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CHE 4341 Baylor (Dr. Jonklaas) > Biochem Exam 1 > Flashcards

Biochem Exam 1 Flashcards

1
Q
A

Alkane

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2
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Alkene

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3
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Alkyne

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4
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Benzene Ring

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5
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Amine

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6
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Alcohol

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7
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Ether

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8
Q
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Alkyl Halide

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9
Q
A

Thiol

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10
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Aldehyde

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11
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Ketone

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12
Q
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Ester

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13
Q
A

Carboxylic Acid

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14
Q
A

Amide

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15
Q
A

Nitrile

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16
Q
A

Epoxide

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17
Q
A

Imine

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18
Q
A

Acid Chloride

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19
Q
A

Anhydride

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20
Q
A

Sulfide

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21
Q
A

Guanidino

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22
Q

What kind of molecules to condensation reactions make?

A

Biopolymers

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23
Q

What is a hydrolysis reaction? What reaction is it the opposite of?

A

Hydrolysis breaks down biopolymers. It is the opposite reaction of a condensation rxn.

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24
Q
A

Acyl Group

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25
Q

What kind of compounds contain stored chemical energy?

A

Anhydrides

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26
Q
A

Phosphoanhydride

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27
Q
A

Phosphoester

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28
Q

What kinds of bonds are considered “low energy”?

A

Phosphoesters

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29
Q

Who proposed the theory of the “warm little pond” for the basis of chemical evolution?

A

Charles Darwin

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30
Q

What are the steps of Prebiotic (Chemical) Evolution?

A
  1. Creating Monomers
  2. Making polymers
  3. Making Self-Replicating Systems
  4. Making Life
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31
Q

Who proposed the idea for the experiment that modeled the “warm little pond”, that was meant to replicate the conditions of the planet before life was formed?

A

Oparin and Haldane

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32
Q

Who conducted the experiment that mimicked the conditions of earth that were thought to give rise to life?

A

Urey and Miller

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33
Q

What was the purpose of Urey and Miller’s experiment?

A

To create a spark discharge apparatus for demonstration of abiotic formation of organic compounds

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34
Q

What are the possible explanations for how polymers were assembled in prebiotic evolution?

A
  1. Building blocks accumulated in solution
  2. Condensation reactions to form polymers (possibly catalyzed by metal ions in clay)
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35
Q

By what fundamental principle does DNA replicate?

A

Complementarity

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36
Q

What is complementarity, and why is it important?

A

Complementarity is the specific pairing of functional groups. It allows macromolecules to replicate.

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37
Q

What are the levels of the hierarchical assembly of life?

A
  1. Small organic molecules (amino acids)
  2. Macromolecules (proteins and DNA)
  3. Supramolecular structures (membranes)
  4. Organelles
  5. Cells
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38
Q

Who proposed the “RNA World Hypothesis”?

A

Proposed independently by Woese, Crick, and Orgel

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39
Q

What does the “RNA World Hypothesis State”? Why is RNA unique compared to DNA?

A

States that the initial biopolymer was RNA (rather than DNA). This is becuase RNA is capable of both carrying genetic information and catalysis.

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40
Q

Who discovered the first RNA enzyme? (ribozyme)

A

Thomas Cech

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41
Q

What are 2 many scientists have with the accepted theory for evolution?

A
  1. Low concentration of building blocks
  2. Hydrolysis is the favored reaction (condensation reaction required for production of biopolymers)
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42
Q

Why is hydrolysis favored over condensation reactions?

A

Most biopolymers are unstable at high temperatures. Also, biopolymers do not replicate reliably by themselves when longer than 40-60 units.

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43
Q

How does the homochirality of Amino Acids relate to the theory of chemical evolution?

A

When a chiral center is formed, a 50:50 racemic mixture is made of each enantiomer of that molecule. However, in nature, we see that proteins are made of only L-enantiomer amino acids.

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44
Q

Which enantiomer of amino acids is favored in nature?

A

L-enantiomer

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45
Q

What has new discoveries in astrobiology taught us about Urey and Miller’s experiment?

A

Recent studies show earth’s atmosphere was never as reducing as Urey & Miller’s experiment presumed (i.e. not enough H) = Even lower production of organic compounds.

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46
Q

Where do some people say that the amino acids and nitrogen-containing bases might have been formed?

A
  1. Deep sea thermal vents
  2. Delivered by interstellar dust, meteorites, or comets
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47
Q

Who proposed the theory of “Directed Panspermia”?

A

Francis Crick

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48
Q

What does the theory of “Directed Panspermia” state?

A

That life on earth originated from aliens

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49
Q

What is traditional phylogeny based on?

A

Taxonomy (the way something looks)

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50
Q

Who proposed using ribosomal RNA sequences as an evolutionary measure?

A

Carl Woese

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51
Q

What is the more modern approach to the phylogeny tree based on?

A

Ribosomal RNA sequences are used as an evolutionary measure (rather than taxonomy)

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52
Q

Who discovered the 3rd superkingdom of life?

A

Carl Woese

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53
Q

How was the third superkingdom of life discovered?

A

By using ribosomal RNA sequences as an evolutionary measure, rather than using taxonomy

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54
Q

What is the third superkingdom of life?

A

Archaea (prokaryotes); More related to eukaryotes than bacteria

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55
Q

Compare the sizes of prokaryotic and eukaryotic cells.

A

Prokaryotes are generally much smaller than eukaryotes

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56
Q

Compare the genomes of prokaryotes and eukaryotes.

A

Prokaryotes have DNA with non-histone proteins, while eukaryotes have DNA with both histone and non-histone proteins

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57
Q

Compare cell division in prokaryotes and eukaryotes.

A

Prokaryotic cells divide by fission or budding. Eukaryotic cells divide by mitosis.

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58
Q

Compare the cytoskeleton of prokaryotes and eukaryotes.

A

Prokaryotes do not have a cytoskeleton. Eukaryotes have a very complex cytoskeleton, consisting of microtubules, intermediate filaments, and actin filaments.

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59
Q

compare intracellular movement in prokaryotic and eukaryotic cells.

A

Prokaryotes have no intracellular movement. Eukaryotes have cytoplasmic streaming, endocytosis, phagocytosis, mitosis, and vesicle transport.

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60
Q

Who proposed the endosymbiotic origin of mitochondria and chloroplasts?

A

Lynn Margulis

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61
Q

What is one major source of evidence that supports the endosymbiotic theory?

A

Both mitochondria and bacteria have circular DNA

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62
Q

What is the definition of living system?

A

A living system must have the ability to extract energy from its environment, transform it and to use that transformed energy to be productive.

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63
Q

What is the definition of a system?

A

Any part of the universe chosen for study. Must have defined boundaries.

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64
Q

What is an open system?

A

Able to exchange energy and matter with surroundings

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65
Q

What is a closed system?

A

Allows only exchange of energy with its surroundings and disallows exchange of matter

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66
Q

Are cells open or closed systems?

A

Open systems

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67
Q

What is the definition of Internal Energy (U)?

A

Any system contains a certain amount of internal energy, which includes all forms of energy that can be exchanged via simple physical processes or chemical reactions.

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68
Q

What are some examples of Internal Energy (U)?

A
  1. The kinetic energy of motion of the system’s atoms and molecules
  2. The vibrational and rotational energy of the system’s atoms and molecules
  3. All the energy stored in the chemical bonds between atoms
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69
Q

What is the 1st Law of Thermodynamics?

A

Energy is conserved, it cannot be created nor destroyed.

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70
Q

What is a State Function?

A

State functions are independent of path. Their values depends only on initial and final state of the system.

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71
Q

What is Enthalpy (H)?

A

Delta H refers to the change in heat of a reaction, which for biochemical pathways, is synonymous with the change in energy of a reaction

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72
Q

What is Entropy (S)?

A

A measure of the randomness or disorder in a system. Only used for closed systems.

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73
Q

What do you have to do in order to create order in a system?

A

Expend energy

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74
Q

What is the 2nd Law of Thermodynamics?

A

The entropy of the universe is always increasing

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75
Q

What is Gibbs Free Energy (G)?

A

For open systems, Gibbs Free Energy (G) is the State function used. It describes the free energy change for a process at a constant temperature and pressure.

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76
Q
A

G = Gibbs Free Energy
H = Enthalpy (heat)
T = Temperature (in Kelvin)
S = Entropy (randomness)

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77
Q

Classify a reaction where ∆G < 0

A

Exergonic reaction (spontaneous); Energy released

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78
Q

Classify a reaction where ∆G > 0

A

Endergonic reaction (reverse rxn favored); Energy consumption

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79
Q

Classify a reaction where ∆G = 0

A

Reaction at equilibrium (energy neither consumed or released)

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80
Q

Where does ∆H come from in Gibbs Free Energy reactions?

A

∆H comes from numerous weak interactions; H-bonds + other non-covalent interactions within the protein and with the solvent.

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81
Q

Is ∆G concentration dependent?

A

Yes

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82
Q
A

∆G = Change in Gibbs Free Energy
∆G°’ = Biochemical Standard ∆G
R = 8.3145 J/mol*K
T = Temperature (in Kelvin)
Q = Rxn Quotient (products/reactants)

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83
Q

Are living things ever at equilibrium?

A

No, living things are at a steady state in a constant rate of FLUX

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84
Q
A

Henderson-Hasselbalch Equation

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85
Q

What is a buffer?

A

A “pH shield”; Able to minimize the change in pH

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86
Q

When does a buffer occur?

A

Buffer occurs when weak acid (HA) and its conjugate base (A-) are PRESENT IN NEARLY EQUAL PROPORTIONS

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87
Q

How do you make a buffer?

A

Need weak acid and conjugate base in close to equal proportions

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88
Q

What are the 3 ways to make a buffer?

A
  1. Start with weak acid & no conjugate base, and add a measured amount of strong base
  2. Start with weak base & no weak acid, and add a measured amount of strong acid
  3. Mixing a specific amount of a weak acid HA and its conjugate base A- together
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89
Q

What percentage of the mass of the human body is water?

A

About 2/3

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90
Q

What does the reactivity of many functional groups in biological molecules depend on?

A

pH

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91
Q

Rank the various types of chemical bonds from strongest to weakest.

A
  1. Covalent
  2. Ionic
  3. Hydrogen bond
  4. Dipole-dipole interaction
  5. London dispersion forces (van der Waals)
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92
Q

What is the bond strength of a Hydrogen Bond?

A

20 kJ/mol

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93
Q

What is the general structure of a Hydrogen bonding interaction?

A

A hydrogen is “shared” between two highly electronegative elements (often N, O, F, Cl, or sometimes S)

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94
Q

Describe Ice. What allows for the formation of Ice?

A

Ice is a hexagonally packed (honeycomb shaped) netweork that fully satisfies the H-bonding potential of H2O molecules.

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95
Q

What is the maximum number of H-bonds a water molecule can have?

A

4

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96
Q

At what temperature is water the most dense?

A

At 4°C, where it is in the liquid state

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97
Q

What is water a good solvent for?

A

Water is a very polar solvent. This makes it good for dissolving Salts and other charged/polar species.

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98
Q

What happens when a small amount of a non-polar solute gets immersed in water?

A

Water molecules must “organize” in an H-bond network around the nonpolar solute to accomodate it. This is not favorable, because entropy is decreasing! Water molecules do not like organization!

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99
Q

What is a Clathrate?

A

A compound in which molecules of one component are physically trapped within the crystal structure of another

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100
Q

Describe a hydrophobic interaction?

A

Entropically driven by the overall increase in entropy that water molecules achieve by excluding a non-polar solute.

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101
Q

What is an Amphipathic Molecule?

A

Molecules that contain both polar and nonpolar groups. (ex. fatty acids or SDS)

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102
Q

What is a micelle?

A

All hydrophobic groups are sequestered from water, and hydrophobic tails are clustered together, with hydrophillic heads pointed outwards.

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103
Q

What drives micelle formation?

A

Entropically driven (hydrophobic effect)

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104
Q

What do two-tailed amphiphiles form in high concentration?

A

Bilayer vesicles (cell membrane)

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105
Q

What is Osmosis?

A

The movement of water from a region of high water concentration to low water concentration

106
Q

What does osmotic pressure depend on?

A

Solute concentration

107
Q

How many inflection points does a titration of a molecule with 3 acidic hydrogens have?

A

3 titration points (there is 1 titration point for every acidic hydrogen)

108
Q

Where are buffers the best? (relative to pH)

A

At pH = pKa

109
Q

Describe a respiratory alkalosis

A

Hyperventilation expels too much Co2 so not enough acid, blood pH goes up!

110
Q

What controls Co2 concentration in the human body’s blood?

A

Breathing rate

111
Q

What is the reference compound in the Fischer Convention?

A

L & D glyceraldehyde

112
Q

Does the Fischer Convention describe optical rotation?

A

No, optical rotation (designated d & l or +/-) can only be determined empirically.

113
Q

L or D enantiomer? Why?

A

This is the D enantiomer, because the most oxidized carbon is at the top, the R group is at the bottom, and the non-H group is pointing to the RIGHT

114
Q

L or D enantiomer? Why?

A

This is the L-enantiomer of the amino acid, because using the CORN method, CO, R, N is spelled clockwise, all while the H is pointing out towards you (giving you a hug). Therefore, this is the L-enantiomer.

115
Q

What 2 components do all amino acids have?

A

An amine (weak base), and a carboxylic acid (weak acid)

116
Q

Describe what it means for amino acids to be ampholytic.

A

Ampholytic means that they can act as both acids and bases

117
Q

Define a Zwitterion

A

A neutral molecule carrying both a positive and a negative charge

118
Q

Describe the melting point of Amino Acids. What makes them this way?

A

Have a HIGH melting point, due to their ionic nature

119
Q

What makes something acidic?

A

The better you can stabilize the conjugate base, the more acidic the molecule in the first place

120
Q

What makes the pKa of the carboxylic group on an amino acid lower than that of a normal carboxylic acid?

A

Due to the proximity to the NH3+, the positive charge further stabilizes the conjugate base

121
Q

Is the carboxylic acid group on an amino acid a stronger or a weaker acid than a normal carboxylic acid.

A

Stronger acid. This is due the proximity of the amino group that stabilizes the conjugate base.

122
Q

Is the amino group on an amino acid a stronger or weaker base than a normal amino group?

A

Weaker base. It is affected by the carboxylic acid on the other side of the alpha carbon.

123
Q

What is the pK1 of an amino acid? (COOH)

A

2.5

124
Q

What is the pK2 of an amino acid? (NH3+)

A

9.5

125
Q

How many amino acids are chiral?

A
  1. Glycine is not chiral.
126
Q

What is unique about d- amino acids? Where can they be found in nature?

A

They are poisonous. They can be found in bacterial cell walls. This is a defense mechanism, so that proteases cannot hydrolyze bacteria.

127
Q

Describe the rules of the CORN method

A
  1. With the H pointing out towards you, if CORN is spelled CLOCKWISE, then it is the L-enantiomer
  2. With the H pointing out towards you, if CORN is spelled COUNTERCLOCKWISE, then it is the d-enantiomer
    Opposite if H is pointing AWAY from you
128
Q

Which L- amino acids are in the R configuration?

A

18 of the 19 chiral L-amino acids have the S-configuration. Cysteine is the ONLY one with the R- configuration

129
Q

How is a weakly acidic group defined?

A
130
Q

How is a weakly basic group defined?

A
131
Q

Is an Amide group basic? Why not?

A

No, the lone pair on the Nitrogen is involved in RESONANCE

132
Q

At what pH did we memorize the amino acids?

A

pH = 7.0

133
Q

Which amino acid has an isopropyl group on the side chain?

A

Valine

134
Q

Which amino acid has an isobutyl group?

A

Leucine

135
Q

Which amino acid has a sec-butyl group?

A

Isoleucine

136
Q

Which amino acid has a thioether group?

A

Methionine

137
Q

What is a diastereomer?

A

2 chiral carbons in the same molecule. They are stereoisomers that are not mirror image isomers.

138
Q

Which are the two amino acids that have diastereomers?

A

Isoleucine and Threonine

139
Q

Which amino acid has an indole side chain?

A

Tryptophan

140
Q

Which amino acid has a mercaptan (thiol) (sulfahydryl)
side chain?

A

Cysteine

141
Q

Describe Oxidation in an O-chem sense.

A

Removal of bonds to H &/or Adding bonds to O

142
Q

Describe Reduction in an O-chem sense.

A

Gaining of bonds to H &/or Removing bonds to O

143
Q
A

Cysteine

144
Q

Which amino acid has an imidazole group?

A

Histadine

145
Q

Which amino acid has a guanidino group?

A

Arginine

146
Q

Which amino acids strongly absorb uv light?

A

Phenylalanine, Tyrosine, Tryptophan (FYW)

147
Q

What is the average maximum absorbance of the FYW?

A

280 nm

148
Q

What are Hydropathy values?

A

A measure of polarity. The smaller the Hydropathy score, the more polar the side chain of the amino acid.

149
Q

What is an essential nutrient?

A

Cannot be synthesized by the organizm and therefore must be supplied as part of its diet?

150
Q

Which amino acids are notably essential for humans?

A

The branched amino acids (Val, Leu, Ile)

151
Q
A

Selenocysteine

152
Q

What is Gamma-aminobuturate (GABA)?

A

A cheif inhibitory neurotransmitter in the mammalian central nervous system (CNS)

153
Q

What is a peptide bond?

A

The amide bond between two amino acids

154
Q

By convention, what side is the N-terminus generally shown on?

A

The left side

155
Q

What is Bradykinin used for?

A

Causes blood vessels to dilate, and therefore causes blood pressure to lower

156
Q

What is Oxytocin used for?

A

Stimulates uterine contractions during childbirth (also, along with Vassopressin, is a mammalian GLUE HORMONE)

157
Q

What drives protein folding?

A

The Hydrophobic effect

158
Q

What is the definition of a Protein?

A

Large biological polypeptide (>40 amino acids long)

159
Q

What are the primary roles of proteins?

A
  1. Synthesis/Breakdown (enzymes catalyze chemical reactions)
  2. Signaling
  3. Structure
  4. Transport
160
Q

What happens if a protein denatures?

A

A protein has to have the correct 3 D structure to function; If it denatures, it loses structure & thus function

161
Q

How is chirality important in enzymes?

A

Proteins have active sites that may only be receptive to a certain enantiomer

162
Q

What is Thalidomide?

A

Supposed to be a “wonder drug” to combat morning sickness; Given as a RACEMIC MIXTURE; S-isomer causes horrible birth defects (flipper arms)

163
Q

Describe the rules for net charge at a given pH for WEAK ACIDS

A
164
Q

Describe the rules for net charge at a given pH for WEAK BASES

A
165
Q

What is the pKa of the amino group whenever it is part of a polypeptide?

A

8.5

166
Q

What is the pKa of the carboxylic acid group whenever it is part of a polypeptide?

A

3.5

167
Q

Which group will get titrated first on an amino acid?

A

The group with the lowest pKa will be titrated first (carboxylic acid)

168
Q

What can you look at on a titration curve to determine how many weak acid/base groups there are?

A

The number of equivalents of strong base added

169
Q

What is the Isoelectric point?

A

The pH where AA/polypeptide carries a 0 net charge (occurs between the -1 and +1 charge state)

170
Q

What is the formula for pI in amino acids without a pKr?

A

Also used for Tyrosine!

171
Q

What is the formula for pI in amino acids with weakly acidic side chains?

A
172
Q

Which amino acids have acidic side chains?

A

D, E, C, Y

173
Q

What is the formula for pI in amino acids with weakly basic side chains?

A
174
Q

What is the primary structure of a protein?

A

The linear sequence of amino acids and the location of any disulfide bridges

175
Q

Who was the first person to sequence a protein (bovine insulin)

A

Frederick Sanger

176
Q

Who won 2 Nobel Prizes?

A

Frederick Sanger

177
Q

What are the 3 steps involved in protein sequencing?

A
  1. Separate into individual polypeptide chains
  2. End Terminus analysis
  3. Sequencing
178
Q

What are the 2 methods to Cleave disulfide bonds? (separate into individual chains)

A
  1. OXIDIZE with Performic Acid
  2. A.) React with XS Thiol to REDUCE -S-S- bonds
    B.) “Cap” with iodoacetate (prevents reverse rxn)
179
Q

What are carboxypeptidases?

A

Exopeptidases that cleaves Amino Acids at the C-terminus

180
Q

What is the process for C-Terminus Analysis (protein sequencing)

A

Carboxypeptidases cleave protein peptide bonds from end, inwards

181
Q

What does Carboxypeptidase B cut? (end terminus analysis)

A

ONLY Lys & Arg (“Basic”)

182
Q

What does Carboxypeptidase A cut? (end terminus analysis)

A

All the others! (except Lys & Arg) (“All the others”)

183
Q

What are the 3 reagents that reach with NH2-terminus of a polypeptide? (N-terminal analysis)

A

Fluorodinitrobenzene, Dabsyl Chloride, Dansyl Chloride

184
Q
A

Fluorodinitrobenzene (N-terminus analysis)

185
Q
A

Dansyl Chloride (N-terminus analysis)

186
Q
A

Dabsyl Chloride (N-terminus analysis)

187
Q

Which Amino Acid will notably give you a false positive with N-terminus analysis? Why?

A

Lysine, because of its NH2 side chain (mimics N-terminus)

188
Q

What is a disadvantage of using Dansyl Chloride and Dabsyl Chloride for N-terminus analysis?

A

Presence of STRONG ACID in the last step hydrolyzes the whole peptide (destroys sequence)

189
Q

Describe the steps of the reaction that involves Dabsyl/Dansyl chloride to conduct N-terminus analysis.

A
  1. Use weak base to bind Dabsyl/Dansyl to the polypeptide
  2. Use STRONG ACID to cleave Dansyl/Dabsyl amino acid from the polypeptide
190
Q

By what mechanism does Fluorodinitrobenzene (DNFB) react during N-terminus analysis?

A

Nucleophillic Aromatic Substitution (NAS)

191
Q

What is significant about the Edman Reagent (PITC)?

A

The Edman reagent can be used for N-terminus analysis. However, it is unique because it does not require the use of strong acid. Therefore, it can also be used to sequence proteins directly, by cleaving 1 AA at a time, with a 98% yield. “Rinse and Repeat”

192
Q

Summarize the steps of the Edman Reagent reaction in N-terminus analysis.

A
  1. PITC (Edman) + Polypeptide
  2. Anhydrous TFA (rearranges molecule)
  3. Weak Base
    = PTH - Amino Acid
193
Q

What is the maximum length of a polypeptide that Edman Reagent can be used to sequence?

A

100 Amino Acid upper limit!

194
Q

What must you do, if your polypeptide is longer than 100 Amino Acids in length?

A

Fragmentation!!!!!!

195
Q

What is an Endoprotease?

A

Cleave proteins at sites within the chain (used for fragmentation)

196
Q

What are the 3 endoproteases used for fragmentation? Describe their specificity.

A
197
Q

What can endoproteases NOT CUT AFTER? Why? (fragmentation)

A

Proline (ring causes steric hindrance)

198
Q

What are the 2 Fragmentation Methods?

A
  1. Endoproteases (3)
  2. Cyanogen Bromide (CNBr)
199
Q

What does Cyanogen Bromide (CNBr) cut after?

A

Methionine

200
Q

How do you figure out where disulfide bonds occur in a polypeptide chain?

A

Sequence protein as normal, then repeat fragmentation without breaking disulfide bonds to identify the Cys-containing sequences involved in the disulfide linkages

201
Q

What are the methods for sequencing proteins?

A
  1. Fragment and use CNBr
  2. Determine protein sequence via DNA/RNA sequenceing (indirect sequencing)
  3. Sequence by Mass Spectrometry (MS)
202
Q

Summarize the steps of Protein Sequencing by Mass Spectrometry (MS)

A
  1. Use Trypsin digestion to determine protein sequence in a technique called Peptide Mass Fingerprinting (PMF)
  2. Use MALDI/ESI to gently vaporize protein fragments
  3. Time of Flight (TOF) uses a long tube where each ion has the same electrostatic force applied, so that smallest fragments move fastest (and biggest move slowest)
    = A “fingerpring” of fragment masses
203
Q

What does MALDI-TOF stand for?

A

Matrix Assisted Laser Desorption Ionization
Time of Flight

204
Q

What does ESI-TOF stand for?

A

Electrospray Ionization
Time of Flight

205
Q

Summarize the technique of Tandem Mass Spectrometry

A

Used for Protein Sequencing. Useful for PROTEOMICS. First step separates individual proteins by mass. Helium Collision chamber then fragments each individual protein. Second step separates the fragments by mass.

206
Q

In the realm of bioinformatics, why was it significant that scientists determined that the x-ray structures of proteins in different species appear to be very similar, despite their sequence of AA being different?

A

This lead evolutionary biochemists to believe that the essential structural elements of proteins (rather than their amino acid residues) are conserved during evolution

207
Q

Define Invariant Regions, and describe what makes them significant.

A

Amino Acids DO NOT CHANGE
Thus, a protein must have each of these AA’s in the correct place for stable structure/proper function

208
Q

Define Conserved Regions, and describe what makes them significant.

A

Amino Acids have the same POLARITY
Thus, AA polarity is important for structure/function

209
Q

Define Hyper-variable regions, and describe what makes them insignificant

A

Amino Acids not conserved
Thus, polarity is not important for structure/function

210
Q

Describe how a Phylogenetic Tree is a “molecular clock”?

A

Amino acid difference between 2 species shows how long ago they diverged in their evolutionary history. Shows that Amino Acid differences survived natural selection.

211
Q

Are all proteins capable of accepting a constant rate of mutation?

A

No, some proteins cannot accept mutations because the mutations kill the function of the protein. Other proteins are significantly more resilient.

212
Q

What is a protein family?

A

A group of evolutionarily-related proteins. Share a common ancestor and typically have similar 3D structures, functions, and have a significant sequence similarity.

213
Q

Define Homologous

A

Share a common ancestor!

214
Q

What are 3 methods of Cell Disruption for Protein Purification

A
  1. Osmotic Lysis (hypotonic lysis)
  2. Mechanical Disruption (blender, french press)
  3. Chemical Disruption (detergents, enzymes)
215
Q

Whenever you are purifying a protein, why do you want to carry out the purification in as few steps as possible?

A

To avoid denaturing

216
Q

What strategy of Purification separates proteins by CHARGE?

A

Ion Exchange, Electrophoresis, Isoelectric Focusing

217
Q

What strategy of Purification separates proteins by POLARITY?

A

Paper Chromotography

218
Q

What strategy of Purification separates proteins by SIZE?

A

Dialysis, Gel Electrophoresis, Gel Filtration, Ultracentrifugation

219
Q

What strategy of Purification separates proteins by SPECIFICITY?

A

Affinity Chromatography

220
Q

What strategy of Purification separates proteins by SOLUBILITY?

A

Salt Precipitation

221
Q

When centrifugal force is applied to an aqueous mixture, which components will sediment faster?

A

Larger mass will sediment faster

222
Q

Summarize the fundamental process of Liquid Chromatography techniques

A
  1. A protein mixture is dissolved in a liquid (mobile phase)
  2. A column that contains a porous solid matrix (stationary phase)
  3. Mobile phase is eluted through stationary phase
    = Each stationary phase (column) separates based on different characteristics.
223
Q

What are the 2 types of columns used for Ion Exchange Chromatography (IEC)?

A

Carboxymethyl (CM) (-)
Diethylaminoethyl (DEAE) (+)

224
Q

In what order do proteins elute through an IEC column? (generally stated)

A

Zero and like-charge proteins elute 1st, TOGETHER (followed by opposite-charge proteins)

225
Q

By what 2 methods can you “wash” an IEC column?

A
  1. Increasing the Salt Concentration (ions will trade places with protein of interest bound to column)
  2. Altering the pH of the mobile phase (this will change the protein’s charge, which will release it from column)
226
Q

How do you determine the charge of a protein at a given pH, when given the protein’s pI (useful for protein purification to determine what will “stick” to column)

A
227
Q

By what property does Gel Filtration Chromatography (GFC) (i.e. size-exclusion, molecular exclusion, molecular sieve) separate proteins? Which proteins will elute through the column first?

A

Separates by size (molecular weight). Largest proteins will elute through the column first, because they are too big to get “lost” in the Gel’s small pores.

228
Q

For what technique does a plot of log(MW) vs. elution volume (Ve) give a straight line?

A

Gel Filtration Chromatography (GFC); Can be used to determine MW of an unknown protein.

229
Q

Summarize how Affinity Chromatography (AC) works.

A

“Fishing” for protein of interest!! Ligand is the “bait” that attracts the protein of interest (which has a specific affinity for the ligand); Ligand is able to “capture” the protein of interest.

230
Q

How is the absorbed protein eluted in Affinity Chromatography?

A
  1. Changing the conditions towards dissociation (salt conc./pH)
  2. Adding excess of free ligand/ligand analog
231
Q

Describe the process of His Tagging (affinity chromatography)

A

Attach His to Protein of interest. Imidazole rings have a high affinity for Nickel, so you bind Nickel containing substrates to silica beads in column. This nickel “fishes” for protein of interest (which has imidazole groups attached)

232
Q

What is HPLC?

A

The use of a high pressure system to push a liquid mobile phase solution through a column, allowing a separation of complex mixtures with high resolution

233
Q

What does HPLC stand for?

A

High Pressure Liquid Chromatography

234
Q

What is the difference between Normal Phase HPLC and Reverse Phase HPLC?

A

Normal Phase: Silica Beads (polar column)
Reverse Phase: Silica Beads with alkyl groups (non-polar column)

235
Q

What is Amino Acid Analysis (AAA)? (HPLC application)

A

A process by which proteins are hydrolyzed, and then each amino acid is quantitatively analyzed by HPLC

236
Q

What happens during Amino Acid Analysis (AAA) that is important to note when determining the amino acid isolated by the column?

A

Whenever the peptide bonds are hydrolyzed (by either strong acid or strong base), Gln is converted to Glu, and Asn is converted to Asp.
Thus, you cannot tell the difference between Gln/Glu and Asn/Asp, so the abbreviations Glx and Asx are used.

237
Q

What 3 factors affect protein solubility?

A
  1. pH
  2. Solvents
  3. High temperature
238
Q

At what pH are proteins the least soluble?

A

At pH = pI (no charge, hydrophobic)

239
Q

Describe what is means to “salt in” proteins. What does it mean to “salt out” proteins?

A

Salting in: At low concentrations, added salt increases the solubility of proteins (because the salt ions get in between proteins, preventing them from “sticking” together)
Salting out: At high concentrations, salt lowers the solubility of proteins because it pulls water away from proteins (thus, high salt conc. causes proteins to “crash” out of solution)

240
Q

What is the best salt for salting in/out? Why?

A

Ammonium sulfate (because it can dissolve to high M)

241
Q

What is Dialysis?

A

Uses osmosis to change ions in solution

242
Q

What does Gel Electrophoresis (GE) separate biomolecules by?

A

Charge and size

243
Q

What is the charge of the anode? What about the Cathode? (gel electrophoresis)

A

Anode (+) “Anions run to the anode”
Cathode (-)

244
Q

In Gel Electrophoresis, which molecules move the farthest through the gel? (size and charge)

A

Small, negative (anions run to the anode)

245
Q

At what pH is Gel Electrophoresis (GE) typically run? Why?

A

At pH = 9, because this gives most proteins neg. (-) charge

246
Q

What does PAGE stand for? (SDS-PAGE)

A

Polyacrylamide Gel Electrophoresis

247
Q

What is this molecule? What is it used for?

A

Coomassie Blue
Used to stain and visualize proteins on Gel (SDS-PAGE)

248
Q

What does SDS stand for? (SDS-PAGE)

A

Sodium Dodecylsulfate (detergent)

249
Q

Describe what happens during SDS-PAGE.

A
  1. SDS coats proteins uniformly and DENATURES them
  2. Due to its negative charge, SDS gives each protein the same charge:mass ratio (ideally)
  3. This allows separation only by MOLECULAR WEIGHT (ideally)
250
Q

How does SDS-PAGE separate proteins? Explain.

A

> Separates by SIZE ONLY …. largest move least! (opposite of GFC)
Since proteins are DENATURED, it gives SUBUNIT MW (whereas GFC gives total MW)
Like GFC, LogMW vs. relative migration = Straight line

251
Q

What is the primary use of SDS-PAGE for biochemists?

A

Used to track protein purification (one band most likely means pure protein)

252
Q

Describe Paper Electrophoresis and Paper Chromatography

A

Cellulose Paper is used as matrix (polar!)

253
Q

What compound is used to detect proteins when using paper electrophoresis/chromatography?

A

Ninhydrin

254
Q

What molecule is this? What is it used for?

A

Ninhydrin. Stains amino acids and proteins, particularly used for paper chromatography/electrophoresis

255
Q
A

Ruhemann’s Purple; Used to stain AA and proteins for visualization

256
Q

What does Isoelectric Focusing (IEF) separate proteins by?

A

Separates proteins by their ISOELECTRIC POINTS (where pI = pH)

257
Q

Summarize 2D Gel Electrophoresis

A
  1. Run IEF gel in one dimension to separate by pI
  2. Run SDS-PAGE in 2nd dimension to separate by size
    ***Can be very useful for PROTEOMICS
258
Q

What does ELISA stand for?

A

Enzyme-Linked Immunosorbent Assay

259
Q

How does ELISA work?

A

Uses Antibodies to detect proteins of interest

260
Q

Why is ELISA particularly useful?

A

Has a very HIGH SENSITIVITY (makes it useful to detect viruses like AIDS, food allergens, etc.)

261
Q

Define the concept of Blotting and explain why it is important.

A

Blotting is the process of transferring from gel to membrane. It is useful because gels are very flimsy, which makes them hard to work with. Membranes are much more DURABLE

262
Q

What are the 3 different kinds of blotting?

A

Southern blotting = DNA
Northern blotting = RNA
Western Blotting = Proteins

CHE 4341 Baylor (Dr. Jonklaas) (14 decks)

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