Lab Activity 9 Precipitation Of Proteins Lab activity

Lab Activity 9 Precipitation Of Proteins

: Lab activity to day 1. 2. 3. 4. 5. Effect of strong Acids and Alkali Precipitation by Metallic Salts Precipitation by Alkaloidal Reagents Precipitation by K 4[Fe(CN)6] · 3 H 2 O Fractional Precipitation of Proteins by Conc. Salt solutions 6. Precipitation of Proteins at the Isoelectric Point

1. Effect of strong Acids and Alkali • Reagents: • 1% solution of egg albumin, • conc. HNO 3, H 2 SO 4, HCl, and acetic acids, • conc. Na. OH 3

Procedure 1. Place 2 ml of conc. HNO 3 in a test tube, inlcine the tube and add the dilute albumin slowly from a pipette, allowing the solution to run down the side of the tube and form a layer over the nitric acid. Note the appearance of a protein precipitate at the zone of contact between the two fluids. 2. Now mix the contents of the tube thoroughly by careful shaking. Is protein precipitated by conc. HNO 3? 4

3. Repeat the above steps using conc. H 2 SO 4, conc. HCl, acetic acid, and Na. OH. - Observe what happens in each case, then compare it with the first experiment with HNO 3. 5

2. Precipitation by Metallic Salts • Reagents • 1% Solution of egg albumin, 5% solution Hg. Cl 2, CH 3 COOPb, Cu. SO 4, • 1% solution of Ag. NO 3 6

Procedure 1. Prepare 4 test tubes, each containing 2 to 3 ml of dilute albumin sol. , to the first test tube add Hg. Cl 2 sol. , drop wise slowly until an excess of the reagent has been added. 2. Record your observations. Unless the reagent is added very slowly, the formation of the precipitate may not be noted, due to it’s solubility in excess of the reagent. 3. Repeat the above exp. With CH 3 COOPb, Cu. SO 4, and Ag. NO 3 7

3. Precipitation by Alkaloidal Reagents • Reagents • 1% albumin solution, sat. sol. Of picric acid, 10% trichloroacetic acid solution (TCA), tannic acid sol. , Phosphotungstic acid sol. 8

Procedure 1. Prepare 4 test tubes each containing 2 to 3 ml of albumin sol. 2. To the first test tube add picric acid drop wise, until an excess of the reagent has been added. 3. Note any change that may occur. 4. Repeat the exp. With Trichloroacetic acid sol. , Tannic acid sol. , phosphotungstic acid sol. 5. Are these precipitates soluble in excess of the reagent? (acidify with hydrochloric acid before testing with the last 2 reagents). 9

4. Precipitation by K 4[Fe(CN)6] · 3 H 2 O Reagents 1% Albumin sol. , 1% potassium ferrocyanide sol. 10

Procedure 1. To 5 ml of albumin sol. , in a test tube add 5 to 10 drops of acetic acid, mix well and add potassium ferrocyanide drop by drop, until a precipitate forms. 2. Write down your observation 11

5. Fractional Precipitation of Proteins by Conc. Salt solutions • Reagents • Dilute egg white solution (mix 1 volume of raw egg with 4 volumes of 1 % Na. Cl and filter). • Sat. sol. of (NH 4)2 SO 4, 1% acetic acid solution. 12

Procedure 1. To a portion of the dilute egg-white sol. add an equal volume of a saturated sol. of (NH 4)2 SO 4 and mix. Does the egg white contain a portion which precipitates by half-saturated (NH 4)2 SO 4? 2. If a precipitate is formed, then filter the content, and to the filtrate add an excess of solid (NH 4)2 SO 4 and stir until the solution is saturated with salt. 3. Record what happens? If a precipitate formed, filter and to the filtrate add 2 to 3 drops of 1% acetic acid sol. And heat to boiling. 4. If there is no precipitate formed, it indicates the absence of protein in the last filtrate. 13

6. Precipitation of Proteins at the Isoelectric Point Reagents • Casein powder, • Solutions of: 1 N Na. OH, 1 N CH 3 COOH, H 2 Od, • Volumetric flask 14

Procedure 1. Into a 50 ml volumetric flask add 20 ml of water. 2. Add 0. 25 g of pure casein, followed by the addition of 5 ml of 1 N Na. OH solution. 3. Once casein is dissolved, add 5 ml of 1 N acetic acid solution, then dilute with H 2 O to 50 ml and mix well. The resulted solution is a 0. 1 N casein acetate sodium. 15

4. Setup a series of 9 test tubes as shown in the table below. 5. In the first test tube put 3. 2 ml 1 N CH 3 COOH, and 6. 8 ml H 2 O and mix thoroughly. 6. In each of the other test tubes (2 -9) put 5 ml H 2 Od. 7. From the test tube 1 transfer 5 ml to the test tube 2, and mix thoroughly. 16

8. Repeat step 7 for the rest of test tubes (3 - 9). 9. Now to each test tube (1 -9) add 1 ml of the casein acetate sodium solution, and shake the test tubes immediately. 10. Let the samples stand for 30 min, and note the turbidity in the 9 test tubes. 11. Use + / – signs to describe the turbidity in the different test tubes. 12. You should observe the most precipitation in the test tube which has the p. H around 4. 7 (close to the isoelectric point of casein). 17

TUBE 1 2 1 N CH 3 COO 1. 6 0. 8 H PH 3. 5 3. 8 3 4 5 6 7 8 0. 4 0. 2 0. 1 0. 05 0. 02 0. 01 5 2 0. 00 6 5. 3 5. 6 5. 9 4. 1 4. 4 4. 7 5. 0 9 TURBID ITY 18