Exam 1 Flashcards
1
Q
What can an alcohol be oxidized into?
A
alcohol - aldehyde - carboxylic acid
2
Q
What are three differences between prokaryotic and eukaryotic cells?
A
Prokaryotic: - no membrane bound organelles - free circular DNA - 1-10 um Eukaryotic - membrane bound organelles - chromatin bound DNA - 10 -100um
3
Q
What is the role of the nucleus?
A
carries genetic information, site of transcription
4
Q
What is the role of the endoplasmic reticulum?
A
secretory protein and lipid synthesis
5
Q
What is the role of the golgi?
A
glycosylation, distribution center
6
Q
What is the role of the mitochondria?
A
respiration (TCA cycle), electron transport, ATP synthesis
7
Q
What is the role of the lysosome?
A
acidic compartments with proteases that break down proteins
8
Q
What is the role of the peroxisome?
A
lipid breakdown
9
Q
What is kinetics?
A
will the reaction happen, involves activation energy from the reactants to the transition state
10
Q
What is thermodynamics?
A
change in energy between the reactants and the products, spontaneous or nonspontaneous process
11
Q
What is the first law of thermodynamics?
A
energy is conserved, cannot be created or destroyed
12
Q
What is the second law of thermodynamics?
A
entropy is always increasing (spontaneous process -> order to disorder)
13
Q
What is delta H
A
enthalpy
14
Q
What is delta S
A
entropy
15
Q
What does delta H > 0 indicate?
A
endothermic
16
Q
What does delta H< 0 indicate?
A
exothermic
17
Q
What does delta G < 0 indicate?
A
spontaneous process
18
Q
What does delta G > 0 indicate?
A
non-spontaneous
19
Q
What does delta G = 0 indicate?
A
the reaction is at equilibrium - forward and reverse are the same, concentrations of A and B not changing
20
Q
What does delta S > 0 indicate?
A
disorder is increasing
21
Q
What does delta S less than zero indicate?
A
disorder is decreasing
22
Q
What are biochemical reactions initiated by?
A
changes in concentrations of reactants and products
23
Q
What does Keq > 1 indicate?
A
products > reactants
24
Q
What does Keq < 1 indicate?
A
products < reactants
25
What is le chatlier's principle?
deviation from equilibrium stimulates processes to restore equilibrium
26
Describe activation energy at equilibrium.
Activation energy is the same in both directions at equilibrium
27
Why is water so important in biology?
- controls the structure of biomolecules
- hydrogen bonding and hydrophobic effect
- active participant in biological reactions
28
What is the vanderwaals radius of an H and an O atom?
H - 1.2 angstrom
| O - 1.4 angstrom
29
What is the bond length between H - O in a water molecule?
0.958 angstrom
30
What is the bond angle between the two hydrogens in a water molecule?
104.5 degrees
31
What is the bond angle between two Hs in CH4?
109.5
32
What is the polarity of a water molecule?
O: -.66e
H: +.33e
33
What is e-?
the charge of an electron
34
What are the two components of a hydrogen bond?
a donor: electron poor (H)
| an acceptor: electron rich (O)
35
What is the length of a typical H-bond?
1.8Å
36
What are the five types of bonds in strength order?
```
Covalent bond - 400kj/mol
Ionic bond - 80kj/mol
H - bond - 20kj/mol
Dipole-dipole - 9kj/mol
London dispersion forces- 0.3kj/mol
```
37
What is a permanent dipole?
between two polar molecules
38
What is an induced dipole?
one polar molecule, one non-polar molecule with an induced dipole
39
What are london dispersion forces?
between two non-polar molecules
40
How many h-bonds can a water molecule form?
4 - Hbonds
Has 2 acceptors (electron pairs on O)
and 2 donors (H atoms)
41
What is ice made up of?
- a tetrahedral array of H-bonds
| - open structure - 6-membered rings
42
What happens molecularly when water freezes?
- water expands when freezing, water molecules are pushed far apart and fixed
- this means ice is less dense than water
43
What is the density of water vs density of ice?
Density of water = 1g/ml
| Density of ice = .92g/ml
44
Describe the bonding/ structure of liquid water.
- irregular, dynamic H-bonding
- break/reform every 2 x 10 -11
- 3-7 member rings
45
Define solubility.
Ability of the solvent to interact with a solute
46
When will a substance dissolve?
a substance will dissolve when solute-solvent interaction is stronger than solute-solute interaction
47
What does hydrophilic mean?
polar/ionic - solvated in H2O
48
What does hydrophobic mean?
non-polar - does not interact w/ H2O (does not necessarily mean no hydrogen bonds)
49
What is the hydrophobic effect driven by?
entropy
50
What is the hydrophobic effect?
- Non-polar solutes aggregate in water to minimize the amount of surface area the solutes are exposed to, therefore decreasing the number of ordered H2O molecules
51
How does entropy change when a hydrophobic molecule is dissolved?
- Entropy changes in this reaction: H2O becomes more ordered and forms cages when a hydrophobic/non-polar molecule is dissolved, making this less favorable
52
What are amphiphiles?
contain polar and non-polar groups
53
What structures do amphiphiles form in nature?
micelles and bilayers
54
What is osmosis?
movement of water across a membrane from high to low concentration
55
What is osmotic pressure?
pressure applied to prevent inward flow of water
56
Describe osmotic pressure in cells.
- in cells the osmotic pressure is equal inside and outside
| - need balanced salt concentrations
57
What is dialysis?
- a membrane removes salt from proteins to purify
- kidney dialysis removes salt from the blood
- solutes diffuse through the membrane until equilibrium is reached (not osmosis because water is not crossing a membrane)
58
What is a solvent?
H2O
59
What is a solute?
NaCl, what is being dissolved
60
Describe the ionization of water.
- protons can easily jump from one H2O to another
- H3O and OH have high mobility in water
- acid - base reactions are rapid in H2O
61
What does 1 pH unit indicate?
10 -fold difference in [H+]
62
What is a bronsted lowry acid?
a substance that can donate H+
63
What is a bronsted lowry base?
a substance that can accept H+
64
What does a high or low pka indicate?
- high pKa = less likely to dissociate
| - low pKa = more likely to dissociate
65
When do species have multiple pKas?
- when there are multiple ionization states
66
What is the relationship between pH and pKa?
- pKa is the pH at which 50% of HA has dissociated
| - [HA] = [A-] when pH = pKa
67
What is the buffering capacity?
1 pH unit above/below pKa
68
What is the difference between the sugar of RNA and DNA?
- RNA - ribose
| - DNA - 2'deoxyribose (no OH at the 2' carbon)
69
What are the purines?
Adenine and Guanine
70
What are pyrimidines?
Cytosine, Thymine, Uracil
71
How are bases of DNA linked?
-phosphodiester linkages
72
What is the pKa of O- in a phosphodiester linkage?
pKa = 0 at pH = 7 (prefers deprotonated form)
73
Why are the phosphodiester linkages of RNA polymers weaker?
RNA is less stable because of an additional 2' hydroxyl, if a base deprotonates it the oxygen can attack the phosphodiester linkage - this makes RNA more prone to cleavage
74
Describe the structure of DNA.
- double helix
- 2 polynucleotide strands
- chains are antiparallel
- right-handed helix
- bases are hydrogen bonded to bases on opposite strands
- major and minor groove
75
Describe how the hydrophobic effect is at play in the structure of DNA.
- bases are at the core of the helix and phosphates are exposed to water
- this is because the bases are non polar and phosphates are negatively charged
76
How many hydrogen bonds are there between the bases of DNA?
A - T (two)
| G - C (three)
77
Describe the structure of RNA.
- formation of stem loop structure
- single stranded
- intramolecular -bonding
78
What is the central dogma?
DNA is transcribed into RNA and translated into protein
79
What are the building blocks of DNA replication?
- deoxynucleotide triphosphates are the building blocks (dNTPs)
80
Which direction is DNA replicated in in nature? vs in the lab?
5' - 3' in nature
| 3' - 5' in the lab
81
Describe DNA replication.
- RNA primers - necessary for DNA polymerase to bind to
- generate by primases
- helicase and topoisomerase unwind DNA
- leading strand - continuous
- lagging stranf okazaki fragments
82
Describe the chemical reaction of DNA replication
3' hydroxyl is nucleophile (deprotonated by a base) and phosphate is electrophile
83
What are the two strands of DNA in transcription?
coding strand and template strand (rna identical to codinf strand)
84
Describe the process of transcription.
- DNA template
- catalyzed by RNA polymerase
- synthesis occurs 5'-3'
- building blocks ---> NTPs
- no primer required
85
Which direction are proteins translated in?
N-term to C-term
86
What are the three types of RNA?
messenger RNA - encodes for protein sequence
tRNA - transfer
rRNA - makes up the ribosome
87
Describe tRNA.
- 3' hydroxyl of tRNA binds to O- of an amino acid
- when it has an amino acid tRNA is called "amino acylated"
- contains an anticodon loop that carries 3 nucleotides that read the codon of RNA
- enzymes called tRNA synthetases add the amino acids to tRNA
88
What are the steps of classic Sanger Sequencing?
- Attach a primer that contains a radiolabel or fluorescence (Why?)
- Run 4 PCR reactions, with DNA polymerase, and ddNTPs
- Run the 4 reactions on a gel - (remember a primer was added, so first few nucleotides are known)
- Read from bottom to top of the gel (5' - 3')
- Reverse complement is the actual template strand
- Cant get info on the first base (of actual template) using this method (Why?)
89
What are the steps of one pot sanger sequencing?
- No radiolabel/fluorescence on the primer
- dNTPs and ddNTPs with fluorescent dyes are all added to the same reaction with DNA polymerase
- Reaction is run on a gel (one lane), colors indicate the places where specific bases were added
- Read from bottom to top of the gel (5' - 3')
- Reverse complement is the actual template strand
- (info can be gotten on the first nucleotide)
90
What are two other methods of sequencing (other than sanger)?
- Replace the gel with capillary electrophores - this gives us faster results and better resolution
- Next gen seqeuncing
91
What are ddNTPs?
dideoxynucleotides - deoxyribose lacks -OH at 3' carbon that is required for elongation of a DNA strand
92
What is PCR?
Polymerase chain reaction
93
What does PCR do?
amplify (create many copies of) a specific DNA sequence
94
What are the components of a PCR reaction?
- primers - forward and reverse, bind specifically to the region you want amplified
- DNA polymerase - must be heat stable (Taq which was isolated from a thermophile)
- dNTPs
- template strand
95
What is the process of a PCR reaction?
- design primers
- heat - denature
- cool - anneal to primers
- extension
- 20 cycles will create 2^20 amplification
96
What is the process of cloning?
- Restriction digest
- Ligation
- Transformation
- select for cells with plasmid
97
What are two components of a vector/plasmid in cloning?
- MCS - multiple cloning site - short segment of DNA that contains many restriction sites
- Amp^R - antibiotic (ampicillin) resistance gene
98
What is site directed mutagenesis?
- uses a primer that carried desired mutation to introduce a specific mutation into the DNA strand
99
What is the pKa of the amine group in an amino acid?
9-10
100
What is the pKa of the carboxyl group in an amino acid?
3.1
101
What is the alpha carbon of an amino acid?
central carbon to which the amine and carboxyl group are attached (R group and H also attached)
102
What is the stereochemistry of the alpha carbon?
- s - stereochemistry (chiral center)
- counter clockwise when the hydrogen is facing into the page
- priority goes: amino (N-bonded directly), carboxyl (C=O), then R-group
103
What is the regular configuration of all natural amino acids?
L - amino acids
104
Describe the pKas given for the amino acids. What would occur if they were place in a hydrphobic pocket?
- the pKa values are the pKas when directly facing water
| - if in a hydrophobic pocket, the amino acids would be LESS likely to ionize and pKa would increase
105
What direction is s?
counter clockwise
106
How many bonds between each set of base pairs?how does that impact denaturation/ melting temp?
- G-C 3
- A-T 2
- harder to denature, more H- bonds
- higher G-C content = harder to denature and higher melting temp
107
What are the two other amino acids, not naturally occuring?
selenocystein and pyrolysine
108
What character does an amide bond possess?
some double bond character, little rotation
109
What are some possible post-translational modifiactions of proteins?
phosphoserine - add (po4)
| methy - can be added wherever there is an NH
110
What conformation of an amide bond is favored?
trans is always favored to cis 1000:1
| exception: proline trans favored 4:1
111
What is a method for protein quantification?
ELIZA
112
What is an ELIZA?
enzyme- linked immunosorbent assay
113
Describe the steps of an ELIZA?
- have a plate with an immobilized antibody that recognizes your protein of interest
- lyse cell and add mixture of proteins to plate
- wash away unbound proteins
- use a secondary antibody (HRP) with an enzyme attached
- can be amplified to produce a fluorescent product
114
What are four methods to purify proteins?
- charge
- hydrohpobicity
- size
- affinity chromatography
115
Describe the process to separate proteins by size.
- Ion exchange chromatography
- resins / beads the opposite charge of protein of interest
- anion (-) cation (+)
- charged proteins bind to the column, strength of charge determines how tightly
- elute with changing pH
116
Describe the process to separate proteins by hydrophobicity.
Hydrophobic interaction chromatography
- hydrophobic proteins stick to the column
- elute with detergents
117
Describe the process to separate proteins by size.
- size exclusion chromatography
- beads with pores
- smaller = longer time to elute
118
What is affinity chromatography?
- specific antibody
- HIS tag - Ni column
- FLAG tags HA tags with specific antibodies
119
What is a strategy for protein sequencing?
Edman degradation
120
Describe Edman degradation.
- requires a pure peptide
- plucks off single amino acid at a time from end terminus
- PITC (N=C=S)
- Creates PTH amino acid
- Repeat cycles to figure out the sequence
121
What is the process of mass spec?
- protease degradation of peptide mixture with Trypsin
- (cleaves at K - lysine and R - arginine)
- run mass spec and isolate a peptide
- fragmentation of peptide binds at low enough energy that only a certain percentage of the bonds fall apart
- b fragments left
- y fragments right
