LAB(3) - PROTEIN DENATURATION Flashcards
Denaturation
When a protein loses it function because of a change in its secondary,
tertiary, or quaternary structure
Denature proteins
have lower solubility and often precipitate from solution.
What is the goal of protein folding?
To achieve the LOWEST energy state
Can we unfold proteins once they are folded?
- 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
Proteins can be denatured by?
Heat, detergents, mechanical/whipping action and certain acids/bases
During digestion, dietary proteins are denatured and then hydrolyzed to form
amino acids as shown here. (STUDY THE PICTURE ON PPT)
heat, agitation, and urea, guanidine HCl = H-bonds, hydrophobic interactions
pH = salt bridges
Mercaptoethanol = disulfide bridges
detergents (SDS) = hydrophobic interactions
What are the other examples and applications of denaturization? (study the meaning via the ppt)
- 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
Denaturation can be reversible
- heat treatment usually is not reversible
what did Christian Anfinsen do to win the Nobel Prize in 1972?
renaturation of the protein RIBONUCLEASE (an enzyme that cleaves DNA)
Experiment:
1. Denatured pure Ribonuclease
* UREA = breaks H-bonds and hydrophobic interactions
* beta-mercaptoethanol = reduce disulfide bonds
- removed the denaturants and exposed the protein to air
- protein folded back into its original 3D shape and activity was restored
Amino acid sequence determines protein shape
How to determine the composition? (study the meaning via ppt)
- purify the protein of interest
- estimate the molecular weight of the protein
- establish the composition by complete hydrolysis of the protein under acidic conditions
How to determine the composition? (study the meaning via ppt)
- treat w/ 6M HCl at 110 degrees Celsius (12-36)
- each peptide bond is broken and products are all of the free amino acids
- each amino acid is separated identified, and quantified
- final result = know how MANY of each amino acid present in the original
Protein hydrolysis
(study more on this via the ppt bc confusing)
uses strong solutions of hydrochloric acid (6M HCl)
Peptides and proteins can be hydrolyzed by REFLUXING with moderately concentrated hydrochloric acid for several hours.
Digestion of proteins in food (proteases)
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.
Digestion of proteins in food (proteases) formula to the table:
enzymes = major sites of action = source
trypsin = Arg, Lys = pancreas
chymotrypsin = Trp, Phe, Tyr = pancreas
pepsin = Trp, Phe, Tyr, Met, Leu = pancreas
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
Papain
a major ingredient in meat tenderizers with enzymes similar to pepsin
Edman degradation: how to determine the ORDER:
- 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 - Identify the N-terminal amino acids in order
* SEQUENCING
* often difficult to characterize an intact protein
Edman degradation
- 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
Edman degradation
a lab method used in determining the amino acid sequence in a polypeptide
What are the tests for proteins and amino acids?
Xanthoproteic
Biuret test
Lowry assay
Bradford assay
Ninhydrin test
test = reagent = positive test result
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
