysis per second.

Finally record these rates and the rates (if any) from samples 1, 5, 6, and 7.

Write a brief discussion of your results indicating the use of controls, the effect of pH on
this enzyme’s catalytic activity, and dentify the optimum pH for this assay.

Pour the samples down the sink and carefully wash out the cuvettes using the plastic
wash bottle. Dry the outside carefully and invert on tissue to drain.

Wash your hands before leaving the laboratory.