Biochemistry

Question 1

Hydropathy

Answer 1

the relative hydrophobicity of each amino acid

Question 2

How to prep your protein to analyze amino acids

Answer 2

1. cleave di-sulfide bonds with Mercaptoethanol and acetase
2. Peptide bonds cleared via acid hydrolysis or autoclave
3. Amino acids chromatographied and quantified
4. Derivatize with PITC before HPLC (edman’s reagent)

Question 3

Edman Degradation

Answer 3

Cleaves the N-terminal peptide bond and labels it without disrupting the other amino acids of the chain
1. treat with PITC and form PTC peptide with N terminus
2. treat with TFA to selectively cleave N-Terminal Peptide bond
3. Separate N-terminal derivative from Peptide
4. convert derivative to PTH amino acid
ID with chromatography

Question 4

What are ways to purify your protein

Answer 4

Ammonium sulfate and acetone to “salt” out your protein (precipitation)
Chromatography (ion-exchange, gel-filtration, and affinity chromatography)

Question 5

Ion-exchange chrom

Answer 5

separates based on overall charge of molecule

Question 6

Gel-filtration chrom

Answer 6

separates based on molecular size

Question 7

Affinity chromatography

Answer 7

separation based on specific binding interactions between column matrix and target proteins

Question 8

What are protease enzymes?

Answer 8

Enzymes used to cleave specific peptide bonds
Chymotrypsin
Trypsin
Staph V.8 Protease
CNBR

Question 9

Chymotrypsin

Answer 9

cleaves the carbonyl side of the aromatic or bulky noncharged aliphatic reside (Phe, Tyr, Trp, and Lys)

Question 10

Trypsin

Answer 10

Carbonyl sides for basic residues (Lys, Arg)

Question 11

Staph V8 protease

Answer 11

carbonyl side of charged residues (Glu and Asp)

Question 12

CNBR

Answer 12

cleaves met-terminal

Question 13

Alpha Helix

Answer 13

C=O (residue n) forms H bond with hydrogen on the amide residue n+4
Stabilized by h bonds (parallel to long axis of helix)
C=O groups point towards C-terminus (entire helix is a dipole)

Question 14

B strands

Answer 14

polypeptide chains that are almost fully extended (slightly folded)

Question 15

B sheets

Answer 15

multiple B strands arranged side by side; stabilized by C=O and -NH on adjacent strands

Question 16

What is a motif? and examples?

Answer 16

Motifs -- recurring protein structures (consists of several domains)
helix-loop helix (two helices connected by turn)
Coiled-coil (two ampiphatic alpha helices that interact in parallel thru hydrophobic edges)
Helix bundle (several alpha helices that associate in antiparallel manner to form bundle)
B&B (two parallel B strands linked to intervening alpha helix by two loops)

Question 17

Myoglobin structure

Answer 17

8 alpha helices and is water soluble
heme prosthetic group binds oxygen
His-93 complexed to iron atom; His-64 forms H bond with oxygen

Question 18

Hemoglobin structure

Answer 18

Hb is an A2B2 tetramer (2 alpha globin subunits, 2 B subunits)
Each globin subunit is similar in structure to myoglobin
Each subunit has heme group
Alpha chain has 7 alpha helices, B chain has 8 alpha helices

Question 19

Positive Cooperativity

Answer 19

The O2 affinity of Hb increases as each O2 molecule is bound

Question 20

2, 3 BPG

Answer 20

Allosteric effector of Hb –> LOWERS affinity of deoxyHb for Oxygen by paring with the + charges on the central cavity of DeoxyHb, therefore stabilizing it
Negative charged

Question 21

What are the two conformations of hemoglobin?

Answer 21

Active state (R) and inactive (T) –> they’re in rapid equilibrium in allosteric proteins
activators stabilize R state, shifts equilibrium in R direction
allosteric inhibitors stabilize T state and shift eq to T

Question 22

Bohr Effect

Answer 22

Lowers pH which decreases Hb affinity for oxygen
Based on eq: CO2 + H2O H+ + HCO-
Increasing pH stimulates Hb to bind more O2 at lower O2 pressures