BIOCHEM MODULE 8 EXAM FINAL
COMPREHENSIVE ASSESSMENT TEST
PAPER QUESTIONS AND SOLUTIONS
⩥ FMOC Chemical Synthesis.
Answer: Used in synthesis of a growing amino acid chain to a
polystyrene bead. FMOC is used as a protecting group on the N-
terminus.
⩥ Salting Out (Purification).
Answer: Changes soluble protein to solid precipitate. Protein precipitates
when the charges on the protein match the charges in the solution.
⩥ Size-Exclusion Chromatography.
Answer: Separates sample based on size with smaller molecules eluting
later.
⩥ Ion-Exchange Chromatography.
Answer: Separates sample based on charge. CM attracts +, DEAE
attracts -. May have repulsion effect on like charges. Salt or acid used to
remove stuck proteins.
⩥ Hydrophobic/Reverse Phase Chromatography.
,Answer: Beads are coated with a carbon chain. Hydrophobic proteins
stick better. Elute with non-H-bonding solvent (acetonitrile).
⩥ Affinity Chromatography.
Answer: Attach a ligand that binds a protein to a bead. Elute with harsh
chemicals or similar ligand.
⩥ SDS-PAGE.
Answer: Uses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster.
Visualized with Coomassie blue.
⩥ SDS.
Answer: Sodium dodecyl sulfate. Unfolds proteins and gives them
uniform negative charge.
⩥ Isoelectric Focusing.
Answer: Variation of gel electrophoresis where protein charge matters.
Involves electrodes and pH gradient. Protein stops at their pI when
neutral.
⩥ FDNB (1-fluoro-2,3-dinitrobenzene).
,Answer: FDNB reacts with the N-terminus of the protein to produce a
2,4-dinitrophenol derivative that labels the first residue. Can repeat
hydrolysis to determine sequential amino acids.
⩥ DTT (dithiothreitol).
Answer: Reduces disulfide bonds.
⩥ Iodoacetate.
Answer: Adds carboxymethyl group on free -SH groups. Blocks
disulfide bonding.
⩥ Homologs.
Answer: Shares 25% identity with another gene
⩥ Orthologs.
Answer: Similar genes in different organisms
⩥ Paralogs.
Answer: Similar "paired" genes in the same organism
⩥ Ramachandran Plot.
Answer: Shows favorable phi-psi angle combinations. 3 main "wells"
for α-helices, ß-sheets, and left-handed α-helices.
, ⩥ Glycine Ramachandran Plot.
Answer: Glycine can adopt more angles. (H's for R-group).
⩥ Proline Ramachandran Plot.
Answer: Proline adopts fewer angles. Amino group is incorporated into a
ring.
⩥ α-helices.
Answer: Ala is common, Gly & Pro are not very common. Side-chain
interactions every 3 or 4 residues. Turns once every 3.6 residues.
Distance between backbones is 5.4Å.
⩥ Helix Dipole.
Answer: Formed from added dipole moments of all hydrogen bonds in
an α-helix. N-terminus is δ+ and C-terminus is δ-.
⩥ ß-sheet.
Answer: Either parallel or anti-parallel. Often twisted to increase
strength.
⩥ Anti-parallel ß-sheet.
Answer: Alternating sheet directions (C & N-termini don't line-up). Has
straight H-bonds.
COMPREHENSIVE ASSESSMENT TEST
PAPER QUESTIONS AND SOLUTIONS
⩥ FMOC Chemical Synthesis.
Answer: Used in synthesis of a growing amino acid chain to a
polystyrene bead. FMOC is used as a protecting group on the N-
terminus.
⩥ Salting Out (Purification).
Answer: Changes soluble protein to solid precipitate. Protein precipitates
when the charges on the protein match the charges in the solution.
⩥ Size-Exclusion Chromatography.
Answer: Separates sample based on size with smaller molecules eluting
later.
⩥ Ion-Exchange Chromatography.
Answer: Separates sample based on charge. CM attracts +, DEAE
attracts -. May have repulsion effect on like charges. Salt or acid used to
remove stuck proteins.
⩥ Hydrophobic/Reverse Phase Chromatography.
,Answer: Beads are coated with a carbon chain. Hydrophobic proteins
stick better. Elute with non-H-bonding solvent (acetonitrile).
⩥ Affinity Chromatography.
Answer: Attach a ligand that binds a protein to a bead. Elute with harsh
chemicals or similar ligand.
⩥ SDS-PAGE.
Answer: Uses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster.
Visualized with Coomassie blue.
⩥ SDS.
Answer: Sodium dodecyl sulfate. Unfolds proteins and gives them
uniform negative charge.
⩥ Isoelectric Focusing.
Answer: Variation of gel electrophoresis where protein charge matters.
Involves electrodes and pH gradient. Protein stops at their pI when
neutral.
⩥ FDNB (1-fluoro-2,3-dinitrobenzene).
,Answer: FDNB reacts with the N-terminus of the protein to produce a
2,4-dinitrophenol derivative that labels the first residue. Can repeat
hydrolysis to determine sequential amino acids.
⩥ DTT (dithiothreitol).
Answer: Reduces disulfide bonds.
⩥ Iodoacetate.
Answer: Adds carboxymethyl group on free -SH groups. Blocks
disulfide bonding.
⩥ Homologs.
Answer: Shares 25% identity with another gene
⩥ Orthologs.
Answer: Similar genes in different organisms
⩥ Paralogs.
Answer: Similar "paired" genes in the same organism
⩥ Ramachandran Plot.
Answer: Shows favorable phi-psi angle combinations. 3 main "wells"
for α-helices, ß-sheets, and left-handed α-helices.
, ⩥ Glycine Ramachandran Plot.
Answer: Glycine can adopt more angles. (H's for R-group).
⩥ Proline Ramachandran Plot.
Answer: Proline adopts fewer angles. Amino group is incorporated into a
ring.
⩥ α-helices.
Answer: Ala is common, Gly & Pro are not very common. Side-chain
interactions every 3 or 4 residues. Turns once every 3.6 residues.
Distance between backbones is 5.4Å.
⩥ Helix Dipole.
Answer: Formed from added dipole moments of all hydrogen bonds in
an α-helix. N-terminus is δ+ and C-terminus is δ-.
⩥ ß-sheet.
Answer: Either parallel or anti-parallel. Often twisted to increase
strength.
⩥ Anti-parallel ß-sheet.
Answer: Alternating sheet directions (C & N-termini don't line-up). Has
straight H-bonds.
