The Properties Of Ovalbumin

Property	Detail
Solubility	Soluble in water (especially in mildly alkaline solutions)
Stability	Stable at room temp but denatures with heat
pI (isoelectric point)	Around 4.5–4.7
Thermal behavior	Denatures and coagulates when heated — this is why egg whites solidify when cooked
Allergenic potential	It is a known egg allergen (one of several in egg whites)

Feature	Description
Source	Egg white
Main function	Nutrient storage, structural protein
Applications	Food, lab research, biotech
Allergic?	Yes, in some individuals
Denatures?	Yes, easily with heat or pH changes

General Steps in Purification

Step 1: Preparation of Egg White

1. Separate egg white from yolk carefully.

2. Dilute the egg white with cold distilled water (typically 3–4 times its volume).

   • This reduces viscosity and facilitates further processing.

3. Filter or centrifuge to remove insoluble debris and aggregates.

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Step 2: Fractional Precipitation

The first step is often salting out, which separates proteins based on solubility:

1. Ammonium sulfate precipitation is commonly used:

   • Gradually add ammonium sulfate to the diluted egg white while stirring at 0–4°C.

   • Ovalbumin precipitates at 50–70% saturation of ammonium sulfate.

2. Collect the precipitate by centrifugation.

3. Dissolve the pellet in minimal buffer (e.g., phosphate buffer, pH 7.0).

Salting out reduces protein solubility by changing ionic strength, causing selective precipitation.

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Step 3: Dialysis

• The ammonium sulfate and small impurities are removed by dialysis against a large volume of buffer.

• Dialysis membrane allows salts and small molecules to diffuse out while retaining proteins.

• This step prepares the protein for chromatography.

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Step 4: Chromatographic Purification

Multiple chromatography methods are often combined for high purity:

A. Ion-Exchange Chromatography

• Exploits the net charge of ovalbumin (isoelectric point ~4.6).

• Use anion exchangers (e.g., DEAE-cellulose) because ovalbumin is negatively charged at pH 7.4.

• Steps:

  1. Load the dialyzed protein solution onto a DEAE-cellulose column equilibrated with low-salt buffer.

  2. Wash to remove unbound proteins.

  3. Elute ovalbumin using a gradual salt gradient (e.g., NaCl).

B. Gel Filtration (Size-Exclusion) Chromatography

• Separates proteins based on size.

• Ovalbumin (~45 kDa) can be separated from smaller or larger egg white proteins.

• Common media: Sephadex G-100 or G-75.

C. Affinity Chromatography (Optional)

• Exploits specific binding properties of ovalbumin to lectins (e.g., Concanavalin A) because it is glycosylated.

• Provides high selectivity and purity.

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Step 5: Concentration and Final Preparation

• The purified ovalbumin solution is concentrated using:

  • Ultrafiltration or

  • Lyophilization (freeze-drying) to obtain powdered ovalbumin.

• Purity can be checked by:

  • SDS-PAGE (single 45 kDa band indicates high purity)

  • UV absorbance at 280 nm

  • Isoelectric focusing

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Alternative/Additional Methods

1. Heat Treatment: Ovalbumin is more heat-stable than some minor egg white proteins, allowing partial denaturation of others before chromatography.

2. Precipitation by pH adjustment: Adjusting pH to 4.5 (its isoelectric point) can selectively precipitate ovalbumin.

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Summary of Key Principles

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References / Classic Protocols:

• Nakai, S. & Ohta, Y. (1972). Purification of ovalbumin by ammonium sulfate fractionation and DEAE-cellulose chromatography. Journal of Biochemistry.

• Roy, D., & Kahn, T. (1976). Ovalbumin purification using Sephadex G-100 chromatography.