ACS BIOCHEMISTRY EXAM 2026
QUESTION AND ANSWERS
Henderson-Hasselbach Equation - answerpH = pKa + log ([A-] / [HA])
FMOC Chemical Synthesis - answerUsed 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) - answerChanges soluble protein to solid precipitate. Protein
precipitates when the charges on the protein match the charges in the solution.
Size-Exclusion Chromatography - answerSeparates sample based on size with smaller
molecules eluting later.
Ion-Exchange Chromatography - answerSeparates 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 - answerBeads are coated with a carbon
chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent (acetonitrile).
Affinity Chromatography - answerAttach a ligand that binds a protein to a bead. Elute
with harsh chemicals or similar ligand.
SDS-PAGE - answerUses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster. Visualized with
Coomassie blue.
SDS - answerSodium dodecyl sulfate. Unfolds proteins and gives them uniform
negative charge.
Isoelectric Focusing - answerVariation 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) - answerFDNB 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) - answerReduces disulfide bonds.
Iodoacetate - answerAdds carboxymethyl group on free -SH groups. Blocks disulfide
bonding.
Homologs - answerShares 25% identity with another gene
,Orthologs - answerSimilar genes in different organisms
Paralogs - answerSimilar "paired" genes in the same organism
Ramachandran Plot - answerShows favorable phi-psi angle combinations. 3 main
"wells" for α-helices, ß-sheets, and left-handed α-helices.
Glycine Ramachandran Plot - answerGlycine can adopt more angles. (H's for R-group).
Proline Ramachandran Plot - answerProline adopts fewer angles. Amino group is
incorporated into a ring.
α-helices - answerAla 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 - answerFormed from added dipole moments of all hydrogen bonds in an
α-helix. N-terminus is δ+ and C-terminus is δ-.
ß-sheet - answerEither parallel or anti-parallel. Often twisted to increase strength.
Anti-parallel ß-sheet - answerAlternating sheet directions (C & N-termini don't line-up).
Has straight H-bonds.
Parallel ß-sheet - answerSame sheet directions (C & N-termini line up). Has angled H-
bonds.
ß-turns - answerTight 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 - answerNot highly structured. Not necessary highly flexible, but can occasionally
move. Very variable in sequence.
Circular Dichroism - answerUses UV light to measure 2° structure. Can be used to
measure destabilization.
Disulfide-bonds - answerBonds between two -SH groups that form between 2° and 3°
structure.
ß-mercaptoethanol - answerBreaks disulfide bonds.
α-keratin - answerformed from 2 α-helices twisted around each other. "Coiled coil".
Cross-linked by disulfide bonds.
, Collagen - answerRepeating sequence of Gly-X-Pro. 3 stranded "coiled coil". Contains
gly core.
Myoglobin 4° Structure - answerSymmetric homodimer,
Hemoglobin 4° Structure - answerTetramer. Dimer of dimers. α2ß2 tetramer.
α/ß Protein Folding - answerLess distinct areas of α and ß folding.
α+ß Protein Folding - answerTwo distinct areas of α and ß folding.
Mechanism of Denaturants - answerHighly soluble, H-binding molecules. Stabilize
protein backbone in water. Allows denatured state to be stabilized.
Temperature Denaturation of Protein - answerMidpoint of reaction is Tm.
Cooperative Protein Folding - answerFolding transition is sharp. More reversible.
Folding Funnel - answerShows 3D version of 2D energy states. Lowest energy is stable
protein. Rough funnel is less cooperative.
Protein-Protein Interfaces - answer"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 - answerWeird interaction where aromatic rings stack on each other
in positive interaction.
σ-hole - answerMethyl group has area of diminished electron density in center; attracts
electronegative groups
Fe Binding of O2 - answerFe2+ binds to O2 reversible. Fe3+ has an additional + charge
and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments.
Ka for Binding - answerKa = [PL] / [P][L]
ϴ-value in Binding - answerϴ = (bound / total)x100%
ϴ = [L] / ([L] + 1/Ka)
Kd for binding - answerKd = [L] when 50% bound to protein.
Kd = 1/Ka
High-Spin Fe - answerElectrons are "spread out" and result in larger atom.
Low-Spin Fe - answerElectrons are less "spread out" and are compacted by electron
rich porphyrin ring.
QUESTION AND ANSWERS
Henderson-Hasselbach Equation - answerpH = pKa + log ([A-] / [HA])
FMOC Chemical Synthesis - answerUsed 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) - answerChanges soluble protein to solid precipitate. Protein
precipitates when the charges on the protein match the charges in the solution.
Size-Exclusion Chromatography - answerSeparates sample based on size with smaller
molecules eluting later.
Ion-Exchange Chromatography - answerSeparates 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 - answerBeads are coated with a carbon
chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent (acetonitrile).
Affinity Chromatography - answerAttach a ligand that binds a protein to a bead. Elute
with harsh chemicals or similar ligand.
SDS-PAGE - answerUses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster. Visualized with
Coomassie blue.
SDS - answerSodium dodecyl sulfate. Unfolds proteins and gives them uniform
negative charge.
Isoelectric Focusing - answerVariation 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) - answerFDNB 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) - answerReduces disulfide bonds.
Iodoacetate - answerAdds carboxymethyl group on free -SH groups. Blocks disulfide
bonding.
Homologs - answerShares 25% identity with another gene
,Orthologs - answerSimilar genes in different organisms
Paralogs - answerSimilar "paired" genes in the same organism
Ramachandran Plot - answerShows favorable phi-psi angle combinations. 3 main
"wells" for α-helices, ß-sheets, and left-handed α-helices.
Glycine Ramachandran Plot - answerGlycine can adopt more angles. (H's for R-group).
Proline Ramachandran Plot - answerProline adopts fewer angles. Amino group is
incorporated into a ring.
α-helices - answerAla 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 - answerFormed from added dipole moments of all hydrogen bonds in an
α-helix. N-terminus is δ+ and C-terminus is δ-.
ß-sheet - answerEither parallel or anti-parallel. Often twisted to increase strength.
Anti-parallel ß-sheet - answerAlternating sheet directions (C & N-termini don't line-up).
Has straight H-bonds.
Parallel ß-sheet - answerSame sheet directions (C & N-termini line up). Has angled H-
bonds.
ß-turns - answerTight 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 - answerNot highly structured. Not necessary highly flexible, but can occasionally
move. Very variable in sequence.
Circular Dichroism - answerUses UV light to measure 2° structure. Can be used to
measure destabilization.
Disulfide-bonds - answerBonds between two -SH groups that form between 2° and 3°
structure.
ß-mercaptoethanol - answerBreaks disulfide bonds.
α-keratin - answerformed from 2 α-helices twisted around each other. "Coiled coil".
Cross-linked by disulfide bonds.
, Collagen - answerRepeating sequence of Gly-X-Pro. 3 stranded "coiled coil". Contains
gly core.
Myoglobin 4° Structure - answerSymmetric homodimer,
Hemoglobin 4° Structure - answerTetramer. Dimer of dimers. α2ß2 tetramer.
α/ß Protein Folding - answerLess distinct areas of α and ß folding.
α+ß Protein Folding - answerTwo distinct areas of α and ß folding.
Mechanism of Denaturants - answerHighly soluble, H-binding molecules. Stabilize
protein backbone in water. Allows denatured state to be stabilized.
Temperature Denaturation of Protein - answerMidpoint of reaction is Tm.
Cooperative Protein Folding - answerFolding transition is sharp. More reversible.
Folding Funnel - answerShows 3D version of 2D energy states. Lowest energy is stable
protein. Rough funnel is less cooperative.
Protein-Protein Interfaces - answer"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 - answerWeird interaction where aromatic rings stack on each other
in positive interaction.
σ-hole - answerMethyl group has area of diminished electron density in center; attracts
electronegative groups
Fe Binding of O2 - answerFe2+ binds to O2 reversible. Fe3+ has an additional + charge
and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments.
Ka for Binding - answerKa = [PL] / [P][L]
ϴ-value in Binding - answerϴ = (bound / total)x100%
ϴ = [L] / ([L] + 1/Ka)
Kd for binding - answerKd = [L] when 50% bound to protein.
Kd = 1/Ka
High-Spin Fe - answerElectrons are "spread out" and result in larger atom.
Low-Spin Fe - answerElectrons are less "spread out" and are compacted by electron
rich porphyrin ring.
