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Isolation and Purification of Egg White Proteins

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Introduction

Title : Isolation and Purification of Egg White Proteins Date : 10 May 2011 Session : Morning (9 a.m. - 12 p.m.) Demonstrator's Name : Yan Qi INTRODUCTION OBJECTIVE The aim of this experiment was to isolate and purify ovalbumin, conalbumin, ovoglobulin present in the egg of the domestic fowl for the first week. In week two, the ion exchanger, carboxymethylcellulose column was used to purify the lysozyme at alkaline pH. Besides that, the effect of pH and heat on the state of tyrosine was investigated. In week three, the determination of protein content of conalbumin, ovalbumin, lysozyme A, B C and D was done by a using a standard curve obtained from the Biuret test on a standard protein solution containing bovine serum albumin. Next, the iron binding capacity of conalbumin will be determined by titrating 'iron-free' conalbumin with Fe3+. In week four, the enzymic activity was measured by its action on Micrococcus lysodeikticus using a spectrophotometer. Furthermore, the point of saturation of the conalbumin with iron was determined. RESULTS Week 1 Volume of egg white collected (mL) 64 Volume of egg used for isolation of ovalbumin and conalbumin (mL) 62 Volume of saturated ammonia sulphate added (mL) 62 Volume of 0.25 M H2SO4 added to supernatant (mL) 9.5 Volume of supernatant containing ovalbumin and conalbumin (mL) 7.1 Volume of saturated ammonia sulphate added to supernatant (mL) ...read more.

Middle

mg/ml Absorbance 570nm 0.000 0.107 0.153 0.239 0.322 0.400 Table 3: Respective data of dilution, absorbance, suitability of spectroscopy analysis, mass of protein and protein concentration with different types of protein used. Types of Proteins Dilution Absorbance Suitability of spectroscopy analysis Mass of Protein Protein Concentration Conalbumin Undiluted 0.902 No - 36.57 1/10 0.147 Yes 3.657 Ovalbumin Undiluted 0.900 No - 44.03 1/10 0.177 Yes 4.403 Lysozyme A Undiluted 0.795 No - 21.14 1/10 0.085 Yes 2.114 Lysozyme C Undiluted 0.064 Yes 1.592 1.592 1/10 0.009 Yes 0.224 Lysozyme D Undiluted 0.048 Yes 1.194 1.194 1/10 0.008 Yes 0.199 Lysozyme B Undiluted 0.820 No - 37.31 1/10 0.150 Yes 3.731 1/100 0.122 Yes 3.035 Egg White 1/3 0.647 No - 326.87 1/30 0.438 Yes 10.896 Table 4: Records of results including volume, total mass and percentage obtained for each protein Volume collected after isolation, ml Total mass of protein collected after isolation in 1ml, mg Total mass of protein collected after isolation in whole egg, mg Percentage of protein contained in egg white, % Conalbumin 15 36.57 438.84 2.4 Ovalbumin 19.5 44.03 814.55 4.49 Lysozyme A 132.5 21.14 3382.40 18.63 Lysozyme B 123 37.31 4775.68 26.30 Lysozyme C 75 2.24 176.96 0.97 Lysozyme D 61 1.99 121.39 0.67 Egg White 64 330.18 18159.9 100 *Conalbumin and Ovalbumin protein were obtained from the dialysis reading in week 1's result table * Volume Lysozyme A, B, C, D were recorded in week's result table Calculations: From the standard curve, the equation Absorbance = 0.0402(mass of BSA); Mass of Conalbumin (diluted 1/10) ...read more.

Conclusion

0.159 0.056 0.25 0.072 160 0.159 0.053 0.247 0.067 170 0.159 0.049 0.246 0.063 180 0.159 0.046 0.244 0.06 190 0.159 0.043 0.239 0.056 200 0.159 0.042 0.234 0.054 210 0.159 0.04 0.23 0.051 220 0.159 0.038 0.225 0.048 230 0.159 0.038 0.225 0.048 240 0.159 0.038 0.225 0.048 Figure 1: Graph of Absorbance Reading vs. Time for Lysozyme A, B, C and D Table 7: Lysozyme A Lysozyme B Lysozyme C Lysozyme D Point 1 (X1,Y2) (0, 0.304) (0, 0.304) (0,0.304) (0,0.304) Point 2 (X1,Y2) (0.8, 0.03) (1.2, 0.07) (1.5, 0.12) (1.9, 0.26) Activity in 0.5ml, abs/min -0.342 -0.195 -0.123 -0.023 Activity in 1mL, abs min-1/mL -0.685 -0.390 -0.246 -0.046 Activity in 1ml of undiluted sample abs min-1/ml -6.850 n/a n/a -0.460 Amount of protein in 1mL, mg 21.14 37.31 2.24 1.990 Specific activity abs min-1 mL-1 mg -0.324 -0.010 -0.109 -0.231 Total volume, mL 132.500 123.000 75.000 61.000 Total activity abs/min -907.62 -47.97 -18.5 -28.06 Table 8: Tube 1(blank) 2 3 4 5 6 7 8 9 10 0.001M FeCl3, ml - 0 0.08 0.125 0.25 0.375 0.5 0.75 1.0 1.5 FeCl3 content in �moles - 0 0.08 0.125 0.25 0.375 0.5 0.75 1 1.5 Iron binding buffer,ml - 1 1 1 1 1 1 1 1 1 Conalbumin 10mg/ml - 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 H2O 6.000 2.000 1.920 1.875 1.750 1.625 1.500 1.250 1.000 0.500 Absorbance 450nm 0.000 0.018 0.024 0.032 0.045 0.058 0.069 0.087 0.094 0. ...read more.

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