biochem - exam 1 Flashcards
bio vs chemistry
levels 1,2,3 & 4
levels 1-3 are chemistry
level 1: monomeric units: nucleotides, amino acids, sugars
level 2: macromolecules: DNA, Protein, cellulose
level 3: supramolecular complexes: chromosome, plasma membrane, cell wall
biology is level 4
level 4: the cell and its organelles
what are the parts of a cell
cytoplasm
plasma membrane
ribosomes
nucleus
nuclear membrane
membrane bound organelles
what is principle 1
All cells of the simplest and most complex organismsshare certain fundamental properties, which can beseen at the biochemical level.
what do the parts of the cell do
A place for chemical reactions/hold- everything together- cytoplasm
Something to separate in/out- plasma membrane
Something to make proteins- nucleus
Genetic material- nucleus
“Areas of specificity”- organelles
surface area to volume issue
solves by having many parts make up a whole
all cells have
Plasma membrane (lipids)
Cytoplasm/sol (all kinds of molecules!)
Regions of “specificity”
Organelles (Favorite?)
Inclusions (Pro)
“Supramolecular” structures
: Differences between plant and animal eukaryotic cells!
Animal cells each have a centrosome and lysosomes, whereas plant cells do not. Plant cells have a cell wall, chloroplasts and other specialized plastids, and a large central vacuole, whereas animal cells do not.
Evolutionary and Genetic Foundations:We can/have tracked these differences through time using molecules!
Cell membranes are made of
DNA replication is
Genetic code makes
Glucose is a preferred
ATP is an
Aerobic and anaerobic organisms
Cell membranes are made of lipids
DNA replication is semi-conservative
Genetic code nucleic acids
Glucose is a preferred sugar source for energy
ATP is an energy currency
Aerobic and anaerobic organisms
Biochemistry: Small Differences Matter!
A- bacterial and Archean ribosomes are smaller than eukaryotic ribosomes, but serve the same function: protein synthesis from an RNA message
B- cell envelope: structure differ
C- nucleoid: contains one or several long, circular DNA molecules
D- pili: provide points of adhesion to surface of other cells
E- flagella: propel cell through its surroundings
gram positive bacteria
gram negative bacteria
methanothermus
gram positive bacteria: solid outer layer, glycoprotein, peptidoglycan, plasma membrane
gram negative bacteria: LPS, outer membrane, peptidoglycan, plasma membrane, lipoprotein, porin, periplasm
methanothermus (heat tolerant Archean) solid layer, glycoprotein, pseudopeptidoglycan, plasma membrane
carbons can
make all types of bonds!
what are the main organic elements
CHNOPS
Carbon
hydrogen
nitrogen
oxygen
phosphorus
sulfur
CHNO make up 99% of all atoms of the human body
light elements can form strong covalent bonds in a variety of ways
what re the 4 macromolecules
Carbohydrates
Lipids
Proteins
Nucleic Acids
How do macromolecules work?
functions groups!
what are the functional groups that are are supposed to know
methyl
ether
guanidinium
ethyl
ester
imidazole
phenyl
acetyl
sulfhydryl
carbonyl (aldehyde)
anhydride (2 carboylic acids)
disulfide
carbonyl (ketone)
amino (protonated)
thioester
carboxyl
amido
phosphoryl
hydroxyl (alcohol)
imine
phosphoanhydride
enol
N-substituted imine (schiff base)
mixed anhydride (carboxylic acid and phosphoric acid; also called acyl phosphate)
what functional groups make up acetyl coenzyme A
thioester
amido
hydroxyl
phosphoanhydride
imidazole-like
amino
phosphoryl
what atoms do
Blue
black
Red
gray/White
represent
and what model representations should know
Blue: Nitrogen
Black: carbon
red: oxygen
Gray/White: hydrogen
structural, ball & stick, space-filling
Geometric/Configurational Isomers
Double Bond!
Compare your big groups and your small groups
Cis- and trans- good only for disubstituted double bonds
E/Z for tri and tetra
Z/Cis; E/Trans
Diastereomers!
Optical Isomers
Need: CHIRALITY
Chirality 4 different substituents around the carbon
what should you know about chiral molecules
1- Chiral molecules are optically active - rotate plane-polarized light
2- n chiral centers means 2^n stereoisomers
3- Chiral molecules are not identicalto their mirror image.
4- Achiral molecules are identical to theirmirror image
how do you organize and identify CHIRAL MOLECULES
1- rank atoms attached to choral center according to atomic number
Br > Cl > C > H
2- Rotate the molecule such that the substituent ranked #4 is in the back
3- with #4 in the back, trace the path of the #1, #2 and #3 ranked substituents. if #1, #2 and #3 trace a clockwise oath, assign the chiral center as R
4- if #1, #2 and # trace a counter clockwise path assign the chiral center as S
R/S: L/D: +/-
L/S/(-) = Left (anti)
R/D/(+) = right (clock)
what are enantiomers
differ at all stereocenters, and are therefore mirror images of each other
diasteriomers
Diastereomers are non-mirror image stereoisomers. They occur when a compound has different configurations of one or more (but not all) of the equivalent stereocenters.
why care about Stereospecificity/Stereoselectivity
In nature, most chiral molecules exist in only one configuration
L-amino acids
D-glucose
Proteins and other biomolecules are able to distinguish between (react differently) with different stereoisomers.
Examples to the right have different smells/tastes one is sweet and one is bitter
Racemic Mixture (Racemates)
is an equimolar solution of two enantiomers (50% R/ 50% S)
racemates
The two enantiomers in a racemic mixture are called racemates, rotate plane-polarized light in opposite directions, so there is no net rotation.
Racemic Drugs!
One stereoisomer is usually active, the other inactive (or opposite), or has a different activity than the first form. Why?
Ideally…
So what happens to the dose if you have a racemic mixture, compared to enantiopure?
SSRI Antidepressant
Celexa (trade name) of Citalopram (racemate)
Lexapro (trade name) of Escitalopram (S)-Citalopram
Β2 adrenergic agonist
The (R)-(-) enantiomer is active
The (S)-(+) enantiomer is inactive
Equilibrium does NOT always
mean equal!
Reactions tend to go to completion, notto “equality”
Completion depends
on a specific set of concentrations, described by an Equilibrium Constant, Keq
what is Keq
concentration products/concentration reactants
One can compare where the reaction is going when you used Q and compare it to Keq. Given the concentrations of all of the constituents and the Keq.
put aA + bB goes to cC + dD in Keq form
Q = Keq = [C]^c [D]d / [A]^a[B]^b
Q =
Products/reactants = Keq
what happens if products>reactants
net rxn towards reactants or the left
Q>K
what happens if products<reactants
net rxn towards products or the right
Q<K
what happens if products = reactants
no net rxn
Q=K
What makes a reaction go?
Energetics/Thermodynamics
What is enthalpy
what is its symbol
what does the net change in enthalpy mean
what does it mean when net enthalpy is less than zero
what does it mean when net enthalpy is more than zero
what are the units
enthalpy: number & type of chemical bonds
the net change in enthalpy, delta H, for a rxn depends on the relative strengths of the bonds broken and formed
delta H < 0: exothermic: heat generated/released
delta H > 0: heat energy transformed
measured in kilojoules per mole (kJ/mol)
how do you calculate enthalpy change
Sum Enthalpy (Products)minusSu m Enthalpy (Reactants)
what happens as time moves forward In an isolated or closed system
As time moves forward, the net entropy (degree of disorder) of any isolated or closed system will increase.
It takes a lot of effort (energy?) to decrease entropy.
entropy (S)
randomness
delta S > 0: system becomes more random, less ordered (favored)
delta S < 0: system becomes more ordered, more ordered
what is Gibbs free energy (G)
Delta G < 0: exergonic, rxn releases energy
Delta G > 0: endergonic, must put energy into system to make rxn happen
It is the energy available to do work
exergonic
products predominate at equilibrium (occurs spontaneously as written)
reactants predominate at equilibrium (does not occur spontaneously as written [occurs spontaneously in reverse directional])
Rxn release energy
free energy change and delta H and delta S
Delta G = delta H - T delta S
Free energy change and equilibrium
delta G * = -RT*ln(Keq)
factors that contribute to making delta G more negative (less positive)
-negative delta H (exothermic rxn)
-postive delta S (increasing entropy [more random])
factors that contribute to making delta G more positive (less negative)
-positive delta H (endothermic rxn)
-negative delta S (decreasing entropy [more ordered])
thermodynamics of biosystems
left alone (w/o any energy input), biosystems would fall apart (entropy maximization)
to maintain order, and to grow, energy input is required
to accomplish this, exergonic rxns are couples to endergonic rxns
metabolism
The sum total of all chemical reactions in an organism.
Metabolism = Anabolism + Catabolism
Anabolism
Synthetic reactions.
Normally endergonic (+∆G)
Usually involves reduction (Entropy, too
Catabolism
Degrative Reactions
Normally exergonic (-∆G)
Usually involves oxidation (Entropy?- +)
Reaction Coupling
Some reactions are not energetically favorable. The first reaction of glycolysis, for example, wants to go in reverse.
In living organisms, an energy-releasing reaction can be coupled to an energy-requiring reaction to drive the otherwise unfavorable reactions.
more on rxn coupling
High-energy compounds are used by all organisms to provide a driving force for thermodynamically unfavorable reactions (entropy).
Two reactions are “coupled” when one reaction is energetically favorable and can provide energy which allows the second reaction (unfavorable on its own) to occur.
Energy released by the second reaction drives the first reaction!
Thermodynamically unfavorable reactions (anabolic?; ∆G > 0) create order andrequire work and energy. We gotta get that energy from somewhere.
what is chapter 2 about
water, weak forces and acids/bases
what percentage of an organism is composed of water
water it the universal what
drugs work in what environment
drugs targets are in what environment
how drugs behave is dependent on what and how they interact with what
what bonds do water molecules for with polar solutes (has a dipole)
are the forces of water weak or strong
water is the critical determinant of what
what do non polar things do when in contact with water
≥ 70% of weight of most organisms
Universal solvent Chemical reactions occur in aqueous environment
Drugs work in an aqueous environment (Pharmacy, right?)
Drug targets are in an aqueous environment (Pharmacy, right? Right?)
How drugs behave depends on their electrical charge, and this depends on how they interact with water
Water forms hydrogen bonds with polar solutes
Water and non-polar things have an interesting relationship
Weak forces!
Therefore, water is a critical determinant of the structure and function of proteins, nucleic acids, and membranes! (Basically biochemistry, right here)
non polar elements will ball up in aqueous environment
what kind of Weak Forces/Interactions Exist in Aqueous Systems
what do these forces/interactions allow for
Hydrogen Bonds- strong association bwtn FNO
Ionic Interactions
Hydrophobic Interactions- hide from water and keep to themselves
Van der Waals Interactions
These weak interactions allow for dynamic (rapidly changing/temporary)molecular processes.
what is a hydrogen bond
what elements have
to be connected to a hydrogen bond in order for there to be a hydrogen bond
what is the pneumonic
unequal distribution of charge that results when a hydrogen is covalently bonded to an electronegative atom, such as oxygen or nitrogen interacts with a FNO somewhere else
H-Bonding is for F, O, N
Sulfhydryl (sulfur, below oxygen, behaves like oxygen ) groups too
Directional
Polarity!
A typical Hydrogen bond in bio-molecules is between H and _____
hydrogen bonding is
do not change
what kind of interaction is amphipathic
N or O
FNO
test question
amphipathic is a hydrophobic interaction
A carboxyl group is…
polar
nonpolar
amphipathic
polar
there’s lots of oxygens pulling electro density to itself leaving the hydrogen more positive and so there is a net negative and net positive end
A methyl group is…
polar
nonpolar
amphipathic
non polar
no FON
For the dissolving of a gas in water/in container, the entropy of the system…
decreases
we order it and put it in something
giving it order
not letting it run around me be free
which part of each molecule would make hydrogen bonds?
alcohol
ketone
polypeptides
DNA
alcohol: hydroxyl group and water
ketone: carbonyl and water
polypeptides: peptide groups in polypeptides
DNA: nitrogen and carbonyl
Water and Ionic Interactions
The salt crystal is ordered. It is _____________favorable for water to break it apart and surround the individual components of ____with _______ molecules.
Charge of the electron: _____. Add a negative charge: be ________. If you remove this then become positive
Charge of proton: ________
Opposites ________
________charge repel
This all may change the _______ a molecule so it is needed to know when something might be ________charged or ________
Salt in ________, Na and Cl _________and associate with water—entropy _________
The positive part of water _________Cl, negative part of water surrounds _______—this is a ________shell
Broken up ________ into bits and pieces—this is thermodynamic _________ because entropy increases. The universe tends towards __________
Ionization of water gives us ________—net H+ and OH-
The salt crystal is ordered. It is thermodynamically favorable for water to break it apart and surround the individual components of Na-Cl with water molecules.
Charge of the electron: negative. Add a negative charge: be negative. If you remove this then become positive
Charge of proton: Positive
Opposites attract
Like charge repel
This all may change the shape of something so it is needed to know when something might be negatively charged or positive
Salt in water, Na and Cl dissociate and associate with water—entropy increases
The positive part of water surrounds Cl, negative part of water surrounds Na—this is a hydration shell
Broken up solid into bits and pieces—this is thermodynamically favorable because entropy increases. The universe tends towards disorder
Ionization of water gives us acids and bases—het H+ and OH -
Water and Van der Waals Forces
it broke and _________
even though it is ________the cumulative effect is ________
think of ___________
it broke and reformed
even though it is weak the cumulative effect is great
think of velcro
are
hydrophilic
hydrophobic (lipophilic)
mixed
soluble in water
hydrophilic: yes
hydrophobic (lipophilic): no
mixed: partly
soluble in water
are gases soluble in water
polar gases–yes
non-polar gases—less
rank the Polar/Hydrophilic and Non-Polar/Hydrophobic (Solubility)
highest: polar solids, lipids, and gases
middle: non-polar solids, liquids
lowest: non-polar gases
entropy for:
Any gas in a container
Any gas in the universe
what is the eqn for Gibbs free energy?
Solid or liquid solute: if polar, becomes more ______; if non-polar becomes more_______
Gas
–entropy always_______, because the gas is confined to a small volume
–decrease is smaller if polar (molecules disperse), larger if nonpolar (molecules cluster)
Any gas in the container: entropy decreases because set at a volume
Any gas in the universe: entropy increases, more volume, more random
eqn: deltaG = deltaH - Temp deltaS
Solid or liquid solute: if polar, becomes more disordered; if non-polar becomes ordered
Gas
–entropy always decreases, because the gas is confined to a small volume
–decrease is smaller if polar (molecules disperse), larger if nonpolar (molecules cluster)
Amphipathic
Bulk water has little order what is the entropy
- Water near a hydrophobic solute is highly ordered: low entropy thermodynamically unstable low ______
- Lipids group together, increasing the amount of ______ in the system ___________ are _______ordered around individual lipids.
contain regions that are polar (charged/hydrophilic) and regions that are non-polar (hydrophobic)
Complex hydrophobic/hydrophilic interactions are thermodynamically favorable!
- Lipids group together, increasing the amount of entropy in the system water molecules are less ordered around individual lipids.
Water near hydrophilic solute: water in oil, tends to itself and does not mix—less entropy
Lipid organizes to itself-–water does not have to form hydration shell. Oil and water mixture is thermodynamically favorable—universe tends to thermo. The state, has to have entropy increasing, keep entropy high
do you know all the functional groups yet?
methyl
ether
guanidinium
ethyl
ester
imidazole
phenyl
acetyl
sulfhydryl
carbonyl (aldehyde)
anhydride (two-carboxylic acids)
disulfide
carbonyl (ketone)
amino (protonated)
thioester
carboxyl
amido
phosphoryl
hydroxyl (alcohol)
imine
phosphoannhydride
enol
N-substituted imine (Schiff base)
mixed anhydride (carboxylic acid and phosphoric acid; also called acyl phosphate)
Yes, I know all of them!!!!!! God is good!!!
Nah I don’t but I will go practice them!!!!!!!! God is STILL GOOD!!!!!!!!!
Polar/Hydrophillic and Non-Polar/Hydrophobic (Interactions!).
The association or folding of non-polar molecules in aqueous solution is one of the main factors behind:
Formation of lipid ______ (and membranes)
______folding
Protein-_______association
Binding of ________hormones totheir receptors
Drug/Ligand Receptor ________!
The association or folding of non-polar molecules in aqueous solution is one of the main factors behind:
Formation of lipid micelles (and membranes)
Protein folding
Protein-protein association
Binding of steroid hormones totheir receptors
Drug/Ligand Receptor Binding!
what is the Net Result of all these Forces?
what do weak forces maintain and determine what iinteractions
what do non covalent interactions permits
Dynamic Interactions and Functionality!
Weak forces maintain biological structure and determine biomolecular interactions!
Molecular Complementarity!
Mediated via noncovalent interactions – permits tight, highly specific binding of biomolecules!
Colligative Properties of Water
solutes decrease the concentration of what
what do properties depend on the number of what
what do colligative properties include
what is are the units for osmolarity
what are the units for osmolality
140mmol NaCl/kg = how many osmol
what is osmosis
water follows what
Osmotic pressure
solutes decrease the concentration of water
properties depend only on the number of solute particles not their size
colligative properties: vapor pressure, boiling point, melting& freezing point and osmotic pressure
osmolarity: moles solute particles/L solution
osmolality: moles solute particle/kg solution
140 mol NaCl/kg = 280 mOsmol NaCl/kg
osmosis: diffusion of solvent across a membrane along a solute concentration gradient
water follows salt
Osmotic pressure: forcing water through a membrane in an attempt to equalize concentrations; isotonic, hypertonic and hypotonic solutions
what happens to cells in the following salt solutions
isotonic
hypertonic
hypotonic
isotonic” no net water movement, cell stays at normal size because there is equal amount of water inside that outside
hypertonic: water moves out of cell and cell shrinks because there is more salt outside that inside and water follows the salt outside
hypotonic: water moves in the cell creating outward pressure ; cell swells and may burst because there is more salt inside that outside so water follows salt inside
Water (and Strong Acids) Ionize!
what happens when a water molecule dissociates and what is this called
what Is pH
pH + pOH =
In pure water (neutral), [H+] = [OH] so pH equals what
when [H+] > [OH], pH < 7.0 (____)
when [H+] < [OH], pH > 7.0 (_____)
when a water molecule dislocates: results in a proton and hydroxide
H + H30+ & OH- this is the ionization of water
pH = -log[H+]
pH + pOH = 14
In pure water (neutral), [H+] = [OH] so pH equals 7
when [H+] > [OH], pH < 7.0 (acidic)
when [H+] < [OH], pH > 7.0 (basic)
what are acids
what are bases
Molecules that release hydrogen ions (protons) in solution: acids (H+ donor): H2CO3
Molecules that can accept hydrogen ions: bases (H+ acceptor): HCO3
The osmolarity of a sample of lysosomes is 0.26 M. Relative to these, a 0.10 M sucrose solution is…
hypotonic
the cell is in a hypotonic solution! the solution has LESS salt than the cell
If you immerse the lysosomes (0.26 M osmolarity) in this 0.10 M sucrose solution, what will happen?
the water will go into the lysosomes and the cell may burst
remember: water will move to the solute/salt
water outside will move to solute inside
If the pH of a solution is 6.0, the [OH-] is…
10 ^ -8
pOH = 14 - pH
8 = 14 - 6
pOH = -log[OH-]
8 = -log[OH-]
10^-8 = [OH-]
H+, pH, pOH & OH- converting scales
H+ to pH
pH to H+
pH to pOH
pOH to pH
pOH to OH
OH to pOH
H+ to pH: pH = -log[H+]
pH to H+: [H+] = 10^-pH
pH to pOH: pOH = 14 - pH
pOH to pH: pH = 14 - pOH
pOH to OH: [OH-] = 10^-pOH
OH to pOH: pOH = -log[OH-]
Dissociation of Weak Acids
HA == H+ + A-
because dissociation for a strong acid is complete: what does the H+ of that acid equal
because dissociation for a weak acid is incomplete, what is the extent of the dissociation determined by
what does “p” mean
pKA =
what Ka equal
can we find pH of soln if Ka is known
what is the Ka eqn for a strong acid
-log of Ka =
what is the equilibrium constant
what is the ion product of water
what does pH =
because dissociation for a strong acid is complete: what does the H+ of that acid equal: [H+] - [acid] and pH = -log[H+]
because dissociation for a weak acid is incomplete, what is the extent of the dissociation determined by: Ka
pKA = -logKa
“p” = -log
Ka equal [products]/[reactants]
can we find the pH of soln if Ka is known - yes
Ka = [H+][A-]/[HA]
-log of Ka = pKa
- Equilibrium Constant (Keq = [C][D]/[A][B])
- Ion Product for water: Kw = [H+][OH-] = 1.0 x 10-14
pH = -log [H+]
large Ka means
small pKa means
what does it mean that an acid’s pKa is 2.5
do strong acids dissociate more or less than weak acids
what does a low pH indicate about proton loss
can we change properties of molecules based on environment
what can you think of pKa as
Monoprotic, diprotic (e.g., carbonic), and triprotic (e.g., phosphoric) acids:
large Ka means dissociating more and releasing more protons
small pKa means. strong acid because it is the inverse of Ka
what does it mean that an acid’s pKa is 2.5: the acid loses a proton at pH 2.5 and therefore a strong acid
do strong acids dissociate more or less than weak acids: more!
what does a low pH indicate about proton loss: donates lots of protons, large Ka, low pKa
can we change properties of molecules based on environment - yes
pKa is the pH at which a “ionizable” H is removedfrom the molecule
Monoprotic: 1 proton
diprotic: 2 proton (e.g., carbonic)
triprotic (e.g., phosphoric) acids: 3 protons
Why is pH important?
what does pH effect in biomolecules
what happens if blood pH drops
what happens is blood pH rises
so how do we stay In narrow range of pH?
what does pH effect in biomolecules: effects structure and function
what happens if blood pH drops: CNS becomes depressed resulting in coma and death
what happens is blood pH rises: CNS overexcited, muscles spasms leading to convulsions and respiratory arrest
so how do we stay In narrow range of pH?- buffer
buffers!
what are acids
what are bases
what do buffers have
what do buffers do
at equilibrium, what can conj acid and conj bases do
what does the base do
what does the acid do
what happens when pH = pKa
what can we say about buffering capacity at pH = pKa
are biological processes pH sensitive
when is buffering capacity lost
what do buffers have
where do buffers work best at
what are acids: release hydrogen ions (protons) in soln
what are bases: accept protons in soln
what do buffers have: equal amounts of weak acid and its conjugate (weak) base
what do buffers do: keep pH relatively constant
at equilibrium, what can conj acid and conj bases do: able to neutralize small amounts of other acids and bases when they are added to the soln
what does the base do: gobbles up free H+ when acid is added
what does the acid do: releases H+ when a base is added to the soln
what happens when pH = pKa: 50:50 mixture of acid and anion forms of the compound
what can we say about buffering capacity at pH = pKa: greatest at pH = pKa when donating or accepting proton
are biological processes pH sensitive: yes, very
when is buffering capacity lost: when the pH differs from the pKa by more than 1 pH unit
buffers consist of pairs of weak acids and their conjugate bases (salts)
best at pH = pKa ± 1
pKa 9.6
In what pH range can the glycine amino group be a good buffer?
Buffering region, +1 and -1 of the pKa—buffer works best +1 or -1 of the pKa
8.6 and 10.6
GOOD JOB!
Watch YouTube video to memorize amino acids – (structure, 3 letter code, 1 letter code)
Non-polar, Aliphatic R Groups
glycine
alanine
proline
valine
leucine
isoleucine
methionine
Glycine (Gly, G) = no stearichinderance, least non-polar
Proline (Pro, P) = The ring
decreases the flexibility of the structure (bonds can’t “wiggle” as much), this affectspolypeptide backbone flexibility
MeTHIOnine (Met, M) = containssulfur.
These amino acids as a group:
-hydrophobic core of soluble
proteins
-membrane-spanning region of
transmembrane proteins
Aromatic
phenylalanine
tyrosine (add O to F)
tryptophan
Phenylalanine (Phe, F)
Precursor to Tyrosine, and therefore catecholamines
Tyrosine (Tyr, Y)
Has an acidic proton in the Rgroup (pKR – 10.07)
Tryptophan (Trp, W)
Precursor to serotonin, melatonin, vitamin B3
Polar, Uncharged (at phys pH)
serine
threonine
cysteine
asparagine
glutamine
Electrically neutral at neutral pH
R-groups have hydroxyl (often modified), thiol, amide functionalgroups.
R-groups can form hydrogen bonds!
Cysteine (Cys, C) can form disulfide bonds! (Stay tuned!) pKR = 8.18
AsparagiNe (Asn, N) and
Glutamine (Gln, Q) are have
Amide functional groups!
Positively Charged R Groups (at phys pH)
lysine
arginine
histidine
at physio. group are they depotanted or deprotanetd
Lysine (Lys, K) pKR = 10.53
Arginine (Arg, R – pirate!) pKR = 12.48Has a guanidium group.
Histidine (His, H) pKR = 6.00
Remove the carboxyl group,what do you think we get?
At physiological pH, are these groups protonated or deprotonated?
Negatively Charged R Groups (at phys pH)
aspartate - asparDate
glutamte -E
. Can theR group accept a proton?
Aspartate (Asp, D) pKR = 3.65
Glutamate (Glu, E) pKR = 4.25Primary excitatory neurotransmitter of thecentral nervous system.
Generally, “-ate” refers to a base.
A base is a proton acceptor
. Can theR group accept a proton?–YES! MAYBE EVEN 2!
Measuring Protein Concentration
Beer’s law
absorbance is the inverse of what
Let’s say you have an unknownsolution and you want to know whether it is a protein.
Spectrophotometer time!
Tryptophan and Tyrosine absorb light at 280 nm.
Beer’s Law: The concentration of a solution is directly proportional to absorbance.
Absorbance is the inverse of transmittance.
beer’s law: [ ] = k x A280
Amino Acids are Ionizable!
what is a zwitterion?
amphoteric
whch amino acids have a chrial center
Amino acids are weak polyprotic acids. Each amino acid has at least two titratable groups.
At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form.
At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; suchions are called Zwitterions.
At alkaline (basic) pH, the amino group is neutral –NH2 and, the amino acid is in the anionic form.
Amphoteric”
Acts as an acid AND a base
Also, acts as a buffer
all of them do except for glycine and proline
Amino Acid Titration Curves!
A.A can act as both what
What is a pKa? What does it tell you?
Carboxy group
pKa ~ 2.3
Amino group
pKa ~ 9.6
what is PI
which gets deprotanated first, COOH ro NH3
how do you find PI
Remember, amino acids have different forms at different pH.
can act of both acids and bases
What is a pKa? What does it tell you?- pH where it is deprotanated
pI = Isoelectric point
pH at which a molecule has no net charge
Every amino acid/protein has one
Average of the two pKas
COOH gets deprotanated first
ON EXAM: Have a titration curve, ask what form of the A.A exist at what pKa
For glycine, After pKa1- we have a zwitterion –this is the PI
take average of pKa of both
(2.34 + 9.60)/2 = PI
PI = 0.5(pKa1 + pKaR)
Find electrically neutral molecule nad take the pKa of both to find PI
Histidine can be buffer at physiology pH
Histidine has an ionizable Rgroup! (pKR = 6.0). No other AAside chain has a pKa near neutral pH.
The only AA that can be an effective buffer at physiological pH
pI = Isoelectric point
Average of the two pKas
This time, find the two pKs thatstraddle the neutral species andaverage those.
With reference to glutamate titration curve
at pH 2.19
there is/are…
equal amounts of forms I & II
pKa1 = 2.19
With reference to glutamate titration curve
at pH 2.19
at what pH will the uncharged form be at
3.00
the zwitterionic form!
how do you determine in which solution will the molecule be more soluble in
charged molecules are soluble
so whichever solution will make it charged is where it is more soluble
basic solutions take away protons
acidic solutions give protons
will this make the solution more or less charged and thus make it more soluble
Ion exchange chromatography separates polypeptides on the basis of
net charge
If you have a mixture of proteins that you want to separate based on size, a technique you could use would be
SDS PAGE
what are amino acids are in the non polar aliphatic R groups
GAPMILV
Glycine- Gly, G
alanine- Ala, A
proline- Pro, P
Methionine- Met, M
Isoleucine- Ile, I
Leucine- Leu, L
Valine- Val, V
what are amino acids are in the aromatic R groups
WYF
Phenylalanine- Phe, F
Tyrosine- Tyr, Y
Tryptophan- Trp, W
what are amino acids are in the polar uncharged R groups
CTNQS
Glutamine- Gln, Q
Asparagine- Asn, N
Serine- Ser, S
Threonine- Thr, T
Cysteine- Cys, C
what are amino acids are in the positively charge R groups
HRK
Histidine- His, H
Arginine- Arg, R
Lysine- Lys, K
Negtauvely charged R groups
DE
Aspartate, Asp, D
Glutamate- Glu, E
Titration
To determine the concentration on an (unknown) acid or base by exactly neutralizing it with an acid or base with a known concentration
Can be used to determine the concentration of the unknown and/or pKa
pKa is where the….
When pH < pKa:
when pH > pKA:
equivalence point: what is true about the concentration of acid and base and about pH and pKa
what happens within the buffering region
When pH < pKa: still protonated
when pH > pKA: deprotanated
equivalence point: [acid] = [base] (50/50) AND pH = pkA
in the buffering region, the pH does not change that much because the buffer has a base to gobble up hydrogens if it needs to and an acid to donate hydrogens if need to
what does multiple pKas mean
what does pH determine the amount of
Multiple ionizable H’s
pH determine the amount of each “species”
what is the Henderson - Hasselbach eqn
From here, you should be able to:
Calculate pH, when others are known
Calculate [base]/[acid], when others are known
Calculate pKa, when others are known
What does pKa equal
relates the [acid], [conj base], pH and pKa together
pH = pKa + log [A-]/[HA]
pKa equals the acid dissociation constant (pH at which H falls off)
Biological Buffers Control the pH
an optimal acid-base balance is maintained in body fluids and cells despite large fluxes of metablites. a buffer system protects the body from fluctuations in pH by sacking up excess H+ or OH-
What are the 3 buffering systems in cells
dihydrogenBiological Buffers Control the pH-phosphate buffering system
carbon acid buffering system (blood)
proteins
Biological Buffers Control the pH
base: HCO3-
acid: co2
pKa = 6.1
Why Amino Acids and Proteins?
Proteins are the main agents of biological change
Amino acids are the building blocks “monomers” of proteins
Structure (Chemistry) Function.
Remember the last lecture about chemical interactions and weak forces…
catalysts
transport
structure
motion
Catalysts – Enzymes (as proteins). Change the rate of chemical reactions
Enolase, Amylase, Kinase, Phosphatase, DNA Polymerase, etc
Transport – Ion Channels, Membrane Transporters, etc.
Hemoglobin (transports O2 in the blood), Lactose permease (transports lactose across the cell membrane)
Structure – Need form. Collagen (connective tissue), Keratin (hair, nails, etc)
Motion – Things gotta move around. Myosin (muscle tissue), Actin (muscle tissue, cell motility), Dynein/Kinesin (move components around the cell)
What are Amino Acids and Proteins?
Proteins are linear heteropolymers of α-amino acids (functional groups attached to the CENTRAL carbon
Amino acids have properties that are well-suited to carry out a variety of biological functions:
Capacity to polymerize (dehydration synthesis forms bond between carboxy and amino groups)
Useful acid-base properties (pay attention to where we can deprotonate – pKa!)
Varied physical properties
Various chemical functionality
Amino Acids: L- and D- forms—because they are chiral
how to remember is the amino acid is L or D
For all amino acids (except proline [ring] and glycine [H]) the alpha carbon is bonded to 4 different groups.
The α always has four substituents and is tetrahedral.
Therefore, there are two possible configurations for each amino acid (except for when R = H [glycine]).
All amino acids (except glycine) are chiral! Non-superimposable mirror images
One chiral center, so L/D instead of R/S
COO- to the top
-R group to the bottom
IF:
Amino is to the LEFT = L
Amino is to the RIGHT = D
L- and D- forms Matter!
When synthesizing AA in the lab, racemic mixtures occur
enzyme however are extraordinary precise
the amino acids that occur in natural proteins are the L configurations
D- amino acids do occur in nature
-small peptides in peotodiglycan of bacteria cell walls
-peptide anitbiotics
-neurotranmsitters (D-glutamate)
-platypus venom.
but are not found in protein
Amino Acid Memorization Time!
Common amino acids can be placed in five groups depending on their R substituents
Nonpolar, aliphatic (non-aromatic) – 7: G, A, P, V, L, I, M (all the first letters of the AA)
Aromatic – 3: F, Y, W (kinda weird lettering here)
Polar, uncharged – 5: S, T, C, N, Q (3/5 are the first letters of the AA)
Positively charged – 3: K, H, R (K is random, but H and R (think pirate) make sense
Negatively charged – 2: D, E (I got nothing)
Non-polar, Aliphatic R Groups
Glycine (Gly, G) = no stearichinderance, least non-polar
Proline (Pro, P) = The ring
decreases the flexibility of the structure (bonds can’t “wiggle” as much), this affectspolypeptide backbone flexibility
MeTHIOnine (Met, M) = containssulfur.
These amino acids as a group:
-hydrophobic core of soluble
proteins
-membrane-spanning region of
transmembrane proteins
glycine, proline, alanine, valine, leucine, isoleucine, methionine
Aromatic
Phenylalanine (Phe, F)
Precursor to Tyrosine, and therefore catecholamines
Tyrosine (Tyr, Y)
Has an acidic proton in the Rgroup (pKR – 10.07)
Tryptophan (Trp, W)
Precursor to serotonin, melatonin, vitamin B3
Polar, Uncharged (at phys pH)
Electrically neutral at neutral pH
R-groups have hydroxyl (often modified), thiol, amide functionalgroups.
R-groups can form hydrogen bonds!
Cysteine (Cys, C) can form disulfide bonds! (Stay tuned!) pKR = 8.18
AsparagiNe (Asn, N) and
Glutamine (Gln, Q) are have
Amide functional groups!
serine, threonine, cysteine, asparagine, glutamine
Cysteines and Disulfide Bonds
Disulfide bonds can form under oxidizing conditions (extracellular)
Play an important role in folding and stability of proteins, esp. those secreted to the extracellular medium
Holds two portions of the protein together increasing the concentration of protein residues and decreasing the concentration of water. Stabilizes structure by preventing water from breaking H-bonds
Can be a “seed” for hydrophobic interactions increase folding
Two cysteines that are connected via a disulfide bond become a cysteine
Positively Charged R Groups (at phys pH)
Lysine (Lys, K) pKR = 10.53
Arginine (Arg, R – pirate!) pKR = 12.48Has a guanidium group.
Histidine (His, H) pKR = 6.00
Remove the carboxyl group,what do you think we get?
At physiological pH, are these groups protonated or deprotonated?
lysine, arginine, histidine
Negatively Charged R Groups (at phys pH)
Aspartate (Asp, D) pKR = 3.65
Glutamate (Glu, E) pKR = 4.25The primary excitatory neurotransmitter of thecentral nervous system.
Generally, “-ate” refers to a base.
A base is a proton acceptor. Can theR group accept a proton?
aspartate and glutamate
Measuring Protein Concentration
beers law
Let’s say you have an unknownsolution and you want to know whether it is a protein.
Spectrophotometer time!
Tryptophan and Tyrosine absorb light at 280 nm.
Beer’s Law: The concentration of a solution is directly proportional to absorbance.
Absorbance is the inverse of transmittance.– concentration = k x A280
Non-Standard Amino Acids
hydroxyproline in collagen
carboxyglutamate in prothrombin
Amino Acids are Ionizable!
Amino acids are weak polyprotic acids. Each amino acid has at least two titratable groups.
At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form.
At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; suchions are called Zwitterions.
At alkaline (basic) pH, the amino group is neutral –NH2 and the amino acid is in the anionic form.
Amino Acid Titration Curves!
Remember, amino acids have different forms at different pH.
What is a pKa? What does it tell you?
how do you find pI
Carboxy group
pKa ~ 2.3
Amino group
pKa ~ 9.6
pI = Isoelectric point
pH at which a molecule has no net charge
Every amino acid/protein has one
Average of the two pKas
what is cool about glutamate
Glutamate has an ionizable Rgroup!
pI = Isoelectric point
pH at which a molecule has no net charge
Every amino acid/protein has one
Average of the two pKas
This time, find the two pKs thatstraddle the neutral species andaverage those.
what is cool about histidine
Histidine has an ionizable Rgroup! (pKR = 6.0). No other AAside chain has a pKa near neutral pH.
The only AA that can be an effective buffer at physiological pH
pI = Isoelectric point
Average of the two pKas
This time, find the two pKs thatstraddle the neutral species andaverage those.
A Note on pKas
Why do pKa’s 1 and 2 vary among their precise values?
Remember your organic chemistry. How do electron-withdrawing groups affect the ease at which groups can be extracted?
Levels of Protein Structure
primary
secondary
tertiary
quaternary
Primary (1°) Structure
The primary structure of a protein is the sequence of linked amino acids.
These amino acids are linked together through the formation of peptide bonds via a condensation (dehydration synthesis) reaction (endergonic).
Peptide bonds are covalent bonds formed between the α-COOH and α-NH2 groups of two amino acids
Peptides and proteins are broken down by hydrolysis reactions (exergonic but high Ea).
The primary structure of a protein is the sequence of linked amino acids.
AND cross-links
These amino acids are linked together through the formation of peptide bonds via a condensation (dehydration synthesis) reaction (endergonic).
Peptides
Peptide – two or more amino acids (2-17) joined covalently by a peptide bond.
Polypeptide – many amino acids joined (17-50) together by peptide bonds (M.W. < 10,000)
Protein – macromolecule with one or more polypeptide chains
Numbering and naming starts from the amino (N-) terminus which is usually on the left. The peptide is then read from left toright.
how to find Charge on a Peptide
Identify the ionizable groups (look at your N-terminal, C-terminal, and side chains)
Determine the charge of each at the given pH
Is the pH < or > the pKa?,
Is the charge +2, +1, 0, or -1 – add all charges together
Add all of the charges together!
charge on peptide
Which pKa’s do you need?
Conjugated Proteins
Proteins containing permanently-associated components other than amino acids.
The non-amino acid part is called a prosthetic group.
Peptides Can Have Biological Activity
oxytocin: stimulate uterine contractions
bradykinin: inhibits tissue inflammation
insulin: pancreatic hormones, need for sugar metabolism
glucagon: pancreatic hormone, opposes action of insulin
Protein Purification
It is essential that we know the sequence of the protein we are studying in order for further biochemical analysis
Many drug targets are proteins. We should study the mechanism or pathway the target is involved in. We should determine its structure function.
It’s not easy. Proteins exist in extremely complex mixtures in cells, and to understand the function, gotta isolate. But that isolation cannot affect the structure or function of the protein. Gotta be careful.
You might have to do this one day. Brownie points for sounding like you know what you’re talking about
Protein Purification steps
1- choose protein to purify
2- choose source
3- extract protein from cells
4- separate protein from cellular components
5- purify specific protein or Peoria complex
6- study (activity, structure, mechanism of action, work)
Protein Purification steps 3 & 4
- Extract proteins from cells
Lyse cells by destroying membranes and releasing cytosolic protein mix crude extract
Might have to get physical…
4.. Separate Proteins from other cellular components
Centrifugation!
centrifugation
separates large from small particles
separate sub cellular fractions, isolate specific organelles or isolate all soluble porcine in a cll after cell lysis
once protein-containing solution (supernate) Is separated from the rest of the cellular debris the protein of interest can be purified
protein purification
- Separate Proteins from other cellular components
Dialysis: separating small solutes from proteins
Can be put anywhere in the isolation/purification scheme
Protein Purification step 5
Purify specific protein or protein complex
Separating one protein from the rest using fractionation based on physical and/or chemical properties
Chromatography
all Chromatography requires a
stationary phase (resin/beads) and a mobile phase
clomp Chromatography allows separate of a mixture of protein over a solid phase (porous matrix) using a liquid phase to mobilize the proteins
protein with lower affinity for the solid phase will wash off first, proteins with higher affinity will retain on th exodus longer and wash off later ]
fractions are collect and keep those with protein of interest
Size Exclusion Chromatography
Sometimes the first round of chromatography.
Column packed with porous beads
Separates molecules by size (and shape)
Small proteins pass through the porous beads. Longer net distance to travel. Elute later.
Large proteins move around the porous beads. Smaller net distance to travel. Elute first.
Ion-Exchange Chromatography
To elute, just change the pH. Why?-
Separates by charge: all proteins have a net charge(or neutral) at any given pH (depends also on pI)
Column packed with a charge resin of a synthetic polymercontaining bound (+) or (-) groups.
Lets say the resin is (-) charged…
(-) charged proteins will move through faster and elutefirst. Why? because the resin is (-) charged and thus will repel the (-) charged protein
(+) will move through slower and elute later. Why? because the resin is (-) charged and thus will attract the (+) charged protein
To elute, just change the pH. Why?- Change pH, change charge and makes it remove from resin
Affinity Chromatography
Separates by specific binding. Beads in the column are complementary to the protein of interest. The protein will bind to the ligand on the beads.
Other proteins flow right through.
The most specific/efficient form of chromatography.
How to elute?
Add chemical that breaks the bonds.
Add a high concentration of the ligand competes for binding of the protein with the column (stay tuned)
Protein Purification - Electrophoresis
Separates based on size!
Smaller proteins get through the tangles of the gel
easier than larger proteins move farther
Migration rate depends only on molecular weight
SDS – Sodium dodecyl sulfate
PAGE - Polyacrylamide Gel Electrophoresis
Isoelectric Focusing (IEF)
Electrophoresis
Separates based on charge (pI – isoelectric point) and pH!
pI – isoelectric point. pH at which amino acid has no charge.
Separates proteins by allowing them to reach the pH that matches their pI (net charge is zero).
what are the 2 types of Electrophoresis
Isoelectric Focusing (IEF)
(charge based)
SDS PAGE (size-based)
how to measure total protein
how do you measure the real thing
BCA
A280 - mass (mg)
some activity - the protein’s ability to carry out some concrete activity
BCA: color, absorbance and amount
Quantification
Total activity:
Specific activity:
Total activity: the total units of enzyme in a solution
Specific activity: the number of enzyme units per mg of total protein
This is really just showing that
Specific activity is an extension of signal:noise ratio.
The other proteins in the impure sampleare noise (total activity).
The protein of interest is the signal (specific activity).
As we purify, noise decreases, signal increases.
x
factors that contribute to making delta G more negative (less positive)
factors the contribute to making delta G more positive (less negative)
factors that contribute to making delta G more negative (less positive)
- negative delta H (exothermic rxn)
- positive delta S (increasing entropy [more random])
factors the contribute to making delta G more positive (less negative)
- positive delta H (endothermic rxn)
- negative delta S (decreasing entropy [more ordered])
is the rxn spontaneous at high or low temps
delta G is negative
- at all T
- at low T
+ at high T
+ at low T
- at high T
+ at all T
- at all T: spontaneous at all T
- at low T: spontaneous at low T
+ at high T: non-spontaneous at high T
+ at low T: non-spontaneous at low T
- at high T: spontaneous at high T
+ at all T: non-spontaneous at all T
IF: Keq»_space; 1, ∆G° is large and negative meaning?
IF: Keq «_space;1, ∆G° is large and positive meaning?
IF: Keq»_space; 1, ∆G° is large and negative meaning? - spontaneous
IF: Keq «_space;1, ∆G° is large and positive meaning?- non-spontaneous
at what absorbance does Tyr and Trp absorb light
- Tyr and Trp absorb UV light at 280nm – a way of determining protein concentration
Configurational isomers _____ be interconverted without breaking bonds.
A. Can
B. Cannot
what is the difference between Configuration and conformation
B. Cannot
Configuration differs from conformation by a fact that conformations are only due to the orientation of the atoms in molecule around the covalent bond considering the bond as an axis. And changing the configuration causes the cleavage and formation of new chemical bonds
These two molecules are what type of isomers?
A. Configurational
B. Conformational
what makes Configurational different from Conformational isomers
A. Configurational
Configurational - mirror images that cannot be placed on top of each and be the same
Conformational - rotated differently
How many chiral centers does threonine have?
A. 1
B. 2
C. 3
D. 4
B. 2
A molecule with 3 chiral centers has _____ stereoisomers
A. 2
B. 4
C. 6
D. 8
E. 10
D. 8
Molecules “A” and “D” are
A. Conformational Isomers
B. Diastereomers
C. Enantiomers
B. Diastereomers
Which of the following is/are true of enantiomers?
A. They are mirror images of each other
B. They rotate the plane of plane-polarized light in opposite directions
C. They require a chiral center
D. They are named with the E/Z system
A. They are mirror images of each other
B. They rotate the plane of plane-polarized light in opposite directions
C. They require a chiral center
A solution with equal amounts of two enantiomers is called a:
A. Buffer
B. Racemic Mixture
C. Redox Pair
B. Racemic Mixture
A chemical reaction that generates heat is almost certainly…
A. Endergonic
B. Exergonic
C. Endothermic
D. Exothermic
D. Exothermic
When salt is added to water, the highly structured NaCl crystals are randomly dispersed throughout the solution. In this process, the entropy of the system…
A. Increases
B. Decreases
A. Increases
An exothermic reaction in which the entropy increases is…
A. Endergonic
B. Exergonic
C. We need more information to answer this question.
B. Exergonic
An endothermic reaction in which the entropy increases is…
A. Endergonic
B. Exergonic
C. We need more information to answer this question.
C. We need more information to answer this question.
For a given reaction, ∆H = -10.6 kJ/mol and ∆S = + 7.8 kJ/mol-K. This reaction is clearly…
A. Endothermic
B. Endergonic
C. Exergonic
D. None of the above
C. Exergonic
A typical Hydrogen bond in bio-molecules is between H and _____
A. C or O
B. C or P
C. N or O
D. N or P
E. O or P
C. N or O
A carboxyl group is…
A. Polar
B. Non-polar
C. Amphipathic
A. Polar
A methyl group is…
A. Polar
B. Non-polar
C. Amphipathic
B. Non-polar
For the dissolving of a gas in water, the entropy of the system…
A. Does not change
B. Decreases
C. Increases
B. Decreases
The osmolarity of a sample of lysosomes is 0.26 M. Relative to these, a 0.10 M sucrose solution is…
A. Isotonic
B. Hypertonic
C. Hypotonic
C. Hypotonic
If you immerse the lysosomes (0.26 M osmolarity) in this 0.10 M sucrose solution, what will happen?
A. The lysosomes will stay the same size.
B. The lysosomes will expand and may burst.
C. The lysosomes will shrink.
B. The lysosomes will expand and may burst.
If the pH of a solution is 6.0, the [OH-] is…
A. 106 M
B. 108 M
C. 6 μM
D. 10-6 M
E. 10-8 M
10^-8
In what pH range can the glycine amino group be a good buffer?
A. pH 1.0-7.0
B. pH 7.0-14.0
C. pH 7.6-9.6
D. pH 8.6-10.6
E. pH 9.6-11.6
D. pH 8.6-10.6
The pKa of the amino acid carboxyl group is typically…
A. 1-2.5
B. 4-5
C. 6-7
D. 8-10
E. 11-12
A. 1-2.5
The pKa of the amino acid amino group is typically…
A. 1-2
B. 4-5
C. 6-7
D. 8-10
E. 11-12
D. 8-10
With reference to the glutamate titration curve, at pH 2.19, there is/are…
A. More of Form I than of any other.
B. Equal amounts of Forms I & II.
C. Equal amounts of Forms II & III.
D. Equal amounts of Forms III & IV.
E. More of Form IV than any other.
what do you have to know about this problem
B. Equal amounts of Forms I & II.
you have to know (for example) at Pka1, the molecule is in limbo which means that half is still the first form of the molecule and half is the second form of the molecule
Pka is also the pH at which a proton is removed
With reference to the glutamate, at what pH will an uncharged form be predominant?
A. pH 1.00
B. pH 3.00
C. pH 4.25
D. pH 7.00
E. pH 11.00
B. pH 3.00
it is like you are finding the pI :)
With reference to glutamate, what is the approximate pI?
A. 2.2
B. 3.2
C. 4.3
D. 7.0
E. 9.7
B. 3.2
This amino acid is…
A. M
B. C
C. T
D. W
E. Y
C. T
This amino acid is…
A. A
B. N
C. D
D. E
E. G
B. N
Using standard methods (e.g. Merrifield Synthesis) it is possible to synthesize peptides up to about _____ AA in length.
A. 10
B. 20
C. 40
D. 100
E. 200
D. 100
Ion exchange chromatography separates polypeptides on the basis of…
A. Molecular weight.
B. Net charge.
C. Ability to bind a ligand.
D. pH
B. Net charge.
If you have a mixture of proteins that you want to separate based on size, a technique you could use would be…
A. Ion exchange chromatography.
B. Affinity chromatography.
C. SDS Polyacrylamide Gel Electrophoresis
D. Isoelectric Focusing
C. SDS Polyacrylamide Gel Electrophoresis
Consider 2 proteins with the following properties:
Protein A: pI = 7.6, MW = 82,000, Does NOT bind DNA Protein B: pI = 7.8, MW = 22,000, Does NOT bind DNA
Which technique would be best to separate these?
A. Ion-exchange chromatography
B. Size-exclusion chromatography
C. DNA-affinity chromatography
D. Edman Degradation
B. Size-exclusion chromatography
Which prep has the highest specific activity?
how do you do this again?
A. 1
B. 2
C. 3
D. 4
E. 5
D. 4
Edman degradation can be used to sequence peptides up to _____ amino acids long.
A. 10
B. 20
C. 40
D. 100
E. 200
C. 40
