ACS BIOCHEMISTRY Exam Study
Guide Questions and Correct Detailed
Answers 2025
Henderson-Hasselbach Equation - Ans ✅✅-pH = pKa + log ([A-] / [HA])
FMOC Chemical Synthesis - Ans ✅✅-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) - Ans ✅✅-Changes soluble protein to solid precipitate.
Protein precipitates when the charges on the protein match the charges in the solution.
Size-Exclusion Chromatography - Ans ✅✅-Separates sample based on size with
smaller molecules eluting later.
Ion-Exchange Chromatography - Ans ✅✅-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 - Ans ✅✅-Beads are coated with a
carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent
(acetonitrile).
Affinity Chromatography - Ans ✅✅-Attach a ligand that binds a protein to a bead.
Elute with harsh chemicals or similar ligand.
SDS-PAGE - Ans ✅✅-Uses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster. Visualized with
Coomassie blue.
SDS - Ans ✅✅-Sodium dodecyl sulfate. Unfolds proteins and gives them uniform
negative charge.
Isoelectric Focusing - Ans ✅✅-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) - Ans ✅✅-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) - Ans ✅✅-Reduces disulfide bonds.
Iodoacetate - Ans ✅✅-Adds carboxymethyl group on free -SH groups. Blocks
disulfide bonding.
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,Homologs - Ans ✅✅-Shares 25% identity with another gene
Orthologs - Ans ✅✅-Similar genes in different organisms
Paralogs - Ans ✅✅-Similar "paired" genes in the same organism
Ramachandran Plot - Ans ✅✅-Shows favorable phi-psi angle combinations. 3 main
"wells" for α-helices, ß-sheets, and left-handed α-helices.
Glycine Ramachandran Plot - Ans ✅✅-Glycine can adopt more angles. (H's for R-
group).
Proline Ramachandran Plot - Ans ✅✅-Proline adopts fewer angles. Amino group is
incorporated into a ring.
α-helices - Ans ✅✅-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 - Ans ✅✅-Formed from added dipole moments of all hydrogen bonds in
an α-helix. N-terminus is δ+ and C-terminus is δ-.
ß-sheet - Ans ✅✅-Either parallel or anti-parallel. Often twisted to increase strength.
Anti-parallel ß-sheet - Ans ✅✅-Alternating sheet directions (C & N-termini don't line-
up). Has straight H-bonds.
Parallel ß-sheet - Ans ✅✅-Same sheet directions (C & N-termini line up). Has angled
H-bonds.
ß-turns - Ans ✅✅-Tight u-turns with specific phi-psi angles. Must have gly at position
3. Proline may also be at ß-turn because it can have a cis-omega angle.
Loops - Ans ✅✅-Not highly structured. Not necessary highly flexible, but can
occasionally move. Very variable in sequence.
Circular Dichroism - Ans ✅✅-Uses UV light to measure 2° structure. Can be used to
measure destabilization.
Disulfide-bonds - Ans ✅✅-Bonds between two -SH groups that form between 2° and
3° structure.
ß-mercaptoethanol - Ans ✅✅-Breaks disulfide bonds.
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, α-keratin - Ans ✅✅-formed from 2 α-helices twisted around each other. "Coiled coil".
Cross-linked by disulfide bonds.
Collagen - Ans ✅✅-Repeating sequence of Gly-X-Pro. 3 stranded "coiled coil".
Contains gly core.
Myoglobin 4° Structure - Ans ✅✅-Symmetric homodimer,
Hemoglobin 4° Structure - Ans ✅✅-Tetramer. Dimer of dimers. α2ß2 tetramer.
α/ß Protein Folding - Ans ✅✅-Less distinct areas of α and ß folding.
α+ß Protein Folding - Ans ✅✅-Two distinct areas of α and ß folding.
Mechanism of Denaturants - Ans ✅✅-Highly soluble, H-binding molecules. Stabilize
protein backbone in water. Allows denatured state to be stabilized.
Temperature Denaturation of Protein - Ans ✅✅-Midpoint of reaction is Tm.
Cooperative Protein Folding - Ans ✅✅-Folding transition is sharp. More reversible.
Folding Funnel - Ans ✅✅-Shows 3D version of 2D energy states. Lowest energy is
stable protein. Rough funnel is less cooperative.
Protein-Protein Interfaces - Ans ✅✅-"Core" and "fringe" of the interfaces. Core is
more hydrophobic and is on the inside when interfaced. Fringe is more hydrophilic.
π-π Ring Stacking - Ans ✅✅-Weird interaction where aromatic rings stack on each
other in positive interaction.
σ-hole - Ans ✅✅-Methyl group has area of diminished electron density in center;
attracts electronegative groups
Fe Binding of O2 - Ans ✅✅-Fe2+ binds to O2 reversible. Fe3+ has an additional +
charge and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments.
Ka for Binding - Ans ✅✅-Ka = [PL] / [P][L]
ϴ-value in Binding - Ans ✅✅-ϴ = (bound / total)x100%
ϴ = [L] / ([L] + 1/Ka)
Kd for binding - Ans ✅✅-Kd = [L] when 50% bound to protein.
Kd = 1/Ka
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Guide Questions and Correct Detailed
Answers 2025
Henderson-Hasselbach Equation - Ans ✅✅-pH = pKa + log ([A-] / [HA])
FMOC Chemical Synthesis - Ans ✅✅-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) - Ans ✅✅-Changes soluble protein to solid precipitate.
Protein precipitates when the charges on the protein match the charges in the solution.
Size-Exclusion Chromatography - Ans ✅✅-Separates sample based on size with
smaller molecules eluting later.
Ion-Exchange Chromatography - Ans ✅✅-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 - Ans ✅✅-Beads are coated with a
carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent
(acetonitrile).
Affinity Chromatography - Ans ✅✅-Attach a ligand that binds a protein to a bead.
Elute with harsh chemicals or similar ligand.
SDS-PAGE - Ans ✅✅-Uses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster. Visualized with
Coomassie blue.
SDS - Ans ✅✅-Sodium dodecyl sulfate. Unfolds proteins and gives them uniform
negative charge.
Isoelectric Focusing - Ans ✅✅-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) - Ans ✅✅-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) - Ans ✅✅-Reduces disulfide bonds.
Iodoacetate - Ans ✅✅-Adds carboxymethyl group on free -SH groups. Blocks
disulfide bonding.
1|Page
,Homologs - Ans ✅✅-Shares 25% identity with another gene
Orthologs - Ans ✅✅-Similar genes in different organisms
Paralogs - Ans ✅✅-Similar "paired" genes in the same organism
Ramachandran Plot - Ans ✅✅-Shows favorable phi-psi angle combinations. 3 main
"wells" for α-helices, ß-sheets, and left-handed α-helices.
Glycine Ramachandran Plot - Ans ✅✅-Glycine can adopt more angles. (H's for R-
group).
Proline Ramachandran Plot - Ans ✅✅-Proline adopts fewer angles. Amino group is
incorporated into a ring.
α-helices - Ans ✅✅-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 - Ans ✅✅-Formed from added dipole moments of all hydrogen bonds in
an α-helix. N-terminus is δ+ and C-terminus is δ-.
ß-sheet - Ans ✅✅-Either parallel or anti-parallel. Often twisted to increase strength.
Anti-parallel ß-sheet - Ans ✅✅-Alternating sheet directions (C & N-termini don't line-
up). Has straight H-bonds.
Parallel ß-sheet - Ans ✅✅-Same sheet directions (C & N-termini line up). Has angled
H-bonds.
ß-turns - Ans ✅✅-Tight u-turns with specific phi-psi angles. Must have gly at position
3. Proline may also be at ß-turn because it can have a cis-omega angle.
Loops - Ans ✅✅-Not highly structured. Not necessary highly flexible, but can
occasionally move. Very variable in sequence.
Circular Dichroism - Ans ✅✅-Uses UV light to measure 2° structure. Can be used to
measure destabilization.
Disulfide-bonds - Ans ✅✅-Bonds between two -SH groups that form between 2° and
3° structure.
ß-mercaptoethanol - Ans ✅✅-Breaks disulfide bonds.
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, α-keratin - Ans ✅✅-formed from 2 α-helices twisted around each other. "Coiled coil".
Cross-linked by disulfide bonds.
Collagen - Ans ✅✅-Repeating sequence of Gly-X-Pro. 3 stranded "coiled coil".
Contains gly core.
Myoglobin 4° Structure - Ans ✅✅-Symmetric homodimer,
Hemoglobin 4° Structure - Ans ✅✅-Tetramer. Dimer of dimers. α2ß2 tetramer.
α/ß Protein Folding - Ans ✅✅-Less distinct areas of α and ß folding.
α+ß Protein Folding - Ans ✅✅-Two distinct areas of α and ß folding.
Mechanism of Denaturants - Ans ✅✅-Highly soluble, H-binding molecules. Stabilize
protein backbone in water. Allows denatured state to be stabilized.
Temperature Denaturation of Protein - Ans ✅✅-Midpoint of reaction is Tm.
Cooperative Protein Folding - Ans ✅✅-Folding transition is sharp. More reversible.
Folding Funnel - Ans ✅✅-Shows 3D version of 2D energy states. Lowest energy is
stable protein. Rough funnel is less cooperative.
Protein-Protein Interfaces - Ans ✅✅-"Core" and "fringe" of the interfaces. Core is
more hydrophobic and is on the inside when interfaced. Fringe is more hydrophilic.
π-π Ring Stacking - Ans ✅✅-Weird interaction where aromatic rings stack on each
other in positive interaction.
σ-hole - Ans ✅✅-Methyl group has area of diminished electron density in center;
attracts electronegative groups
Fe Binding of O2 - Ans ✅✅-Fe2+ binds to O2 reversible. Fe3+ has an additional +
charge and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments.
Ka for Binding - Ans ✅✅-Ka = [PL] / [P][L]
ϴ-value in Binding - Ans ✅✅-ϴ = (bound / total)x100%
ϴ = [L] / ([L] + 1/Ka)
Kd for binding - Ans ✅✅-Kd = [L] when 50% bound to protein.
Kd = 1/Ka
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