ACS BIOCHEMISTRY EXAM

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Henderson-Hasselbach Equation

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252 Terms
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Henderson-Hasselbach Equation

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

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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.

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Salting Out (Purification)

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

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Size-Exclusion Chromatography

Separates sample based on size with smaller molecules eluting later.

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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.

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Hydrophobic/Reverse Phase Chromatography

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

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Affinity Chromatography

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

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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.

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SDS

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

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Isoelectric Focusing

Variation of gel electrophoresis where protein charge matters. Involves electrodes and pH gradient. Protein stops at their pI when neutral.

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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.

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DTT (dithiothreitol)

Reduces disulfide bonds.

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Iodoacetate

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

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Homologs

Shares 25% identity with another gene

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Orthologs

Similar genes in different organisms

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Paralogs

Similar "paired" genes in the same organism

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Ramachandran Plot

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

<p>Shows favorable phi-psi angle combinations. 3 main &quot;wells&quot; for α-helices, ß-sheets, and left-handed α-helices.</p>
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Glycine Ramachandran Plot

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

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Proline Ramachandran Plot

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

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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.

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Helix Dipole

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

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ß-sheet

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

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Anti-parallel ß-sheet

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

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Parallel ß-sheet

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

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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.

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Loops

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

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Circular Dichroism

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

<p>Uses UV light to measure 2° structure. Can be used to measure destabilization.</p>
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Disulfide-bonds

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

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ß-mercaptoethanol

Breaks disulfide bonds.

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α-keratin

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

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Collagen

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

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Myoglobin 4° Structure

Symmetric homodimer,

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Hemoglobin 4° Structure

Tetramer. Dimer of dimers. α2ß2 tetramer.

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α/ß Protein Folding

Less distinct areas of α and ß folding.

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α+ß Protein Folding

Two distinct areas of α and ß folding.

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Mechanism of Denaturants

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

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Temperature Denaturation of Protein

Midpoint of reaction is Tm.

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Cooperative Protein Folding

Folding transition is sharp. More reversible.

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Folding Funnel

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

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Protein-Protein Interfaces

"Core" and "fringe" of the interfaces. Core is more hydrophobic and is on the inside when interfaced. Fringe is more hydrophilic.

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π-π Ring Stacking

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

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σ-hole

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

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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.

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Ka for Binding

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

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ϴ-value in Binding

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

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Kd for binding

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

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High-Spin Fe

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

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Low-Spin Fe

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

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T-State

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

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R-State

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

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O2 Binding Event

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

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Hemoglobin Binding Curve

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

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

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Homotropic Regulation of Binding

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

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Heterotropic Regulation of Binding

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

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Hill Plot

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

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

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CO2 binding in Hemoglobin

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

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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.

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Michaelis-Menton Equation

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

<p>V0 = (Vmax[S]) / (Km + [S])</p>
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Km in Michaelis-Menton

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

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Michaelis-Menton Graph

knowt flashcard image
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Lineweaver-Burke Graph

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

<p>Slope = Km/Vmax Y-intercept = 1/Vmax X-intercept = - 1/Km</p>
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Lineweaver-Burke Equation

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

<p>Found by taking the reciprocal of the Michaelis-Menton Equation.</p>
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Kcat

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

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Chymotripsin

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

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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.

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

<p>Does not change slope. Changes Km and Vmax. Results in vertical shift up and down. Y-intercept becomes α&apos;/Vmax X-intercept becomes -α&apos;/Km</p>
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Mixed Inhibition Graph

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

<p>Allosteric inhibitor that binds either E or ES. Pivot point is between X-intercept and Y-intercept.</p>
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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'.

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Ionophore

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

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ΔGtransport Equation

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

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Pyranose vs. Furanose

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

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Mutarotation

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

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Anomeric Carbon

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

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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.

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Starch

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

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Amylose Chain

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

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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.

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Reducing Sugar

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

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Glycogen

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

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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.

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Chitin

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

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Glycoproteins

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

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