Characterization of Egg White Proteins
- Methods and Materials
- Isolation of lysozyme
- Activity and Assay of Lysozyme
- Iron binding Capacity of Conalbumin II
Egg white or albumin is the biggest fraction of whole egg and contains 88% water, 11% proteins, 0.2% fats and 0.2% minerals. The major constituent of egg white proteins are 60% of ovalbumin, 12% of conalbumin, 11% of ovomucoid, and 3.5% of lysozyme. It also contains small amount of ovoinhibitor, ovoglycoprotein, flavoprotein, avidin and ovoglobulins (Brown 2010).
Ovalbumin is the most abundant egg white protein with isoelectric point of 4.58. It has molecular weight of 44.5kDa (Coultate 2009). Ovalbumin is found to have antimicrobial effect, due to its ability to bind iron. This causes deprivation of irons essential for growth of microorganisms (Abdou et al. 2013). It is also suggested that the hydrophobicity and its positive charge contributes to ovalbumin's bactericidal actions against Gram-negative bacteria (Belitz et al. 2009). It also acts as a storage proteins and major nutrient source of amino acids for embryo in late developing stage (Mine & Yang 2008). In food industry, the amphilic behavior of ovalbumin makes it suitable to be used as foaming agent. It is used mainly as stabilizer in bakery products where their texture and structure have to be maintained during and after processing (Du et al. 2002). In medical field, ovalbumin is administered when there is suspected heavy metal poisoning to chelate the metal ions, preventing them to be further absorbed through digestive system.
[...] Lastly, centrifuging purifies the lysozyme fractions recovered and removes other proteins from solution Lysozyme D will be expected to contain highest concentration of purified lysozyme. Lysozyme A will contain all proteins present in egg white such as ovalbumin, conalbumin and lysozyme as no separation is done. Lysozyme B and C fractions will contain a number of mixed proteins in egg white except lysozyme, since most of the lysozyme has already been bound to the CM exchanger. Lysozyme D however contains purely only lysozyme without mixture of other protein fractions. [...]
[...] Undiluted fractions would have very high lysozyme activities. Amberlite IR 120 was a strong cation exchange resin that had negative charge. It would bind to positively charge iron (III) ions. Conalbumin consisted of two lobes linked by ?-helix structure and each lobe could reversibly bind to iron (III) ions (Mine 2010). Bicarbonate ions further enhanced binding of conalbumin to iron ions. Theoretically one mole of conalbumin would bind to two moles of iron since conalbumin consisted of two metal ion binding sites (Belitz et al. [...]
[...] Mechanical agitation of centrifugation enhaced and quickened the process of precipitation by ammonium sulfate. Dialysis removed excess ammonium sulfate from the isolated ovalbumin and conalbumin solution respectively. The porous membrane of dialysis bag allowed ammonium sulfate to move out of the protein solution down its concentration gradient, but large protein molecules could not passed through. Running water ensure equilibrium in ammonium sulfate concentration gradient could not be established and allowed ammonium sulfate to be continuously removed until it was of negligible concentration. [...]
[...] The experiment also studied the iron-binding capacity and saturation point of conalbumin using spectrometry in the presence of bicarbonate ions. Methods and Materials: Week Isolation of Ovalbumin and Conalbumin. Figure Flow chart for isolation of ovalbumin and conalbumin Sample calculation for mass of ammonium sulfate solid measured: Mass of ammonium sulfate required = Concentration x Volume of solution = (8/100) g/mL x 90mL = 7.2000 g Week Isolation of lysozyme Figure Flow chart for Isolation of lysozyme. Week State of tyrosine in ovalbumin: Figure Flow chart for determination of effect of pH and temperature on tyrosine state. [...]
[...] Different dilutions of sample proteins were prepared (shown in Table 3). Sample calculations To prepare 1mL of 1/10 diluted conalbumin, For 1/10 dilution: The volume of undiluted conalbumin sample required = (dilution factor) x (final volume to be prepared for spectrophotometry analysis) = 1/10 x 1ml = 0.1 ml The volume of distilled water added = 1.0 ml 0.1 ml = 0.9 ml Similar calculations were applied to prepare other egg white proteins with dilution factor of (1/100, 1/3 and 1/30). [...]