proteins paula yurkanis bryce

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)

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.

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)

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.

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.

A: To separate amino acids based on their net charge, which depends on pI vs pH.

A: The pH at which it has no net charge (zwitterion).

A: The amino acid is positively charged.

A: The amino acid is negatively charged.

A: Cathode (–)

A: Anode (+)

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.

A: The one with larger molecular weight, because it’s harder to mobilize.

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

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

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.

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

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

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 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.

A: Adds a bromine atom to the α-carbon

A: It forms an α-amino acid

A: SN2 substitution

A: An α-keto acid