proteins paula yurkanis bryce Flashcards
What is Electrophoresis?
Electrophoresis is a separation technique that distinguishes molecules based on their charge, which in turn depends on:
The pH of the buffer
The molecule’s isoelectric point (pI)
Principle of Separation of separation
Each amino acid has a pI, which is the pH at which it has no net charge (zwitterion).
At a pH:
Below its pI → amino acid is positively charged.
Above its pI → amino acid is negatively charged.
Experiment Setup of electrophoresis
A drop of a mixture of amino acids is applied at the center of a strip of filter paper or gel.
The paper/gel is soaked in a buffer (in this case, pH = 5).
An electric field is applied across two electrodes:
Cathode = negative (left)
Anode = positive (right)
Movement Based on Charge in electrophoresis
Molecules with pI > pH of buffer (net positive charge) move toward cathode (−).
Molecules with pI < pH of buffer (net negative charge) move toward anode (+).
The further the pI is from the buffer pH, the stronger the net charge → the faster it migrates.
Additional Insight in electrophoresis
Aspartate moves farthest toward anode because it has the greatest difference between pH and pI.
Arginine moves farthest toward cathode for the same reason.
Alanine barely moves because it’s almost neutral at pH 5.
What is electrophoresis used for in amino acid analysis?
A: To separate amino acids based on their net charge, which depends on pI vs pH.
What is an amino acid’s pI?
A: The pH at which it has no net charge (zwitterion).
What happens if pH < pI?
A: The amino acid is positively charged.
What happens if pH > pI?
A: The amino acid is negatively charged.
Toward which electrode do positively charged amino acids migrate?
A: Cathode (–)
Toward which electrode do negatively charged amino acids migrate?
A: Anode (+)
Reaction of Amino Acids with Ninhydrin — Step-by-Step Breakdown
This is a classic reaction used in protein chemistry and forensic science (e.g., fingerprint development).
🔹 Ninhydrin (Reagent)
Chemical name: Triketohydrindene hydrate
Acts as an oxidizing agent and condensation partner
It reacts specifically with α-amino groups of amino acids
🧪 Step 1: Formation of Imine
The α-amino group of the amino acid attacks the central carbonyl of ninhydrin.
A Schiff base (imine) forms after loss of water.
Mechanism:
Nucleophilic attack of NH₂ on carbonyl
Elimination of H₂O → imine intermediate
🧪 Step 2: Decarboxylation
The carboxylic acid group of the amino acid loses CO₂.
Electrons are pushed toward the imine, forming a resonance-stabilized structure.
🧪 Step 3: Hydrolysis and Deamination
The intermediate undergoes tautomerization and hydrolysis, releasing:
A deaminated α-keto acid
An amine
CO₂
🧪 Step 4: Formation of the Purple-Colored Product
The released ammonia or amine reacts with another molecule of dehydrated ninhydrin.
This forms a highly conjugated imine, known as Ruhemann’s purple.
🌈 Final Product
A deep purple compound forms, indicating the presence of a primary amino acid.
Caveat: This does not occur with secondary amines (e.g., proline), which yield yellow/orange products instead.
If two amino acids have the same charge, which moves slower?
A: The one with larger molecular weight, because it’s harder to mobilize.
What is Paper Chromatography?
It’s a technique used to separate compounds based on differences in their polarity.
It uses:
Stationary phase: Polar filter paper (typically cellulose)
Mobile phase: A non-polar or moderately polar solvent that moves up the paper
How Separation Occurs with
A small spot of an amino acid mixture is placed near the bottom of the paper.
The paper is placed in a solvent chamber; solvent moves up via capillary action.
Amino acids partition between:
Stationary phase (paper) — polar
Mobile phase (solvent) — less polar
The less polar the amino acid:
The less it sticks to the paper
The farther it travels
The more polar the amino acid:
The more strongly it adsorbs to the paper
The less it moves with the solvent front
What is IEC?
Ion-Exchange Chromatography separates amino acids based on their overall charge at a given pH.
It uses a charged resin in a column.
Amino acids are passed through the column and are retained or repelled based on ionic interactions.
What is the HVZ Reaction?
HVZ = Hell–Volhard–Zelinsky reaction
It selectively brominates the α-carbon of a carboxylic acid
Reagents:
Br
2
,
PBr
3
followedbyH
2
O
Br
2
,PBr
3
followedbyH
2
O
What is the HVZ Reaction?
HVZ = Hell–Volhard–Zelinsky reaction Step-by-Step Mechanism
Step-by-Step Mechanism
Bromination of α-carbon:
The carboxylic acid is converted to an acid bromide, then enolized, and brominated at the α-carbon.
Hydrolysis back to acid:
Converts the α-brominated acid bromide back to the α-bromo carboxylic acid.
SN2 substitution with ammonia:
The α-bromo is displaced by NH₃, yielding an α-amino acid.
Overall:
Carboxylic Acid → α-Bromo Acid → α-Amino Acid
Reductive Amination of α-Keto Acids
What is Reductive Amination?
Converts α-keto acids into α-amino acids
Mimics biological amino acid biosynthesis
Conditions:
1.ExcessNH
3
,
traceacid
2.H
2
/
Pd-C(catalytichydrogenation)
1.ExcessNH
3
,traceacid
2.H
2
/Pd-C(catalytichydrogenation)
Mechanism
Reductive Amination of α-Keto Acids
Mechanism
Imine Formation:
The amino group (NH₃) reacts with the keto group to form a Schiff base (imine)
Reduction of the imine:
Catalytic hydrogenation with H₂/Pd reduces the imine to a primary amine, yielding an α-amino acid.
What does the HVZ reaction do to a carboxylic acid?
A: Adds a bromine atom to the α-carbon
What happens when you treat the α-bromo acid with NH₃?
A: It forms an α-amino acid
What kind of mechanism is the NH₃ step in HVZ synthesis?
A: SN2 substitution
What is the starting compound in reductive amination?
A: An α-keto acid
