ISOLATION & HYDROLYSIS OF PROTEINS

Question 1

are essential constituents of all cells known as polymers of amino acids

Answer 1

• Proteins

Question 2

is the disruption of the native protein conformation affecting its structure and function

Answer 2

• Denaturation

Question 3

give an example of albumin

Answer 3

• Egg white

Question 4

enumerate non-polar amino acids

Answer 4

o Glycine
o Alanine
o Valine
o Cysteine
o Proline
o Leucine
o Isoleucine
o Methionine
o Tryptophan
o Phenylalanine

Question 5

enumerate polar amino acids

Answer 5

o Serine
o Threonine
o Tyrosine
o Asparagine
o Glutamine

Question 6

enumerate positively charged amino acids

Answer 6

o Lysine
o Arginine
o Histidine

Question 7

enumerate negatively charged amino acids

Answer 7

o Aspartic acid
o Glutamic acid

Question 8

REAGENTS & MATERIALS used for CASEIN

Answer 8

• Powedered, non-fat milk
• 10% CH COOH
• Thermometer
• pH meter/indicator
• Funnel
• Filter Paper
• Hot plate

Question 9

REAGENTS & MATERIALS used for MYOGLOBIN

Answer 9

• Minced beef (or steak)
• Cheesecloth
• Centrifuge
• Centrifuge tubes
• (NH4) 2SO crystals
• 70% buffer-diluted
• (NHa)SO solution, ph 7.5

Question 10

REAGENTS & MATERIALS used for ALBUMIN

Answer 10

• Filtrate from the isolation of casein
• Thermometer
• Hot plate

Question 11

REAGENTS & MATERIALS used for GLUTEN

Answer 11

• 1 cup of wheat flour
• Cheesecloth
• 0.01 M iodine solution

Question 12

PROCEDURE FOR: CASEIN FROM SKIMMED MILK

Answer 12

1. Place 20.0 g of powdered non-fat milk and 50.0 ml of water into a 100-ml beaker. Mix well.
2. Heat the mixture about 40 °C
3. When the mixture reaches 40 °C, add 10% CHCOOH dropwise. Stir the solution gently after every 5 drops.
4. Continue adding CHCOOH until the pH reaches 4.6 (acidic).
5. Filter of the congealed casein by gravity (or vacuum) filtration. Set aside the filtrate for the isolation and quantitative analysis of albumin.
6. Dry the casein residue and calculate the weight % of casein isolated from the powdered milk.

Question 13

PROCEDURE FOR: ALBUMIN FROM SKIMMED MILK

Answer 13

1. Transfer half of the filtrate to a small beaker. Set aside the remaining half for quantitative protein analysis.
2. Heat the filtrate in the beaker to 57 °C for about 5 minutes using a hot plate.
3. Decant off the liquid from the precipitated albumin.

Question 14

PROCEDURE FOR: GLUTEN FROM WHEAT FLOUR

Answer 14

1. Add enough water to one cup of wheat flour to make a thick dough.
2. Wrap the dough with the cheesecloth. Place the dough under running water until the starch is removed. Test the dough washings with an iodine solution until a negative result is obtained.
3. Collect the gluten (the insoluble material) for hydrolysis and qualitative protein analysis.

Question 15

PROCEDURE FOR: MYOGLOBIN FROM MUSCLE

Answer 15

1. Place 6.0 g of minced beef (or steak) and 6 ml of the 70% (NH4)2SO2 solution in a small beaker.
2. Gently stir the mixture for 1 minute to release the myoglobin.
3. Express the dark-red extract into a new beaker using the cheesecloth.
4. Centrifuge the extract at 13, 000 x g for 5 minutes.
5. Transfer 1.5 ml of the supernatant into another empty centrifuge tube.
6. Add ~0.3-0.35 g of the (NH4)2SO2 crystals ground to fine powder. Mix gently until the solid dissolves. Avoid frothing (overflowing).
7. Centrifuge the sample again for at least 5 minutes.
8. Decant of the supernatant. Describe the appearance of the isolated myoglobin residue.

Question 16

PROCEDURE FOR: ACID HYDROLYSIS OF PROTEIN

Answer 16

1. Add 5 ml of 6 M HCl to 0.5 g of the isolated protein in a hard glass test tube.
2. Cover the tube with cotton and submit it to the instructor for autoclaving at 15 psi (pounds per square inch) for 5 hours. Note: Alternatively, the protein sample may be placed in a sealed container containing 6 M HCl. The whole container is then placed in a microwave temperature up to 5 -30 minutes (depending on the protein) with temperatures up to 200 °C. The hydrochloric acid vaporizes, comes in contact with the protein sample, and hydrolyses it.
3. Take note of the appearance of the mixture after autoclaving. Add 10 ml of distilled water. Transfer the mixture into a 250-ml beaker.
4. Neutralize the mixture with 1M NaOH. Use the neutralized mixture as a sample for the characterization tests and chromatography.

Question 17

TEMPERATURE FOR AUTOCLAVE

Answer 17

121 °C

Question 18

PROCEDURE FOR: ACID HYDROLYSIS OF PROTEIN

Answer 18

1. Add 10 ml of the 4 M NaOH to 0.5 g isolated protein in a hard glass test tube. Label the tube.
2. Cover the tube with cotton and submit it to the instructor for autoclaving (15 psi for 5 hours).
3. Take note of the appearance of the mixture after autoclaving. Add 10 ml of distilled water. Transfer the mixture into a 250-ml beaker.
4. Neutralize the mixture with 1M HCl. Use the neutralized mixture as a sample for the characterization tests and chromatography.

Question 19

PROCEDURE FOR: ENZYMATIC HYDROLYSIS OF PROTEIN

Answer 19

1. Prepare 1 g / 100 ml of distilled water protein mixture.
2. Mix 10 ml of the protein mixture and 10 ml of the saturated protease solution. Alternatively, 0.050g of protease may be added directly to 50 ml of the protein.
3. Add 10 ml of the 0.1 M phosphate buffer, pH 7.5.
4. Incubate the tube in a water bath with a temperature range of 35 °C – 40 °C (depending on the source of the enzyme) for 60 minutes. Alternatively, digestion may be carried out overnight at room temperature.
5. Allow the mixture to cool before using it in the procedures for qualitative color reactions on pages 23-25 and for the separation and identification of amino acids by thin-layer chromatography on pages 26-27.

Question 20

TEST OBJECTIVE FOR ISOLATION OF CASEIN

Answer 20

• To isolate casein by using acid to adjust the pH to 4.6, the isoelectric pH of casein

Question 21

ISOLATION OF CASEIN: PRINCIPLE

Answer 21

• Caseins, lactalbumins, and lactose globulins are globular proteins found in milk. Casein exists in milk as calcium caseinate.
• Proteins tend to be insoluble at their isoelectric point, acetic acid can be used for isolation from their natural sources. On addition of acid, the negative charges on casein micelles are neutralized, by protonation of the phosphate groups and thus casein precipitates out.

Question 22

TEST OBJECTIVE FOR ISOLATION OF ALBUMIN

Answer 22

• To detect the presence of albumin through a process of denaturation by heat.

Question 23

ISOLATION OF ALBUMIN: PRINCIPLE

Answer 23

• Albumins are globular proteins that are soluble in water and in dilute salt solutions. They are, however, denatured and coagulated by heat.

Question 24

ISOLATION OF GLUTEN: TEST OBJECTIVE

Answer 24

• To isolate gluten from the starch of wheat flour

Question 25

ISOLATION OF GLUTEN: PRINCIPLE OF THE TEST

Answer 25

• Gluten is isolated from wheat flour by washing the dough with water. This process removes the starch: the insoluble material is gluten.

Question 26

ISOLATION OF MYOGLOBIN: TEST OBJECTIVE

Answer 26

• To isolate gluten from the muscle

Question 27

ISOLATION OF MYOGLOBIN: PRINCIPLE OF THE TEST

Answer 27

• Myoglobin and hemoglobin are vital to oxygen transport and storage in vertebrates. The former is present in large concentrations in muscles and is responsible for the red color of the tissue. Myoglobin can be isolated by using ammonium sulfate precipitation on the buffered muscle extract.

Question 28

color of:
- Myoglobin
- Hemoglobin

Answer 28

• Myoglobin: clear red
• Hemoglobin: hazy red

Question 29

why hemoglobin is hazy

Answer 29

because hemoglobin contains denatured RBC)

Question 30

HYDROLYSIS OF PROTEIN: TEST OBJECTIVE

Answer 30

• To hydrolyze intact protein by treating it chemically or enzymatically

Question 31

HYDROLYSIS OF PROTEIN: PRINCIPLE OF THE TEST

Answer 31

• Hydrolysis of the protein and analysis of the products are done to obtain information about its composition.
• Proteases (peptidases or proteolytic enzymes) occur naturally in all organisms.
• Currently, there are six classes of peptidases specific to either the N- or C-side of the following amino acid residues: serine, threonine, cysteine, aspartate, glutamate, and metallopeptidases.

Question 32

lysis or -lytic means …

Answer 32

breaking down of bonds