Biochem Lab 8 - Protein Purification

Biochemistry Lab 8: Protein Purification Introduction Experiments involving the study of proteins usually require pure samples of protein in order to ensure that the results are due to the effect of the protein and not due to the presence of contaminants. Because of this, protein purification, the separation of one specific protein from all other large and small biomolecules, is often performed on the basis of differences of physical and chemical properties between proteins. In this lab, we performed the extraction and purification of lysozyme enzyme from egg white using centrifugation and CM-cellulose chromatography. Lysozyme, which causes the lysis of bacteria, is a relatively small protein enzyme with an usually high isoelectric point of pH 11.0; therefore, we purified the homogenized egg white on the basis of lysozyme’s solubility and pI. At each step of the purification, we used the nephelometric assay to measure lysozyme activity and calculated the specific activity, purification factor, and percent yield. Materials & Methods In this lab, we first homogenized 3.0 mL of egg white with 21 mL of cold 0.117 M glycine-NaOH buffer, pH 9.5 with a cold Teflon-glass homogenizer. We poured the mixture into a 50 mL orange-capped tube and mixed by inverting. We centrifuged the homogenate at 5000 x g for 5 minutes and decanted the supernatant solution into a clean 50 mL orange-capped tube. We measured the volume of the supernatant. For the CM-cellulose chromatography, we used a transfer pipet to load 6 mL volumes onto the pre-packed CM-cellulose column equilibrated in 0.100 glycine buffer, pH 9.5. We loaded 6 mL volumes of the sample onto the column and collected the flow-through in test tubes labeled F1 through F4. We then washed the column with 6 mL volumes of 0.100 glycine-NaOH buffer, pH 9.5 and collected the flow-through in test tubes labeled F5 through F9. We eluted the column with 6 mL volumes of 0.100 glycine-NaOH buffer, pH 10.5, 0.3 Na2CO3 and collected the elutions in test tubes labeled E1 through E4. We measured the A280 of the column fractions using the UV Spec 21 Spectrophotometer. At each step of the purification, we also measured the lysozyme activity using the nephelometric assay. Since lysozyme lyses bacterial cells in suspension, which decreases the turbidity, we measured the decrease in the A450 of 3 mL solutions of protein sample, dilution buffer (0.100 M NaPO4 buffer, pH 7.0), and bacterial suspension over two minutes using a Spec 20 Visible Spectrophotometer, making sure that the change per minute was between 0.02 and 0.05. Results 1. Table 2: Lysozyme Purification data Lysozyme Activity Fraction 1. Egg White Homogenate 2. Supernatant CM Column Fraction, 0.1 M glycine buffer, pH 9.5 CM Column Fraction, 0.1 M glycine buffer, pH 10.5 Dil 0 min 0.5 min 1 min 20 0.72 0.69 8 0.690 20 0.74 0.73 0.71 1 4 3 4 5 4 7 8 - E1 1 E2 1 E3 2 E4 4 0.44 3 0.42 5 0.34 0 0.28 2 - 0.32 5 0.27 5 - 0.19 5 0.18 9 0.59 0 0.50 0 0.44 1 0.58 4 0.48 0 0.41 5 0.412 0.313 0.264 0.185 0.580 0.452 0.394 A280 for CM column 1.5 min 2 min 0.68 5 0.70 0.67 8 0.70 0.39 9 0.38 4 0.30 4 0.25 8 - 0.29 6 0.25 8 - 0.18 1 0.17 8 0.57 9 0.43 0 0.37 5 0.57 5 0.40 7 0.35 2 Average ΔA450/min per 0.1 mL Units/mL 0.021 4200 0.020 Protein Conc. Biuret for 1 &2 4000 0.0295 1180 0.011 0.022 880 1.08 0.012 480 1.11 - - 1.26 0.743 0.0085 85 0.054 0.0075 75 0.020 0.0465 930 0.233 0.0445 1780 0.673 Table 3: Purification of Lysozyme from Egg White Enzyme Fraction Fraction Volume (mL) Enzyme Activity+ (units/mL) Enzyme Units Total Egg White Homogenate 24 4200 100800 Supernatant 21 4000 84000 Protein Concentration (mg/mL) Protein Total (mg) Specific Activity (units/mg) Purification Factor Yield (% units of starting material) 1 100% 83.3% CM-column peak 6 1780 10680 0.461 2.766 38611.71 10.6% 2. Sample calculations (for CM-column peak): 0.441−0.352 =0.0445Δ A450/minper0.1mL 2 min 0.0445ΔA450 ∗1lysozymeunit min per0.1mL =44.5lysozymeunits per 0.1mL Δ A450 0.001 min 44.5lysozymeunits=445lysozymeunits/mL 0.1mL 445lysozymeunits ∗4 dilutionfactor=1780lysozymeunits/mLundiluted mLdiluted 6 mL∗1780lysozymeunits =10680totalunits 1 mL % Yield= 10680units ∗100%=10.6% yield 100800units A=εcl ( 0.673= 1.46 c=0.461 ) mL c (1 cm) mg∗cm mg mL 6 mL∗0.461 mg =2.766mg mL Specific Activity= 106800units =38611.71units/mg 2.766mg 3. Fraction Number 1 3 5 7 8 A280 Enzyme Units/mL 0.011 1.08 1.11 1.26 0.743 1180 880 480 - E1 E2 E3 E4 0.054 0.020 0.233 0.673 85 75 930 1780 Elution Profile of CM-Cellulose Column 1.4 2000 1800 1600 1400 1200 1000 800 600 400 200 0 1.2 1 0.8 A280 0.6 0.4 0.2 0 0 2 4 6 8 10 12 Enzyme Units/mL 14 Fraction Number Conclusion In this lab, we used centrifugation and CM-cellulose chromatography to purify lysozyme from homogenized chicken egg white on the basis of solubility and pI. The lysozyme activity was calculated in units/mL with the assumption that one unit is the amount of lysozyme that will result in an absorbance change per minute of 0.001. We made calculations for three steps in the purification process: egg white homogenate, supernatant, and CM-column peak. The enzyme activity (units/mL), total enzyme units, and percent yield all decreased in each step (see Table 3). The percent yield after centrifugation was 83.3%, but after CM-column chromatography, the yield was only 10.6%. This makes sense because some lysozyme enzyme may have been lost in the pellet during centrifugation and some enzyme may have been lost in the CM-cellulose column or in the other elution column test tubes during CM-column chromatography. We used the elution column with the greatest change in absorbance per minute since we assumed that to be the one with the greatest purity of lysozyme; however, the other elution columns may have also contained a significant amount of lysozyme, but with greater impurities. In general, as purity increases, yield decreases. As the protein of interest is separated from the other large and small biomolecules, some of it is lost in the process. For the elution columns, the enzyme activity increased from the first to the last column, which had the highest enzyme activity (see graph of elution profile of CM-cellulose column). This is an unexpected result since it was expected that the second and third columns have the highest enzyme activity. This may be because it was harder to elute the enzyme off the column. Perhaps if we conducted further elutions, we would be able to collect more lysozyme units. This result may have also been due to experimental or instrumental errors; the column may have had cracks, which would have affected the data. Some of the dilutions also did not have absorbance changes in a suitable range, which may have also skewed the data. Generally, the yield decreased while the purification factor should increase.