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BIOCHEM LAB > ISOLATION & HYDROLYSIS OF PROTEINS > Flashcards

ISOLATION & HYDROLYSIS OF PROTEINS Flashcards

1
Q

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

A
  • Proteins
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2
Q

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

A
  • Denaturation
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3
Q

give an example of albumin

A
  • Egg white
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4
Q

enumerate non-polar amino acids

A

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

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5
Q

enumerate polar amino acids

A

o Serine
o Threonine
o Tyrosine
o Asparagine
o Glutamine

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6
Q

enumerate positively charged amino acids

A

o Lysine
o Arginine
o Histidine

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7
Q

enumerate negatively charged amino acids

A

o Aspartic acid
o Glutamic acid

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8
Q

REAGENTS & MATERIALS used for CASEIN

A
  • Powedered, non-fat milk
  • 10% CH COOH
  • Thermometer
  • pH meter/indicator
  • Funnel
  • Filter Paper
  • Hot plate
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9
Q

REAGENTS & MATERIALS used for MYOGLOBIN

A
  • Minced beef (or steak)
  • Cheesecloth
  • Centrifuge
  • Centrifuge tubes
  • (NH4) 2SO crystals
  • 70% buffer-diluted
  • (NHa)SO solution, ph 7.5
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10
Q

REAGENTS & MATERIALS used for ALBUMIN

A
  • Filtrate from the isolation of casein
  • Thermometer
  • Hot plate
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11
Q

REAGENTS & MATERIALS used for GLUTEN

A
  • 1 cup of wheat flour
  • Cheesecloth
  • 0.01 M iodine solution
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12
Q

PROCEDURE FOR: CASEIN FROM SKIMMED MILK

A
  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.
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13
Q

PROCEDURE FOR: ALBUMIN FROM SKIMMED MILK

A
  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.
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14
Q

PROCEDURE FOR: GLUTEN FROM WHEAT FLOUR

A
  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.
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15
Q

PROCEDURE FOR: MYOGLOBIN FROM MUSCLE

A
  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.
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16
Q

PROCEDURE FOR: ACID HYDROLYSIS OF PROTEIN

A
  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.
17
Q

TEMPERATURE FOR AUTOCLAVE

A

121 °C

18
Q

PROCEDURE FOR: ACID HYDROLYSIS OF PROTEIN

A
  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.
19
Q

PROCEDURE FOR: ENZYMATIC HYDROLYSIS OF PROTEIN

A
  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.
20
Q

TEST OBJECTIVE FOR ISOLATION OF CASEIN

A
  • To isolate casein by using acid to adjust the pH to 4.6, the isoelectric pH of casein
21
Q

ISOLATION OF CASEIN: PRINCIPLE

A
  • 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.
22
Q

TEST OBJECTIVE FOR ISOLATION OF ALBUMIN

A
  • To detect the presence of albumin through a process of denaturation by heat.
23
Q

ISOLATION OF ALBUMIN: PRINCIPLE

A
  • Albumins are globular proteins that are soluble in water and in dilute salt solutions. They are, however, denatured and coagulated by heat.
24
Q

ISOLATION OF GLUTEN: TEST OBJECTIVE

A
  • To isolate gluten from the starch of wheat flour
25
Q

ISOLATION OF GLUTEN: PRINCIPLE OF THE TEST

A
  • Gluten is isolated from wheat flour by washing the dough with water. This process removes the starch: the insoluble material is gluten.
26
Q

ISOLATION OF MYOGLOBIN: TEST OBJECTIVE

A
  • To isolate gluten from the muscle
27
Q

ISOLATION OF MYOGLOBIN: PRINCIPLE OF THE TEST

A
  • 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.
28
Q

color of:
- Myoglobin
- Hemoglobin

A
  • Myoglobin: clear red
  • Hemoglobin: hazy red
29
Q

why hemoglobin is hazy

A

because hemoglobin contains denatured RBC)

30
Q

HYDROLYSIS OF PROTEIN: TEST OBJECTIVE

A
  • To hydrolyze intact protein by treating it chemically or enzymatically
31
Q

HYDROLYSIS OF PROTEIN: PRINCIPLE OF THE TEST

A
  • 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.
32
Q

lysis or -lytic means …

A

breaking down of bonds

BIOCHEM LAB (1 decks)

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