ACS BIOCHEMISTRY EXAM

Henderson-Hasselbach Equation

pH = pKa + log ([A-] / [HA])

FMOC Chemical Synthesis

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)

Changes soluble protein to solid precipitate. Protein precipitates when the charges on the protein match the charges in the solution.

Size-Exclusion Chromatography

Separates sample based on size with smaller molecules eluting later.

Ion-Exchange Chromatography

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

Beads are coated with a carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent (acetonitrile).

Affinity Chromatography

Attach a ligand that binds a protein to a bead. Elute with harsh chemicals or similar ligand.

SDS-PAGE

Uses SDS. Gel is made from cross-linked polyacrylamide. Separates based off of mass with smaller molecules moving faster. Visualized with Coomassie blue.

SDS

Sodium dodecyl sulfate. Unfolds proteins and gives them uniform negative charge.

Isoelectric Focusing

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)

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)

Reduces disulfide bonds.

Iodoacetate

Adds carboxymethyl group on free -SH groups. Blocks disulfide bonding.

Homologs

Shares 25% identity with another gene

Orthologs

Similar genes in different organisms

Paralogs

Similar "paired" genes in the same organism

Ramachandran Plot

Shows favorable phi-psi angle combinations. 3 main "wells" for α-helices, ß-sheets, and left-handed α-helices.

Glycine Ramachandran Plot

Glycine can adopt more angles. (H's for R-group).

Proline Ramachandran Plot

Proline adopts fewer angles. Amino group is incorporated into a ring.

α-helices

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

Formed from added dipole moments of all hydrogen bonds in an α-helix. N-terminus is δ+ and C-terminus is δ-.

ß-sheet

Either parallel or anti-parallel. Often twisted to increase strength.

Anti-parallel ß-sheet

Alternating sheet directions (C & N-termini don't line-up). Has straight H-bonds.

Parallel ß-sheet

Same sheet directions (C & N-termini line up). Has angled H-bonds.

ß-turns

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

Not highly structured. Not necessary highly flexible, but can occasionally move. Very variable in sequence.

Circular Dichroism

Uses UV light to measure 2° structure. Can be used to measure destabilization.

Disulfide-bonds

Bonds between two -SH groups that form between 2° and 3° structure.

ß-mercaptoethanol

Breaks disulfide bonds.

α-keratin

formed from 2 α-helices twisted around each other. "Coiled coil". Cross-linked by disulfide bonds.

Collagen

Repeating sequence of Gly-X-Pro. 3 stranded "coiled coil". Contains gly core.

Myoglobin 4° Structure

Symmetric homodimer,

Hemoglobin 4° Structure

Tetramer. Dimer of dimers. α2ß2 tetramer.

α/ß Protein Folding

Less distinct areas of α and ß folding.

α+ß Protein Folding

Two distinct areas of α and ß folding.

Mechanism of Denaturants

Highly soluble, H-binding molecules. Stabilize protein backbone in water. Allows denatured state to be stabilized.

Temperature Denaturation of Protein

Midpoint of reaction is Tm.

Cooperative Protein Folding

Folding transition is sharp. More reversible.

Folding Funnel

Shows 3D version of 2D energy states. Lowest energy is stable protein. Rough funnel is less cooperative.

Protein-Protein Interfaces

"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

Weird interaction where aromatic rings stack on each other in positive interaction.

σ-hole

Methyl group has area of diminished electron density in center; attracts electronegative groups

Fe Binding of O2

Fe2+ binds to O2 reversible. Fe3+ has an additional + charge and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments.

Ka for Binding

Ka = [PL] / [P][L]

ϴ-value in Binding

ϴ = (bound / total)x100%
ϴ = [L] / ([L] + 1/Ka)

Kd for binding

Kd = [L] when 50% bound to protein.
Kd = 1/Ka

High-Spin Fe

Electrons are "spread out" and result in larger atom.

Low-Spin Fe

Electrons are less "spread out" and are compacted by electron rich porphyrin ring.

T-State

Heme is in high-spin state. H2O is bound to heme.

R-State

Heme is in low-spin state. O2 is bound to heme.

O2 Binding Event

O2 binds to T-state and changes the heme to R-state. Causes a 0.4Å movement of the iron.

Hemoglobin Binding Curve

4 subunits present in hemoglobin that can be either T or R -state. Cooperative binding leads to a sigmoidal curve.

Binding Cooperativity

When one subunit of hemoglobin changes from T to R-state the other sites are more likely to change to R-state as well. Leads to sigmoidal graph.

Homotropic Regulation of Binding

Where a regulatory molecule is also the enzyme's substrate.

Heterotropic Regulation of Binding

Where an allosteric regulator is present that is not the enzyme's substrate.

Hill Plot

Turns sigmoid into straight lines. Slope = n (# of binding sites). Allows measurement of binding sites that are cooperative.

pH and Binding Affinity (Bohr Affect)

As [H+] increases, Histidine group in hemoglobin becomes more protonated and protein shifts to T-state. O2 binding affinity decreases.

CO2 binding in Hemoglobin

Forms carbonic acid that shifts hemoglobin to T-state. O2 binding affinity decreases. Used in the peripheral tissues.

BPG (2,3-bisphosphoglycerate)

Greatly reduces hemoglobin's affinity for O2 by binding allosterically. Stabilizes T-state. Transfer of O2 can improve because increased delivery in tissues can outweigh decreased binding in the lungs.

Michaelis-Menton Equation

V0 = (Vmax[S]) / (Km + [S])

Km in Michaelis-Menton

Km = [S] when V0 = 0.5(Vmax)

Michaelis-Menton Graph

Lineweaver-Burke Graph

Slope = Km/Vmax
Y-intercept = 1/Vmax
X-intercept = - 1/Km

Lineweaver-Burke Equation

Found by taking the reciprocal of the Michaelis-Menton Equation.

Kcat

Rate-limiting step in any enzyme-catalyzed reaction at saturation. Known as the "turn-over number". Kcat = Vmax/Et

Chymotripsin

Cleaves proteins on C-terminal endof Phe, Trp, and Tyr

Competitive Inhibition Graph

Slope changes by factor of α. Slope becomes αKm/Vmax.
X-intercept becomes 1/αKm
Y-intercept does not change.
Vmax does not change.

Uncompetitive Inhibition Graph

Does not change slope.
Changes Km and Vmax.
Results in vertical shift up and down.
Y-intercept becomes α'/Vmax
X-intercept becomes -α'/Km

Mixed Inhibition Graph

Allosteric inhibitor that binds either E or ES.
Pivot point is between X-intercept and Y-intercept.

Non-Competitive Inhibition Graph

Form of mixed inhibition where the pivot point is on the x-axis. Only happens when K1 is equal to K1'.

Ionophore

Hydrophobic molecule that binds to ions and carries them through cell membranes. Disrupts concentration gradients.

ΔGtransport Equation

ΔGtransport = RTln([S]out / [S]in) + ZFΔΨ

Pyranose vs. Furanose

Pyranose is a 6-membered ring.
Furanose is a 5-membered ring.

Mutarotation

Conversion from α to ß forms of the sugar at the anomeric carbon.

Anomeric Carbon

Carbon that is cyclized. Always the same as the aldo or keto carbon in the linear form.

α vs. ß sugars

α form has -OR/OH group opposite from the -CH2OH group.
ß form has -OR/OH group on the same side as the -CH2OH group.

Starch

Found in plants. D-glucose polysaccharide. "Amylose chain". Unbranched. Has reducing and non-reducing end.

Amylose Chain

Has α-1,4-linkages that produce a coiled helix similar to an α-helix. Has a reducing and non-reducing end.

Amylopectin

Has α-1,4-linkages. Has periodic α-1,6-linkages that cause branching. Branched every 24-30 residues. Has reducing and non-reducing end.

Reducing Sugar

Free aldehydes can reduce FeIII or CuIII. Aldehyde end is the "reducing" end.

Glycogen

Found in animals. Branched every 8-12 residues and compact. Used as storage of saccharides in animals.

Cellulose

Comes from plants. Poly D-glucose. Formed from ß-1,4-linkage. Form sheets due to equatorial -OH groups that H-bond with other chains.

Chitin

Homopolymer of N-acetyl-ß-D-glucosamine. Have ß-1,4-linkages. Found in lobsters, squid beaks, beetle shells, etc.

Glycoproteins

Carbohydrates attached to a protein. Common outside of the cell. Attached at Ser, Thr, or Asn residues.

Membrane Translayer Flip-Flop

Typically slow, but can be sped up with Flippase, Floppase, or Scramblase.

Membrance Fluidity

Membrane must be fluid. Cis fats increase fluidity, trans fats decrease fluidity.

Type I Integral Membrane Protein

Membrane protein with C-terminus inside and N-terminus outside

Type II Integral Membrane Protein

Membrane protein with N-terminus inside and C-terminus outside

Type III Integral Membrane Protein

Membrane protein that contains connected protein helices

Type IV Integral Membrane Protein

Membrane protein that contains unconnected protein helices

Bacteriorhodopsin

Type III integral membrane protein with 7 connected helices.

ß-Barrel Membrane Protein

Can act as a large door. Whole proteins can fit inside.

α-hemolysin

Secreted as a monomer. Assembles to punch holes in membranes.

Cardiolipin

"Lipid staple" that ties two proteins (or complexes) together in a membrane. Formed from two phosphoglycerols.

Hydrolysis of Nucleotides

Base hydrolyzes RNA, but not DNA. DNA is stable in base because of 2' deoxy position.

Chargaff's Rule

Ratio of A:T and G:C are always equal or close to 1

DNA Double-Helix

Opposite strand direction. 3.4Å distance between complementary bases. 36Å for one complete turn.

A-form DNA

Condensed form of DNA. Deeper major groove and shallower minor groove.

B-form DNA

Watson-Crick model DNA. Deep, wide major groove.

Z-form DNA

Left-handed helical form of DNA