Isolation Of Lysozyme From Egg White
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Isolation of Lysozyme from Egg White
Introduction
Lysozyme isolation from the egg white is done to investigate the ideas behind protein purification and determination of enzyme specific activity; lysozyme is prepared from egg white using ion exchange chromatography.
Method
As seen in the schedule except the following changes: pH 4.5 was used instead of pH 4.6 as written in the schedule. More acetate buffer was used contrary to the 18ml to be used in the schedule.
Results
Ion exchange chromatography table
Sample
Acetate buffer pH 4.5
Phosphate buffer at pH 7.0
Carbonate buffer at pH 11.4
1
-0.16
-0.12
-0.106
2
-0.106
-0.123
-0.098
3
0.202
-0.128
-0.036
4
0.477
-0.133
0.39
5
0.778
-0.13
0.092
6
1.047
-0.072
0.031
7
0.7
0.313
-0.051
8
0.246
1.042
-0.032
9
-0.002
0.812
-0.118
10
-0.076
0.427
-0.084
11
-0.124
0.371
-0.141
12
-0.25
0.237
-0.203
13
-0.3
0.03
-0.25
The absorbance table result
Time at;
(s)
Sample containing
lysozyme
Sample containing acetate
buffer at pH4.5
Sample containing
phosphate buffer at pH 7.0
Sample containing
carbonate buffer at pH 11.4
10
0.465
0.673
0.655
0.438
20
0.448
0.672
0.647
0.412
30
0.45
0.671
0.637
0.384
40
0.432
0.67
0.631
0.366
50
0.358
0.67
0.627
0.350
60
0.291
0.67
0.625
0.335
Calculation for the units of lysozyme in the fraction assayed:
Sample containing lysozyme = 0.465 - 0.291(highest minus lowest value)
= 0.174
0.174 ÷ 0.001 (1unit of lysozyme@450nm)
= 174 units of lysozyme
Sample containing acetate buffer at pH 4.5 = 0.673 - 0.67
= 0.003
0.003 ÷ 0.001= 30 units of lysozyme
Sample containing phosphate buffer at pH 7.0 = 0.655 - 0.625
= 0.03
0.03 ÷ 0.001= 30 units of lysozyme
Sample containing carbonate buffer at pH11.4 = 0.438 - 0.335
= 0.103
0.103 ÷ 0.001 = 103 units of lysozyme
Discussion
The globular protein which contains 129 amino acids is called Lysozyme. The process of lysozyme isolation from the egg whites is done by purifying the lysozyme from the egg white through ion exchange chromatography. The monitoring of the purification process is done through SDS polyacrylamide gel electroporesis (SDS-PAGE) and assays of lysozyme activities.
The most popular protein purifying method is the ion exchange chromatography. Each of the proteins has its own Isoeclectric Point (PI). If the pH buffer is larger than the PI, it means that the protein charge is negative. If the pH buffer is less than ...