Exp 2.2: Protein Denaturation, Hydrolysis, and Separation

Question 1

a loss of three-dimensional structure sufficient to cause loss of function

Answer 1

protein denaturation

Question 2

Protein denaturation involves disruption of what levels of structures?

Answer 2

secondary and tertiary
(primary remains intact)

Question 3

Identify the type of denaturation:
biological function/ activity cannot be regained

Answer 3

irreversible denaturation

Question 4

Identify the type of denaturation:
biological function/ activity can be regained

Answer 4

reversible denaturation

Question 5

Determine the denaturing agents:
disrupts ionic interactions

Answer 5

strong acids and bases
heavy metals

Question 6

Determine the denaturing agents:
disrupts hydrophobic interactions

Answer 6

organic solvents
detergents
temperature

Question 7

Determine the denaturing agents:
disrupts disulfide bonds

Answer 7

reducing agents

Question 8

Determine the denaturing agents:
strip off essential layer of water molecule from the protein surface

Answer 8

salts

Question 9

2 methods for salting

Answer 9

1. salting in
2. salting out

Question 10

Identify the method of salting:
low salt concentration: increase solubility of solute
(increasing ionic strength)

Answer 10

salting in

Question 11

Identify the method of salting:
high salt concentration: decrease solubility of solute
(non-electrolyte could be less soluble)

Answer 11

salting out

Question 12

Determine the denaturing agents:
disrupts
1. hydrogen bonds
2. hydrophobic interactions

Answer 12

temperature

Question 13

Complete Hydrolysis:
components used
product/s
types

Answer 13

components used: strong acid/base and high temp
product/s: amino acids
types: acid and alkaline

Question 14

Acid Hydrolysis:
reagent
disadvantages

Answer 14

reagent: 6N HCl
disadvantages:
1. Cys & Tyr: partial destruction
2. Trp: complete destruction
3. Val & Ile: incomplete destruction
4. Ser & Thr: racemization & destruction
5. Asn+Gln: converted to Asp+Glu

Question 15

Alkaline Hydrolysis:
reagent
advantage
disadvantage

Answer 15

reagent: NaOH or KOH
advantage: Trp - not destroyed
disadvantage: Arg, Asn, Gln, Ser - destroyed

Question 16

Incomplete/ Partial Hydrolysis:
components used
product/s
types

Answer 16

components used: enzymes (protease)
product/s: amino acids + oligopeptides
type: enzymatic

Question 17

Identify the type of hydrolysis:
presence of proteolytic enzymes results to partial or selective hydrolysis of polypeptide to yield a mixture of peptide fragments

Answer 17

enzymatic hydrolysis

Question 18

enzymes that hydrolyze peptide bonds at specific sites

Answer 18

proteases/ peptidases

Question 19

Enzymatic Hydrolysis:
reagent
advantages

Answer 19

reagent: protease
advantage: amino acids - not affected;
it requires certain temp & pH conditions for optimum activity of enzymes

Question 20

Enzymatic Hydrolysis:
2 types of enzymes

Answer 20

1. exopeptidases
2. endopeptidases

Question 21

Identify the enzyme:
cleavages at the terminal peptide

Answer 21

exopeptidases

Question 22

Identify the enzyme:
cleavages at the non-terminal peptide

Answer 22

endopeptidases

Question 23

3 types of exopeptidases

Answer 23

1. Carboxypeptidase A
2. Carboxypeptidase B
3. Aminopeptidase

Question 24

3 types of endopeptidases

Answer 24

1. Trypsin
2. Chymotrypsin
3. Papain

Question 25

Identify the specificity: Carboxypeptidase A

Answer 25

C-terminal residues except R, K, and P

Question 26

Identify the specificity: Carboxypeptidase B

Answer 26

R or K at C-terminus

Question 27

Identify the enzyme: Aminopeptidase

Answer 27

most N-terminal except when P is the next residue

Question 28

Identify the specificity: Trypsin

Answer 28

C-side R and K

Question 29

Identify the specificity: Chymotrypsin

Answer 29

C side of F, Y, and W

Question 30

Identify the specificity: Papain

Answer 30

C side of hydrophobic groups/ aromatic-R groups

Question 31

6 properties of proteins being considered in separation or purification

Answer 31

1. charge 2. molecular size 3. shape 4. solubility 5. affinity to a ligand 6. pI/ IpH

Question 32

a procedure in which the pH of the protein mixture is adjusted to the pI of the protein to be isolated to selectively minimize its solubility

Answer 32

isoelectric precipitation

Question 33

Enumerate proteins in cow milk

Answer 33

1. Casein a. alpha-s1 b. alpha-s2 c. beta d. kappa 2. Whey proteins a. alpha-lactalbumin b. beta-lactoglobulin c. serum albumin d. immunoglobulins e. other proteins

Question 34

Identify the protein: present as micelles in milk and acts as a storage for amino acids

Answer 34

casein

Question 35

Identify the protein: major protein component of cow's milk

Answer 35

casein

Question 36

Casein: function source isolation method composition shape isoelectric point

Answer 36

function: storage of amino acids source: skimmed milk isolation method: isoelectric precipitation composition: conjugated (Ca & Ph) shape: globular isoelectric point: pH 4.6

Question 37

Identify the protein: globular proteins that are soluble in water and diluted salt salutions

Answer 37

albumin

Question 38

Identify the protein: second major protein in bovine milk

Answer 38

albumin

Question 39

Albumin: function source isolation method composition shape isoelectric point

Answer 39

function: regulating lactose biosynthesis source: skimmed milk isolation method: denaturation and coagulation by heat composition: conjugated (metalloprotein) shape: globular isoelectric point: pH 4.3

Question 40

Myoglobin: function source isolation method principle composition shape

Answer 40

function: storage of oxygen in muscle and tissues source: beef muscle isolation method: salt-induced precipitation principle: salting in, salting out composition: conjugated (iron & oxygen) shape: globular

Question 41

Identify the protein: small, bright red protein common in muscle cells

Answer 41

myoglobin

Question 42

Identify the protein: a hemoprotein containing a heme group at its center

Answer 42

myoglobin

Question 43

Isolated myoglobin: precipitate color

Answer 43

white

Question 44

gluten: function source isolation method composition shape principle in isolation test

Answer 44

function: dough's strength and elasticity (structure) source: wheat flour (wheat, rye, barley) isolation method: solubility difference composition: simple shape: fibrous and globular (glutenin & gliadin) principle in isolation: - starch: partially soluble in H2O -gluten: insoluble in H2O test: iodine test

Question 45

Gluten: 2 major classes of storage proteins (composites)

Answer 45

1. prolamin 2. glutelin

Question 46

Gluten: 2 major proteins

Answer 46

1. gliadin 2. glutenin

Question 47

Test for isolated gluten free of starch

Answer 47

iodine test result: negative (-)