Purification of Egg White Protein

Topics: Protein, Egg white, Lysozyme Pages: 13 (2864 words) Published: May 1, 2007
Title: Purification of Egg white protein

Name: Michael Johnson
Partner: David Logad & Nandita
Date: 2nd – 9th September 2004
Group: Thursday 11:30am - 3:30pm

Salting Out
In 1888 Hofmeister that it can be possible to dehydrate a protein by adding salt to the solution, salting out. When a protein in a aqueous solution it is surrounded by water, in fact there can be up to 0.35g of water tightly bound to 1g of protein (Simpson 2004). Also the effectiveness of the salting out can be affected by the type of salt used. This is because of the charge the salt can bring on the protein making it either likely or less likely interacted with water. "The protein structure can also have an affect with it hydophobicity with hydrophobic patches on it (caused by the side chains Phe, Tyr, Leu, Ile, Met & Val). So the optimum salt is one that will cause hydration of the polar regions and dehydration of the hydrophobic regions of the protein. With which the salt does not interact directly with the protein. The best salts have a multivalent anions like sulphate or phosphate with cations like sodium or ammonium."(Scopes, 1994)

Ion Exchanges
The Ion exchanges exploit the different net charges on proteins at a given pH and interact with the proteins on electrostatic attraction (Scopes, 1994). The carboxymethycellulose (CMC) is a cation exchange that is chemical weak. It has a negative charged carboxyl groups, they are active above the pH of 4.5.

Gel Electrophoresis
The Sodium dodcecyl sulphate (SDS) gel is based on the principle that the proteins are all given a negative charge. This allows all proteins to migrate to the positive end of the gel. The gel is made of a certain size pore which means the migrtation is based only on the size of the proteins. The smallest proteins will migrate to the positive electrode before the larger ones and the size can be determined by a marker which has a selection of known molecular weight proteins.

Squire Booker
To isolate and purifiey the protein ovalbumin from egg white, by salting out technique To isolate and purify the enzyme lysozyme from egg white using ion-exchange purification technique. To observe the specific and total activity of the Lysozye A, B, C and D. To determiene the purity of the ovalbumin and lysozyme using protein electrophoresis.

Week 1
Isolation of the Ovalbumin
The egg white was extracted from an egg and was passed through a fine cloth to as to allow the membranes to break. The Volume was recorded and 1ml of the placed into a Eppendorf microfuge tube and label this was then stored at 4°C in a refrigerator. A volume of 2ml was removed from the egg white and placed in a 10ml centrifugation tube. A equal volume of ammonia sulphate was solution was added slowly and the solution was stirred with a glass rod. This was allowed to stand 30 minutes and was occasional stirred.

The sample was then centrifuged down (3500rpm for 20minutes), the supernatant that contained the ovalbumin was placed into a new centrifuged tube.

A volume of 2ml of 0.25 H2SO4 was added to bring the solution containg the supernatant to a pH of 4.7 ( a pH indicator paper was used). The volume of H2SO4 was recorded and the same volume of ammonia sulphate was added to solution which was stirred and left for 5 minutes to allow ovalbumin to precipitate out of solution.

The sample was once again centrifuged (3500rpm for 20minutes) to recover the precipitate of ovalbumin.

The sample was mixed with water and was placed into a small dialysis tubing to dialysed off the ammonium sulphate against running water over night. The sample dialysis bag, of which was then stored at 4°C. The solution was then filtered to reclaim the ovalbumin.

Isolation of Lysozyme
The following procedures were carried out in an attempt to keep the Sollution close to 0°C as possible.

A sample of carboxymethylcellose (CMC) was weighed out (0.3grm), the CMC was placed...
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