Salt Precipitation of Lysozyme and Dialysis/desalting of Lysozyme

Salt Precipitation of Lysozyme and Dialysis/Desalting of Lysozyme

Aim & Introduction

The aim of the experiment was to purify lysozyme from an egg. The way this was achieved was to take advantage of the protein isoelectric point. The isoelectric point is the pH where the sum of the positive amino acids of the protein is equal to the sum of the negative amino acids. Therefore the protein has a net overall charge of zero. This information was used to adjust the pH of the solution, containing the lysozyme, towards its isoelectric point (pI 10.5) so that the lysozyme proteins will carry no charge and will no longer be electrostatically repulsed from each other and therefore will aggregate together and finally precipatate out of solution. In this experiment saturated Ammonium sulphate (NH4)2SO4] is the salt used for the precipitation of the lysozyme. Egg white was used as a source of lysozyme. Tris buffer was used to stabilize the environment for lysozyme (alkali buffer).

The method was carried out from part 1 of the lab manual on pages 6 and 7 and consisted of ten steps. The following precautions were also undertaken: (1) When the ammonium sulphate was mixed into the solution in step 3, it was very important to add it drop by drop into the solution. If the (NH4)2SO4) was added all at once, the salt builds up and inaccurate precipitation would be recorded. (2) Also when the magnetic stirrer was used to mix the ammonium sulphate into the solution, it was important to ensure the stirrer was not mixing too fast. If it mixesd too fast a broth would form and that meant the protein was denatured. (3) The volume of S1 obtained was 20 mls, 5mls transferred into a separate container. The volume of P1 was 5.0 mls.

Lysozymes damage bacterial cell walls by catalysing hydrolysis of 1,4-beta-linkages between N-acetlymuramic acids N-acetyl-D-glucosamine resiudes. This causes lysis of bacteria. Therefore, if lysozyme was added to a solution of bacteria, it would kill all the bacteria in the solution. If this solution was placed in the spectrophotometer, lysozyme activity can be detected by the effect it has on the absorbance values. The more live bacteria, the more light emitted and therefore the higher the absorbance values.

An enzyme assay was carried out to determine the % lysozyme activity and therefore lysozyme in each fraction (S1, P1, S2, P2). This theory was carried out as described in the lab