Lab Manual
CBB20303 BIOCHEMISTRY
EXPERIMENT 4
PURIFICATION AND CHARACTERIZATION OF LYSOZYME
INTRODUCTION
Lysozyme is one of the first enzyme for which the complete three-dimensional structure
was determined by X-ray diffraction. Egg white, human milk, spleen and many tissues contain
this enzyme. Its IUPAC name is mucopeptide, N-acetylmuramylhydrolyase. It hydrolyse
linkages in polysaccharides of a variety of organism.
Lysozyme performs an antibiotic function for the human body. This is due to its ability to
destroy invading bacteria by hydrolyzing the mucopolysaccharide of the cell wall. Egg white is
rich in lysozyme and will be our starting material for purification. Lysozyme has a low molecular
mass (14.3 kDa from chicken white egg) than most other proteins, and therefore considerable
purification can be achieve by simple gel-filtration which separates proteins in the basis of size.
OBJECTIVE
To purify lysozyme from white egg using gel-separation chromatography
MATERIALS
1. 0.05 M Tris Base, 0.05 M NaCl (pH 8.2): Tris-NaCl Column Buffer, 250 ml
2. Blue dextran, 2mg/ml in distilled water
4. Sephadex G-75 (prepared)
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Lab Manual
CBB20303 BIOCHEMISTRY
PROCEDURE
Preparation of column
1. Wash column and rinse with deionized water. Ensure the water flows through column.
2. Fill in the column with Tris-NaCl column buffer.
3. Add enough slurry (Sephadex G-75) to fill the column.
4. Check the stopcock is not opened. Leave the column until it is packed. Do not allow the
buffer to go below level of gel to prevent the column cracked and ruined.
5. Apply 1 ml of blue dexran solution into the column. Elute blue dextran from the column
using Tris-NaCl buffer. Determine column void volume (Vo). Void volume is the volume
of solvent which must pass through the column before any solute molecule will be eluted.
6. Ensure the column homogeneity by watching the passage of blue dextran through the
column. If the column is properly packed, blue dextran band will broaden as it passes
through the column but the band will not be skewed.
Preparation of enzyme source
1. Separate the white egg from the yolk. Place a double layer of gauze over a beaker. Gently
filter the white egg.
2. Transfer 4 ml of filtered egg white into microcentrifuge tubes and spin for 5 minutes at
10,000 rpm.
3. Remove the supernatant into a fresh plastic culture tube and add 8 ml of Tris-NaCl
buffer. Mix thoroughly until homogenous.
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Lab Manual
CBB20303 BIOCHEMISTRY
Gel Filtration
1. Label test tubes for collecting column fractions. Mark all test tubes with a line at 2 ml
volume.
2. Allow the Tris-NaCl buffer to run out of column until the buffer first reaches the top of
packed slurry. Stop the flow of column.
3. Pipette 1 ml of the buffered egg white carefully into the column. Avoid disturbing the
top of the column by putting the pipette tip against the side of the column. Allow 1 ml of
the buffered egg white run into the column. As soon as the solution reaches the top of the
slurry, add 1 ml of Tris-NaCl column buffer and the slowly fill the column with the
column buffer.
4. Began collecting 15 sequential 2.0 ml quantities of eluent is separate test tubes. Do not let
the column go dry. Place the fractions on ice.
5. Measure absorbance of each fraction at 280 nm. Determine which fractions have
absorbance value larger than 0.100. Keep the sample with highest absorbance value for
SDS-PAGE experiment.
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