ACS BIOCHEMISTRY EXAM Questions and Answers 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 precipita tes 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 attra cts +, 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-bondin g 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 wit h 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 -
dinitroph enol 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. Homo logs 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.
dinitroph enol 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. Homo logs 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.
