Method and material:

Part A. Fractionation of egg white

Egg white, saturated ammonium sulfate solution, pH 7.5 of 0.1M phosphate buffer, 2M NaCl

and distilled water.

1 mL of egg white was put in a 10mL centrifuge tube and added with 1mL of saturated

ammonium sulfate solution. The contents were mixed well with gentle conversion and were

centrifuged at maximum speed for 2 minutes.

The supernatant was poured into a clean 10mL centrifuge tube and was added with 1mL of

saturated ammonium sulfate solution. The mixture was centrifuged for 5 minutes.

The precipitate was dissolved in 5mL of pH7.5 0.1M phosphate buffer and was used as

ovalbumin stock solution.

5mL of egg white was put in a 10mL centrifuge tube and was added with 9.5mL of distilled

water. The contents were mixed well with gentle conversion and were centrifuged at top speed

for 10 minutes.

The supernatant was discarded and the ovomucin pellet was suspended in 1mL of distilled water

by gentle shaking. The mixtures were transferred to a glass test tube.

3 drops of 2M NaCl were added to the test tube and the precipitate was dissolved. This solution

was used as ovomucin stock solution.

0.2mL of ovomucin solution and 0.2mL of ovalbumin solution was added with 1mL of distilled

water respectively. The observation was recorded.

Result:

Part A. Fractionation of egg white

Summarize the process for the preparation of ovalbumin:

1mL of egg white was added with 1mL of saturated ammonium sulfate solution. The contents

were mixed well with gentle inversion and were centrifuged for 2 minutes. The supernatant was

poured into a clean tube and was added with 1mL of saturated ammonium sulfate solution. The

mixture was centrifuged and the precipitate was dissolved in 5mL of pH7.5 1.0M phosphate

buffer.

Summarize the process for the preparation of ovomucin:

0.5mL of egg white was added with 9.5mL of distilled water. The contents were mixed well with

gentle inversion and were centrifuged for 10 minutes. The supernatant was discarded and the

ovomucin pellet was suspended in 1mL of distilled water. The mixture was transferred to a glass

test tube and the precipitate was dissolved by added with 3 drops of 2M NaCl.

Test Observation

0.2mL ovomucin + 1mL water Cannot dissolve in water

0.2mL ovalbumin + 1mL water Dissolved completely in water

Question:

Part A

From the observed solubility properties of ovalbumin and ovomucin, to which class of protein do

they belong?

Solubility of protein depends on the surface hydrophobicity.

There are many types of globular proteins. The common protein was albumins which soluble in

water after hydrolysis with ammonium sulfate solution. The second protein was globulins which

partially soluble in water but dissolved completely in dilute salt solution. In the process of

preparing ovalbumin stock solution, saturated ammonium sulfate solution was used repeated.

Saturated ammonium sulfate solution is a salt solution which has a high solubility in water which

lowers the solubility of proteins. By adding ammonium sulfate to egg white, the high

concentration of salt will causes the cell wall to break thus releasing the proteins inside. After

centrifugation, supernatant containing proteins was taken and the pellet was discarded. Further

adding of saturated ammonium sulfate solution has lowered the solubility of proteins causing the

proteins to form precipitate. Phosphate buffer saline, PBS was used to dissolve the precipitate

formed by proteins and to store the proteins within the solution. PBS also used to maintain the

pH of proteins. In the process of preparing ovomucin stock solution, the precipitation after

adding water to egg white was ovomucin. After centrifugation, the ovomucin pellet was

dissolved by adding 3 drops of 2M NaCl but not dissolved in water. Excess drops of NaCl will

cause solution to from precipitate again. (Nelson and Cox, 2000; Biologicalworld.com, 2009)

By observation, ovalbumin and ovomucin are globular proteins. Ovalbumin is an albumins

protein while ovomucin is a globulins protein.

Precaution:

A pipette should be used to extract the supernatant instead of pouring it to minimize the mixing

with pellet.

References

Nelson and Cox, 2000. Lehninger Principles of Biochemistry. Amino Acids, Peptides, and

Proteins. © 2000, 1993, 1982 by Worth Publishers. Third Edition. Page 130.

[Accessed on 18.09.2010]

Biologicalworld.com, 2009. Using PBS in Biochemical and Cell Biology Research. [online]

Available from: < http://biologicalworld.com/pbs.htm >

[Accessed on 18.09.2010]

3. Amino acid sequence

Ovalbumin is a glycoprotein with a relative molecular mass of 45 000. The amino acid sequence of hen egg-white ovalbumin comprising 386 amino acids was deduced from the mRNA sequence by McReynolds et al. [22] and is in agreement with sequences of the purified protein [23] and the cloned DNA [24]. The sequence includes six cysteines with a single disulfide bond between Cys74 and Cys121 [25]. The amino terminus of the protein is acetylated [26]. Ovalbumin does not have a classical N-terminal leader sequence, although it is a secretory protein. Instead, the hydrophobic sequence between residues 21 and 47 may act as an internal signal sequence involved in transmembrane location [27]. Two genetic polymorphisms of ovalbumin have been reported: a Glu→Gln substitution at residue 290 [28] and an Asn→Asp substitution at residue 312 [29]. The sequences of chicken, Japanese quail, and common turkey ovalbumins have been determined and are 90% identical. Major differences include: a truncation of three amino acids from P1–P2′ in quail ovalbumin, which supports the theory that ovalbumin is not inhibitory; an extra glycosylation site at 372 in turkey ovalbumin; chicken ovalbumin has six Cys residues while quail and turkey ovalbumin lack Cys31. As more ovalbumin sequences become available it may be possible to determine which domains are required for function through sequence comparison.

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