Midterm 1 Flashcards
(126 cards)
1
Q
What are the 4 biomolecules?
A
- Amino acids/proteins
- Nucleic acids
- Carbohydrates
- Lipids
2
Q
What are covalent bonds?
A
O-H, C-H, and C-C
3
Q
What are noncovalent bonds (strongest to weakest)?
A
Ionic interaction (+/-) - van der Waals forces; hydrogen bond - dipole-dipole interaction - london dispersion forces
4
Q
What are the five general types of reactions in cells?
A
- Redox: oxidation and reduction
- C-C bond formation and cleavage
- Internal rearrangements
- Group transfers
- a) Condensation reactions: loss of water to join subunits together
b) Hydrolysis: gain of water to cleave two units
5
Q
What is a redox reaction?
A
Formation of a disulfide bond
6
Q
What is an internal rearrangement?
A
Sugar isomerization
7
Q
What is a group transfer?
A
Transfer of an amine functional group
8
Q
What is a condensation reaction?
A
Peptide bond formation
9
Q
What is a hydrolysis reaction?
A
Peptide bond cleavage
10
Q
What is the 1st law of energy?
A
Energy is conserved
11
Q
What is endothermic? Number?
A
A positive H where it absorbs heat from its surroundings
ex. melting of ice cubes
12
Q
What is exothermic? Number?
A
A negative H where it releases heat into its surroundings
ex. dissolving KCI in water
13
Q
What is 2nd law of energy?
A
Order to disorder (entropy = S)
14
Q
When is S negative?
A
When the amount of randomness decreases
ex. 2 units to 1
15
Q
When is S positive?
A
The amount of randomness increase
ex. 1 unit to 2
16
Q
What happens if G is negative?
A
The process is spontaneous/exergonic
17
Q
What happens if G is positive?
A
The process is not spontaneous/endergonic
18
Q
What happens if G is 0?
A
The process is at equilibrium
19
Q
Stereoisomers
A
Different molecules in which the bond order is the same but the spatial arrangement of the atoms is different
20
Q
What is the configuration of a molecule determined by?
A
- Double bonds
- Chiral centers
21
Q
Geometric isomers
A
Arrangements differ with respect to double bond (cis and trans)
22
Q
Asymmetric carbon
A
A carbon atom with 4 different substituents, thus chiral centres
23
Q
Enantiomers
A
Stereoisomers that are mirror images of each other and can’t be superimposed on the other
ex. our hands
24
Q
Diastereomers
A
Stereoisomers that are no mirror images of each other
25
What does the presence of 2 or more chiral centers in a molecule result in?
2^n stereoisomers
26
R chirality
Priority goes clockwise (H sticking back)
27
S chirality
Priority goes counterclockwise (H sticking back)
28
Epimers
Sugars that differ only by the configuration about 1 chiral centre (enzymatically interconverted)
29
How are sugars labelled with chirality?
Backbone carbon centre furthest from the carbonyl carbon (aldhyde - COH)
30
Where must the H be in the Fischer projection?
For correct chirality, it must be on the top or bottom
31
How do enantiomers differ?
Rotate plane-polarized light in opposite directions
32
What must amino acids occur as? Sugars?
Amino acids = L isomers
Sugars = D configuration
33
Molecular conformation
Spatial arrangement of substituents that are free to assume different configurations in space by free rotation around single bonds (steric hindrance)
34
What do configuration and conformation lead to?
The biological activity of biomolecules
35
What is the difference between conformation and configuration?
Conformation is the rotation of a single bonds meanwhile configuration (so cis versus trans; diastereomers or enantiomers); broken and reattached
36
Why does oxygen have a dipole?
Due to the side with oxygen two lone pairs being more electronegative and the other with the hydrogens being slightly positive.
37
What is the structure of hydrogen bonding?
D-H----A (D and A are negative while H is positive; the dotted line represents H bonding)
38
What does the D in a H bonding represent?
The weakly acidic donor group
ex. OH or amines
39
What does the A in a H bonding represent?
The weakly basic acceptor group
ex. those with lone pairs of electrons such as nitrogen and oxygen
40
When are H bonding the strongest?
When they are all linerary arranged, so angles of 180 (directional interaction).
41
Why is H2O's solid state less dense than the liquid state?
Because the crystal lattice is bonded to 4 other molecules while the liquid state is bonded to 3.4 other water molecules, thus the solid state occupies more volume than its liquid form.
42
What is the formation of liquid water?
Flickering (short-lived) clusters of H-bonded molecules attached to 3.4 other water molecules
43
How do ions dissolve in water?
Water molecules form hydration shells around ions where no interaction is forced, thus entropy increases and the crystalline substance dissolves
44
Entropy
The disorder or randomness of a system
45
How do water molecules orient around positive ions (cation)?
The oxygen (negative) surround the positive charge
46
How do water molecules orient around negative ions (anion)?
The hydrogens (positive) surround the negative charge
47
How do polar molecules dissolve?
The hydrogen bonds replace solute-solute bonds with solute-water bonds
48
Do polar molecules like water?
Yes they are hydrophilic
49
Do nonpolar molecules like water?
No they are hydrophobic
50
Do amphipathic molecules like water?
It's fifty/fifty
51
How do non-polar solutes dissolve in water?
Highly ordered water molecules form cages around the chains to minimize contact and decreases the entropy of water (low randomness)
52
What happens to the entropy of water if the non-polar solute becomes more clustered?
The entropy is increased as fewer water molecules are ordered
53
What does H bonding between water and polar solutes cause?
Ordered water to form around them
54
How is ordered water released?
An entropy drive force in which the substrate with order water binds to the enzyme with ordered water around it as well
55
What does proton movement help with?
The protons jump quickly from organic molecule or oxygens to result in the fast acid and base reactions
56
What's the difference between a strong acid and weak acid?
A strong acid completely dissociates into its ions while a weak acid doesn't dissociate quickly
57
How does acid strength relate to pKa?
As acid strength increases, pKa decreases.
58
What is the buffering region?
A wide range of pHs where there are small changes (don't react much to the addition of strong acids and bases)
59
What is a zwitterionic compound?
Those that have a neutral charge
60
What are acid-base properties of amino acids?
Zwitterions and amphoteric
61
Zwitterions
Have both positive and negative charges
62
Amphoteric
Act as both an acid and base
ex. amino group acts as a base as it accepts a proton (positive) and a carboxylic acid acts as an acid and loses an H (negative charge)
63
What are the non-polar amino acids?
Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp
64
What amino acid doesn't have a primary amino group?
The amine (N) in proline is secondary as it's bounded to two carbons
65
What are the uncharged polar amino acids (neutral, so pH =7)?
Ser, Thr, Asn, Gln, Tyr, and Cys
66
What are the charged polar amino acids?
Lys, Arg, His, Asp, and Glu
67
What are the non-polar amino acids?
Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, and Trp.
68
How to remember the non polar amino acids?
Grandma Always Visits London In May For Winston's Party.
G = gly, glycine
A = ala, alanine
V = val, valine
L = leu, leucine
I = ile, isoleucine
M = met, methionine
F = phe, phenylalanine
W = trp, tryptophan
P = pro, proline
69
What happens as the amino acid gets more basic?
H ion lost in COOH first, then H in N+H3 (due to lower pka than amine group)
70
What is the pKa of N-terminus in peptide?
8
71
What is the pKa of the C-terminus in a peptide?
3
72
How to name peptides?
Three letter or one letter abbreviations of amino acids
73
How do amino acids link to form peptides?
Condensation reactions to form amino acid residue
74
Dipeptide
Peptide chain of 2 amino acids
75
Tripeptide
Peptide chain of 3 amino acids
76
Oligopeptide
Peptide chain of 3-10 amino acids
77
Polypeptide
Peptide chain of many amino acids
78
Protein
Molecules of one or more polypeptide chains
79
Prosthetic group
A non amino acid group that is permanently associated with a protein
ex. lipids, iron, zinc, etc.
80
How to get the protein out of the cell into solution?
Lysing a cell (rupturing the cell walls and membranes) by crushing or grinding cells and they filtration to remove unwanted components.
81
How to solve altered pH's for a protein?
Use a buffer to get stability
82
How to solve elevated temperatures for a protein?
Lower the temp to 0 degrees Celsius so it doesn't denature
83
How to solve degradative enzymes for a protein?
These proteins are also sensitive to pH and temp (can't denature desired protein to be isolated) so add an inhibitor (bind and block these proteins).
84
How to solve surface absorption for a protein?
Reducing foaming and keep the solution concentrated so proteins don't denature from O2
85
How to solve storage problems for a protein?
Store in a very cold fridge (below -80 degrees Celsius) and in liquid N2
86
What is the amount of light absorbed proportional to?
The concentration of the absorbing species (Beer-Lambert Law)
87
Once you have a solution with the protein, how to we quantify the amount of desired protein?
1. Amount of protein of interest (absorbance spectroscopy or immunoassay)
2. Amount of activity of the protein (if it is an enzyme)
88
What does an immunoassay do?
1. Antibodies bind to solid support
2. Protein sample comes in
3. Add second antibody that is linked to an testable enzyme
4. Wash and examine enzyme activity - amount of substrate converted to product indicates the amount of protein present
89
What happens when the amino terminal or side chain reaches its pKa?
Becomes deprotonated (-1 charge).
90
What happens before the amino terminal or side chain reaches its pKa?
Is protonated (positive or neutral charge).
91
What is the purpose of salting out?
Adding salt ions to decrease the solubility of a specific proteins, thus it's precipitate can be collected (after a couple rounds).
92
When are proteins least soluble?
When pH = pI
93
Why does salting out result in a decreased solubility?
The salt ions attract the water molecules, so the protein doesn't have enough water to remain solvated.
94
What is the purpose of dialysis?
Uses a gradient from the dialysis bag to solvent it's placed in to have small molecules (salts, detergents, etc.) pass out of the bag, thus reducing the amount of contaminants in the partially purified protein.
95
What is the principle of chromatography?
A liquid (mobile) phases moves through the column containing a porous solid (stationary) matrix that interact with the matrix depending on certain charges, weights, and sizes.
96
How is the matrix chosen?
Based on the physicochemical properties of the desired solute.
97
What does ion exchange chromatography do?
Separation based on charge due to the pH or salt concentration of the incoming buffer.
98
Elution
The process of extracting one material from another by washing it with a solvent.
99
How to separate proteins in ion exchange chromatography?
Use a matrix (ion exchanger) that has a positive or negative charge based on its pH and once molecules are attracted to it apply a buffer with a different pH to elute these molecules out (repel out of tube).
100
Cation exchange
The cations (+) of the protein bind to the anions (-) in the matrix
ec. carboxymethyl (CM)
101
Anion-exchange
Anions (-) of the protein bind to the cations (+) in the matrix
ex. diethylaminoethyl (DEAE)
102
What happens to the charge of the protein if pH = pI?
No net charge
103
What happens to the charge of the protein if pH is less than pI?
The protein has a positive charge.
104
What happens to the charge of the protein if pH greater than pI?
Protein is negative
105
What does hydrophobic chromatography use to separate proteins?
Uses hydrophobic interactions (non-polar groups or other regions interact with hydrophobic groups).
106
What is the matrix full of in hydrophobic chromatography?
Octyl or phenyl groups as they are hydrophobic beads.
107
What does affinity chromatography use?
The binding specificity of a chosen ligand (insoluble beads) in the matrix, where proteins bind noncovalently to it (those that can bind stay near the top).
108
How can a ligand exist in a matrix?
Covalently cross linked here
109
How is the protein released in affinity chromatography
By washing the column with a solution containing free ligand or altering conditions that affect binding (pH or salt).
110
Immunoaffinity chromatography
Requires antibodies in the proteins and elutes with a low pH.
111
Glutathion affinity chromatography
Requires GST tag and elutes with glutathione.
112
Metal ion chelate affinity chromatography
Requires 6X-His tag in molecules and elute with a low pH or imidazole.
113
What does gel filtration chromatography rely on?
Molecules separated according to size and shape as the matrix has gel beads with small pores that allow bigger molecules to pass outside faster, than smaller ones longer (gor into internal volume of beads).
114
Elution volume
Volume of solvent required to elute the protein from the column since it first contacted the gel.
115
Relationship between weight and elution volume.
Greater elution volume corresponds to a smaller molecular weight.
116
What are the principles of gel electrophoresis?
Separate based on size and charge.
117
Polyacrylamide gel electrophoresis (PAGE)
Use a gel with a particular pore size.
118
Native (non-denaturing) PAGE
Separates proteins based on size, shape, and charge.
119
Sodium dodecyl sulfate-PAGE (SDS PAGE)
(solution proteins are placed in before electrophoresis)
Separates mostly on molecular weight as one SDS anion binds to the protein per 2 aa residues (becomes negative charge) allowing known proteins to be run against unknown (attracted to positive charge at other end of gel).
120
What does isoelectric focusing use?
Uses the pI of a protein so that it will no longer move in an electric field, as a gradient of pH is established in a gel after application of an electric field. The electric field reapplied after protein solution to show different bands at different areas.
121
What does pairing IEF and SDS-PAGE together allow?
Isoelectric focusing separates proteins based on decreasing pH and then placed on a gel where it can be cut and shown in decreasing molecular weight.
122
Why do you use an elution of decreasing salt concentrations with hydrophobic interaction chromatography?
With a high salt concentration, it forms hydration shells around water molecules, thus hydrophilic proteins don't strongly bind to hydrophobic ligands and elute first. As the salt concentration decreases, the water is unbound and freely moves, thus eluting the most hydrophobic (more polar) molecule last.
123
Why does increasing the salt concentration for ion exchange chromatography elute proteins?
These salts bind to the matrix now, thus reducing the interactions with the protein, so moderate proteins are eluted first with the most charged eluted last.
124
What does mercapotethanol do?
Breaks the bridges of the disulfide bonds between proteins
125
Purpose of mass spectrometry?
Sequences short peptides quickly while also giving an accurate MW based on its mass to charge ratio.
126
What is the purpose of MALDI-TOF?
Proteins are desolvated and ionized. Follow different paths hitting the spectrometer at different times, thus producing different amino acids.
