LAB(3) - PROTEIN DENATURATION

Question 1

Denaturation

Answer 1

When a protein loses it function because of a change in its secondary,
tertiary, or quaternary structure

Question 2

Denature proteins

Answer 2

have lower solubility and often precipitate from solution.

Question 3

What is the goal of protein folding?

Answer 3

To achieve the LOWEST energy state

Question 4

Can we unfold proteins once they are folded?

Answer 4

• Denatured
• lose most levels of structure
• protein adopts a random coil conformation
• primary amino acid sequence is maintained
• loss of protein function (NATIVE = correctly folded, biologically active state to DENATURED & UNFOLDED = loss of organized structure and function)
• use denaturing agents = interfere with the forces that stabilize protein folding

Question 5

Proteins can be denatured by?

Answer 5

Heat, detergents, mechanical/whipping action and certain acids/bases

Question 6

During digestion, dietary proteins are denatured and then hydrolyzed to form
amino acids as shown here. (STUDY THE PICTURE ON PPT)

Question 7

heat, agitation, and urea, guanidine HCl = H-bonds, hydrophobic interactions

pH = salt bridges

Mercaptoethanol = disulfide bridges

detergents (SDS) = hydrophobic interactions

Question 8

What are the other examples and applications of denaturization? (study the meaning via the ppt)

Answer 8

• protein-containing foods are cooked
• Cauterization
• sterilizing surgical instruments and canning foods
• Casein denatures inside stomach when reacted w/ gastric juice (HCl)
• UV from the sun
• Body temp. above 41 degrees Celsius
  *Cheese being made out of milk by growing lactic acid producing bacteria in skim milk
• yogurt
  *clouded cornea
• isopropyl/ethyl alcohol

Question 9

Denaturation can be reversible

• heat treatment usually is not reversible

Question 10

what did Christian Anfinsen do to win the Nobel Prize in 1972?

Answer 10

renaturation of the protein RIBONUCLEASE (an enzyme that cleaves DNA)

Question 11

Experiment:
1. Denatured pure Ribonuclease
* UREA = breaks H-bonds and hydrophobic interactions
* beta-mercaptoethanol = reduce disulfide bonds

2. removed the denaturants and exposed the protein to air
3. protein folded back into its original 3D shape and activity was restored

Question 12

Amino acid sequence determines protein shape

Question 13

How to determine the composition? (study the meaning via ppt)

Answer 13

• purify the protein of interest
• estimate the molecular weight of the protein
• establish the composition by complete hydrolysis of the protein under acidic conditions

Question 14

How to determine the composition? (study the meaning via ppt)

Answer 14

1. treat w/ 6M HCl at 110 degrees Celsius (12-36)
2. each peptide bond is broken and products are all of the free amino acids
3. each amino acid is separated identified, and quantified
4. final result = know how MANY of each amino acid present in the original

Question 15

Protein hydrolysis
(study more on this via the ppt bc confusing)

Answer 15

uses strong solutions of hydrochloric acid (6M HCl)

Question 16

Peptides and proteins can be hydrolyzed by REFLUXING with moderately concentrated hydrochloric acid for several hours.

Question 17

Digestion of proteins in food (proteases)

Answer 17

Many different enzymes are involved in the digestion of
proteins in food since only specific pairs of amino acids could
be cleaved by an enzyme.

Question 18

Digestion of proteins in food (proteases) formula to the table:

enzymes = major sites of action = source

Answer 18

trypsin = Arg, Lys = pancreas

chymotrypsin = Trp, Phe, Tyr = pancreas

pepsin = Trp, Phe, Tyr, Met, Leu = pancreas

Question 19

note to the ysa studying this now: please study on the pictures you don’t understand i.e. enzymes cleaving at the end of the carbonyl of the amino acids

Question 20

Papain

Answer 20

a major ingredient in meat tenderizers with enzymes similar to pepsin

Question 21

Edman degradation: how to determine the ORDER:

Answer 21

1. Determine the C-terminal amino acid
   * USE CARBOXYPEPTIDASE - enzyme that removes the last C-terminal amino acid in a free form by breaking the peptide bond = hydrolyzes the peptide bond nearest the C-terminus
2. Identify the N-terminal amino acids in order
   * SEQUENCING
   * often difficult to characterize an intact protein

Question 22

Edman degradation

Answer 22

• sequencing of the peptides generated y proteases
• react the N-terminal amino acid with phenylisothiocyanate
• derivatized amino acid released as PTH - phenylthiohydantoin
• each PTH amino acid derivative is identified by chrom.
• newly exposed N-terminal residue can be derivatized, removed, and identified sequentially - useful p to 25-50 amino acids

Question 23

Edman degradation

Answer 23

a lab method used in determining the amino acid sequence in a polypeptide

Question 24

What are the tests for proteins and amino acids?

Answer 24

Xanthoproteic
Biuret test
Lowry assay
Bradford assay
Ninhydrin test

Question 25

test = reagent = positive test result

Answer 25

Xanothproteic test = HNO3 = yellow products for proteins with benzene rings

Biuret test = CuSO4 = violet color peptides/proteins with two or more peptide bonds, (test is negative for amino acids/dipeptides)

Lowry assay = CuSO4 and molybdates/tungstates = dark violet-blue for tyrosine/tryptophan containing proteins

Bradford assay = Coomassie brilliant + blue dye = dark blue color for proteins… most sensitive common test or proteins

Ninhydrin test = ninhydrin = blue soln. for all amino acids except proline an hydroxyproline