Chromophores, Amino Acids Flashcards
(40 cards)
When pH>pI, most of the molecules in a solution have a net ____ charge
most molecules have a net negative charge
More = Negative
most of the molecules in a solution will have a net positive charge when
pH___ pI
pH
Why do we use electrophoresis?
How does electrophoresis work?
Helps us to identify and separate amino acids.
A sample is placed into the middle of the tray, and then a current is applied. This is being run at neutral pH. Amino acids (eg, Aspartate) that have a net negative charge (low pI) will go to the anode. Amino acids (eg, Arginine) with a net positive charge (aka, are protonated, high pI) travel to cathode. And the neutral ones will stay in middle.
If a pigment has a lower Rf, where will it be situated on the tablet?
How do you calculate Rf?
Low Rf = lower on plate.
Rf = (distance spot travelled) / (distance solvent front travelled)
If all the points are clumped at the bottom of the plate, what’s wrong?
The solvent was not polar enough.
If all the points are at the top of the plate, what’s wrong?
The solvent was too polar. (just think toooo polar, too high)
Fully protonated state when:
pH is low
pKa1
Protonated NH3+, de-protonated CO2-
What are the proportions of pH and pKas when Protonated NH3+, de-protonated CO2-?
pKa1(carboxyl)
pKa1
Fully de-protonated state
Fully de-protonated state when:
pKa1
What’s the pKa for carboxyl group?
Why does it have this value?
2
Is so low because of the inductive effect of electron density towards the ammonium group.
What’s the pKa for ammonium group?
Why does it have this value?
9 or 10
Very electron-withdrawing
What is Ka?
the acid dissociation constant (how much it takes for the acid to dissociate into its conjugate base)
What happens if pKa=pH?
[Conjugate Base] = [acid]
Calculating pI for neutral side chains.
Calculating pI for acidic/basic side chains.
neutral: avg the strongest NH3 and the strongest CO2H?
acidic/basic: avg the pKas that are the most similar in number
When proteins are denatured, what is lost?
Tertiary and sometimes secondary. Primary not lost unless hydrolyzed.
What is Amino Acid Analysis used for?
What are the 3 steps to this?
To determine the composition and relative amounts of amino acids present in a peptide.
DOES NOT TELL US CONNECTIVITY/no primary structure.
- Acid Hydrolysis of peptide to amino acid
- Separation of AAs by Ion-Exchange HPLC
- based on ionic interactions and AA net charge
- Quantification
- determine how many of each AA is here because after it leaves column it reacts with triketone form of ninhydrin (via Nucleophilic Addition/Imine Formation then Decarboxylation to give an amine again) to produce Ruhemann’s Purple (a chromophore), and its absorption is measured. (intensity proportional to amount)
Primary Structure
Secondary Structure
Tertiary Structure
Quaternary Structure
Primary: sequence/order of AAs – only ruined by hydrolysis
Secondary: folding of proteins into conformations that are stabilized by non-covalent forces (eg, H bonds btwn non-adjacent AAs)
- alpha helix: NH bonded to C=O 4 AAs away
- beta/pleated sheets: NH bonded to C=O on neighbouring B strand
Tertiary: folding into 3D shape/native conformation.
-stabilized by non-covalent interactions (electrostatic, hydrophobic, H bonds) & covalent (disulfide bridges)
Quaternary: aggregate of proteins forming a molecule (ex, hemoglobin). If the protein exists as a single molecule, won’t have a quaternary structure.
How are disulfide bridges formed?
Where do we see them?
Oxidation of thiols in Cysteine creates cyclic disulfide bridges in Tertiary Structure. Can be reduced by using excess of another thiol.
Which amino acids will leave the column first and last?
What is this column made of?
Amino acids that are the same charge of the column will be repulsed and jetted out.
Amino acids that have opposite charge will do a lot of interacting inside the column, and come out slower.
Column filled with insoluble polymer that has permanent charges.
What is Edman Degradation?
It’s N-Terminal Sequencing.
It’s a way to figure out the sequence of AA in a peptide by removing one AA at a time from the N terminus and chemically converting it into a PTH derivative that will have an R group specific to that AA.
What AA’s side chain will react with Sanger’s Reagent?
Lysine
How to protect and deprotect COOH
Using solution-phase synthesis
Protect: introduce benzyl ester through Fischer Esterification
Deprotect: treatment of strong acid without breaking/hydrolyzing amide bond that will be made during synthesis. So yea if not too strong of an acid, then good because esters are more acid-labile (will undergo change readily) than amides.
