Experiment No. 1 EXTRACTION AND CHARACTERIZATION OF PROTEINS Methodology Materials: 95% ethanol 0.3 M sucrose 2% glucose 2% fructose 2.

0 M HCl 2.0 M M NaOH 10% NaOH 0.01 M NaOH 1.0 M HOAc saturated (NH4)2SO4 solution hexane petroleum ether 0.1 M acetate buffer pH 5 5.0% NaCl 0.9% NaCl Equipment: Top loading balance Centrifuge Spectrophotometer Procedure I. Extraction of Proteins A. Invertase from Yeast 1. Immerse a clean 50-mL beaker and 150-ml 95% ethanol in an ice bath. 2. Grind 10 g bakers yeast with 5 g of sand in a clean mortar and pestle until a fine powder is obtained. Add 10 mL hexane to the fine powder. 3. Add 30 mL of water in 3 mL portions and continue grinding for another 10-15 minutes. 4. Filter the ground yeast through cheesecloth to obtain a cell-free extract. 5. Centrifuge the filtrate at 6000 rpm for 5 minutes. Discard the sediment. Repeat centrifugation step until supernatant is clear. (Use longer time if necessary.) 6. Pour the supernatant into the pre-cooled beaker. Slowly add the cold 95% ethanol to equivalent to 4 times the volume of the extract. Do not stir. Set aside in the ice bath until precipitation occurs. 7. Centrifuge the resulting suspension at 3000 rpm for 10 minutes. Discard the supernatant. 8. Transfer the crude extract to a pre-weighed filter paper. Air dry under the hood. Determine the weight of the solid. Have the crude extract checked by the instructor before proceeding to the next step. 9. Dissolve the precipitate in enough 0.1 M acetate buffer pH 5 to make 10% (w/v) solution. 10. Place the solution a well-sealed container and keep in the refrigerator if it cannot be used for part II on the same laboratory session. B. Albumin from Egg

0.1 M HCl Benedicts reagent pH paper 0.1 mg/mL standard BSA solution 0.1 mg/mL standard casein solution 0.1 mg/mL standard globulin solution Bradford Dye Reagent Ice Cheesecloth Bakers yeast Fine sand Eggs Milk Squash/cucumber seeds

1. Immerse a 250-mL Erlenmeyer flask in an ice bath. 2. Measure out 30 mL of egg white and place in beaker. 3. Gently stir the egg whites with a stirring rod to break up the membranes but avoid frothing). 4. Filter the sample through a damp cheesecloth into a 250-mL beaker. Use a stirring rod to press the cloth against the side of the funnel to further break the membranes and release the filtrate. 5. Note the volume of the sample and to each nine volumes, slowly add one volume 1.0 M HOAc with constant stirring. The pH of the mixture should be about 5. Otherwise, add more HOAc dropwise until the desired pH is attained. 6. Centrifuge the mixture at 3000 rpm for 10 minutes and discard the precipitate. 7. Transfer the supernatant to the previously cooled 250-mL Erlenmeyer flask. 8. While continuously stirring the clear yellow supernatant, slowly add along the sides of the flask enough saturated ammonium sulfate to bring the material to 40% saturation. 9. Allow the mixture to stand for about 30 minutes in an ice bath. 10. Centrifuge the mixture. Discard the resulting supernatant and collect the precipitate in a pre-weighed watch glass. Air dry under the hood. Record the weight of the dry precipitate. Have the instructor check the crude extract. 11. Place the precipitate in a well-sealed container and place in the refrigerator if it cannot be used for part II on the same laboratory session. C. Casein from Milk 1. Measure out 20 mL of non-fat milk into a 100-mL beaker. Use a pH paper to test the initial pH of the milk sample. 2. Add 0.1 M HCl drop wise to the sample over a period of 10 minutes until a flocculent precipitate forms. Measure the acidity of the mixture occasionally with a pH paper and the pH must decrease to just about 4.8. Avoid adding excess acid. 3. Centrifuge the mixture at 3000 rpm for 10 minutes and discard the supernatant. 4. Wash the curd by resuspending it in enough cold 95% ethanol. Mix thoroughly to wash the residue. Decant the ethanol washings. Wash the residue with ethanol repeatedly until the ethanol washing is clear. 5. Transfer the moist residue into a pre-weighed filter paper, wash with acetone into a funnel and air dry under the hood. Determine the mass of the crude extract and have it checked by the instructor. 6. Place the precipitate in a well-sealed container and place in the refrigerator if it cannot be used for part II on the same laboratory session. D. Globulin from Squash/Cucumber Seeds 1. Immerse two clean 250-mL beakers and 100-ml each of 2M NaOH and 2M HCl in an ice bath. 2. Wash 50 g of seeds thoroughly with distilled water. Pat dry and press between filter paper to remove most of the water washing. 3. Grind the seeds in a clean mortar and pestle until a fine powder is obtained. Add 50 mL hexane or petroleum ether to the fine powder. Mix vigorously using a stirring rod. 4. Filter the mixture through a cheesecloth. Collect the residue in an evaporating dish and air dry the material under the hood for 30 minutes. 5. Place the air-dried residue into the previously cooled 250-mL beaker and add 100 mL water. Place the beaker in a magnetic stirrer and stir the mixture for an hour at RT. 6. Adjust the pH of the mixture to 9 by adding cold 2M NaOH drop by drop. Stir for

another hour at RT and adjust the pH to 9, if necessary. 7. Filter the material through cheese cloth. Collect the filtrate in the 2nd 250-ml beaker. 8. Adjust the pH of the filtrate to 4.5 by adding cold 2M HCl. Let the mixture stand in an ice bath for an hour or until precipitation is complete. (Alternatively, the air-dried residue from step 4 can be processed by salt extraction with 100 mL 5% NaCl but this will require stirring the mixture for 4 h at RT, instead of using steps 6,7,8) 9. Centrifuge the suspension at 10,000 rpm for 15 mins. 10. Collect the residue in a previously weighed filter paper and wash with a minimal amount of acetone. Let it air dry under the hood. Determine the mass of the crude extract and have it checked by the instructor. 11. Place the precipitate in a well-sealed container and place in the refrigerator if it cannot be used for part II on the same laboratory session. II. Characterization A. Activity Assay for Invertase 1. Take out the stock 10% (w/v) invertase solution from the refrigerator and immerse it in an ice bath. 2. Prepare a set of four test tubes according to the table below: Volume, mL Test tube # 0.3 M H2O Acetate buffer 2% glucose 2% fructose sucrose 1 6.0 4.0 6.0 ----2 6.0 8.0 6.0 ----3 --8.0 6.0 1.0 1.0 4 --10.0 6.0 ----3. Place the test tubes in a water bath maintained at 37C. Allow to equilibrate for 5-10 minutes. 4. Add 4mL of invertase extract to test tubes 1, 3 and 4. Keep tubes in the water bath for 6 more minutes. 5. Stop the reaction by adding 2 mL 10% NaOH to the test tubes. 6. Test for the presence of reducing sugars using Benedicts reagent.* (Recall protocol for Benedicts test in Organic Chemistry) B. Quantification of Protein Concentration by Spectrophotometry 1. Warburg- Christian Method Sample preparation: i. Prepare 20.0 mL of 10% (w/v) solution of albumin in 0.9% NaCl. ii. Prepare 20.0 mL of 1% (w/v) casein solution using 0.01 M NaOH. iii. Prepare 20.0 ml of 1% (w/v) globulin solution in distilled water. Spectrophotometric Analysis i. Measure the absorbance of the protein extracts at 280 and 260 nm against the following blanks: 0.9% NaCl for albumin, 0.01 M NaOH for casein and distilled water for globulin. Compute the A280/A260 ratio and roughly estimate the purity of the protein isolate by subtracting the corresponding %nucleic acid of the computed ratio from 100%. Refer to the table below. A280/A260 % Nucleic Acid A280/A260 % Nucleic Acid 1.75 0.00 0.87 5.00 1.63 0.25 0.85 5.50

1.52 1.40 1.36 1.30 1.25 1.16 1.09 1.03 0.98 0.94

0.50 0.75 1.00 1.25 1.50 2.00 2.50 3.00 3.50 4.00

0.82 0.80 0.78 0.77 0.75 0.73 0.71 0.67 0.64 0.62 0.60

6.00 6.50 7.00 7.50 8.00 9.00 10.00 12.00 14.00 17.00 20.00

ii. Calculate the protein concentration in the crude extracts using the following equation: Protein concentration (mg/mL) = {1.55 A280 - 0.76 A260} x dilution factor 2. Bradford Assay i. Set up 12 test tubes and add protein standards and protein extracts according to the table below. (Tube 1 is used as blank and tubes 2 through 6 for the construction of a standard curve for protein quantitation. Tubes 7 to 12 are duplicates of the three different concentrations of the isolates.) Test tube Standard protein Protein extract Distilled H2O Bradford reagent 1 0.0 --9.8 0.2 2 0.2 --9.6 0.2 3 0.4 --9.4 0.2 4 0.6 --9.2 0.2 Volume, mL 5 6 0.8 --9.0 0.2 1.0 --8.8 0.2 7 --0.3 9.5 0.2 8 --0.3 9.5 0.2 9 --0.5 9.3 0.2 10 --0.5 9.3 0.2 11 --0.7 9.1 0.2 12 --0.7 9.1 0.2

ii. Add enough distilled water to each tube according to the table. iii. Add 0.2 mL Bradford reagent to each test tube and mix thoroughly. iv. After 5 minutes, read the absorbance of each tube at 595 nm. The tubes should be read within an hour after the addition of the dye. v. Construct a calibration curve and determine the protein concentration of the crude extract. vi. If the crude extracts have absorbance readings outside the range established by the standard curve, dilute the extracts with water and redo the absorbance measurements at 595 nm. Consider the dilution factors when calculating for the actual protein concentration of the extracts. POST-LAB QUESTIONS 1. What principle is involved in the extraction of the following? a. invertase b. albumin c. casein 2. State the purpose/s of each step/reagent used in the extraction procedure that your group performed.

3. Compare & contrast the Warburg-Christian method from the Bradford assay as protein concentration determination techniques. Use your results to explain which technique is more efficient. 4. Classify the proteins the proteins studied in this experiment and list their function/s. 5. Show calculations for the concentration of the protein extracted by your group. 6. Tabulate the calculated results obtained by the whole class. Reference Biochemistry Laboratory Manual, UP Diliman, 2002, 24-29.