ammonium sulfate: "salting out" by competing with ammonium
isoelectric precipitation: change pH to create pI, no charge is less water soluble and will precipitate out.
why will a protein be less water soluble at it's pI?
no net charge so nonpolar, less water soluble and will precipitate out, a type of crude separation
examples of sophisticated separation techniques and when to use them
later in purification, use expensive materials and work better on more homogenous substances.
mixture in mobile phase passes over separating material in stationary phase
separation occurs via distribution in between phases
types characterized by nature of separating material
1. TLC (thin layer chromatography) for lipids
2. paper - amino acids and peptides
3. column for amino acids, peptides and proteins
concentrating purification for amino acids, peptides and proteins.
ion-exchange chromatography, separated by net charge. Can be anion exchanger (DEAE-Sephadex, binds - charges) or cation exchanger (Dowex-50, binds + charges)
like charges bind, neutral or opposite charge wash off
increase salt or change pH to elute.
will increasing or decreasing pH elute a protein bound to a cation exchanger.
increasing, makes cation into acid so protein comes off
will a protein with a net negative charge bind to a cation or anion exchanger?
will a negative protein be eluted by increasing or decreasing pH?
decreasing, make anion into base to accept proton, must lower solution to make it acid.
How does a cation exchanger separate net negative from neutral
it can't, they wash off together.
gel filtration chromatography
separation by size and shape, aka size exclusion chromatography, molecular sieve chromatography
VERY SMALL VOLUME (<5%)
stationary phase is porous beads (Sephadex), large molecules go around and elute first, small molecules enter all beads and come out last. Compact molecules move more slowly than elongated, "effective radius"
in gel filtration chromatography, elongated molecules move like they are large spheres with the molecule as the diameter.
They therefore come out earlier/move faster than they should
This is a source of error.
arrange the following in the order they will emerge from a gel filtration column
separates by specific binding to another molecule (or ligand), attached covalently to stationary phase.
substrate/enzyme, hormone/receptor, Ag/Ab.
unbound wash off, then elute with free ligand, dialysis
a molecule that specifically and reversibly binds to a protein
how would you separate the protein from bound ligand once complex is eluted from affinity chromatography column?
High performance liquid chromatography
separation based on polarity. Stationary phase can be polar (bind polar molecules, elute by increasing polarity) or nonpolar (binds nonpolar, elute by decreasing polarity)
tightly packed column, high pressure, great resolution, fast.
enzyme assay as a means of product detection
by rate of reaction, follow appearance of product or disappearance of reactant by spectrophotometer
1 unit enzyme activity = amt of enzyme that converts 1 micromol S to P/min at 25C
after each step acn deterimine recovery, calculate cumulative yield of activity, specific activity, cumulative purification factor per step.
How to assess effectiveness of purification step
stepwise % yield = (units recovered at end of step)/units recovered at end of previous step x 100
stepwise purification factor = specific activity at the end of step/specific activity at end of previous step.
If a substance is pure the specific activity won't increase.
Ways to standardize gel filtration chromatography column
molecular weight markers (proteins of known Mr)
Blue Dextran (large polysaccharide excluded by beads, determines Vo = void volume)
elution volume of your protein via standard curve
Polyacrylamide Gel Electrophoresis (PAGE)
proteins migrate in electric field according to net charge. Stationary is polyacrylamide gel, mobile is proteins dissolved in buffer.
mobility (charge/friction), strength of electric field (higher volts is faster, % acrylamide (higher = smaller pores, slower), net charge (larger moves faster), shape (smaller=faster), shape (elongated is slower)
SDS-PAGE helps determine Mr, bids to make all same charge density and shape, migrate to anode with rate dependant on size.
Run with bromophenol blue, anionic small dye.
Detect via Coomassie Blue
Good for subunit composition, 1 or few bands = pure
Blue dextran is large polysaccharide which skips beads in gel filtration chromatography
bromophenol blue is an anionic small dye run with MW standards on SDS-PAGE
Coomassie Blue binds proteins via peptide/Ab binding for western blot on SDS-PAGE
isoelectric focusing gel has ampholytes, mix of organic acids and bases. When placed in electric field, pH gradient forms
molecules with both acidic and basic groups.
Why will ampholyte with pH 3 migrate to anode and pH 9 migrate to cathode?
@ pH 7 most organic acid = COO-, migrates towards +
@ pH 7 organic bases are mostly protonated, NH3+, migrates to -
why do proteins migrate to position where pH = pI
no net charge won't migrate
equilibrium technique, combines isoelectric focusing (1st) and SDS-PAGE (2nd) to resolve complex mixtures. FIrst separation along one part of tube gel, 2nd by running those bands into slab gel, get 2D protein spots (molecular weight and pI
which method should be applied first in two-dimensional elecrtophoresis?
isoelectric focusing must be first because SDS-PAGE makes everything the same charge.
why is two-dimensional electrophoresis more effective than any one method
tests 2 things, unlikely that two compounds have same pI and Mr
primary structure of proteins
amino acid sequence and disulfide bond location
secondary structure of proteins
local folding, reapeated pattern (alpha or beta)
tertiary structure of proteins
overall folding of molecule
quaternary structure of proteins
best way to determine aa sequence of short polypeptides
6M HCl with no O2, complete hydrolysis, leaving free amino acids (spectophotometry)
Then cation exchange chromatography (aa bind column at low pH when + charged, elute by increasing pH. least + elute first (asp), then hydrophobic (gly-phe), then basic/most +.
N-terminal analysis: reagents react with free alpha amino group, form derivative stable to acid hydrolysis. ID'd by HPLC
Edman degredation: automated sequencing method. Can "walk down" polypeptide chain N--->C up to 50-60 aas.
best way to determine aa sequence of a protein
break disulfide by oxidation or reduction (prevent reformation by covalent block like iodoacetate)
amino acid composition (spectrophotometry)
specific internal cleavages by 2 reagents, overlapping fragments (not Edman beyond 60)
sequence fragments, assemble like a puzzle.
Locate disulfide via 2-D paper electrophoresis
tandem mass spectometry (MS/MS)
used to sequence polypeptides
mass spectrometer separates mix of peptides
one product is cleaved, makes a mixture of singly-cleaved products
cleavate products are separated in second mass spectometer
sequence can be read off spectrum
can be performed off a few 100ng of protein extracted from SDS
leu and ile are hard to distinguish (small molecular mass)
conformation of proteins
the spatial arrangement of groups in a molecule that are free to assume different positions due to free rotation about simple bonds
native conformation of proteins
the conformation of a protein that is biologically active.
Only marginally stable, held together by weak interactions (except S-S), considered DYNAMIC (so that they work)
unfolded proteins that are biologically inactive. Some can refold spontaneously in better conditions.
evidence that amino acid sequence --> conformation --> function (3)
proteins have 1 or few native conformations, those with lowest G/most stable
many synthetic proteins spontaneously form into native conformation
some denatured proteins spontaneously renature
folded polypeptide chains (spherical-ish or globular in shape)
enzymes, Ig's, transport molecules, hormones
polypeptide chains arranged in long sheets or strands
structural proteins such as collagen, keratin, silk fibroin
most significant contributor to delta G folding
interactions that maintain protein conformation
ionic bonds/salt bridges
ionic bonds/salt bridges and H bonds role in delta G of protein folding
occur between polar groups trapped in the middle with nonpolar. These bonds are in lieu of bonds that would be made with water on the outside.
DO NOT ADD TO DELTA G, EVEN EXCHANGE.
Disulfide bonds in proteins
only covalent interaction, strongest bond. Holds loops of proteins in place and chains of insulin together.
bond angles in protein folding
Defined mathematically by Ramachandran, PHI AND PSI
limited due to steric hindrance, otherwise O and H collide
specialized right handed spiral (clockwise when viewed from above), favored by L amino acids.
3.6 acids per turn/0.54 nm axial distance is repeat length.
Each NH is H-bonded to CO
R groups stick out from helix
BACKBONE NOT EXTENDED - springy. Hair or wool
Things that stabilize an alpha helix
asp-arg, phe-leu 3-4 amino acids apart
alpha helix breakers
bulky R near each other (ser, thr, cys, asn)
gly (slips due to free rotation)
pro (ring interferes with NH)
polypeptide chains, almost fully extended in zig-zag
beta pleated sheets when chains line up side by side. Can be PARALLEL OR ANTIPARALLEL
H-bonds between NH-CO, perpendicular in antiparallel, not in parallel
R groups alternate up and down, ROOM FOR BULK
Flexible but inelastic, HIGH TENSILE STRENGTH
silk fibroin, spider web
alpha or beta?
more tensile strength
bulky R groups
bends and loops in proteins and most common type
a reverse in direction in polypeptide chain that permits folding.
beta-turn (hairpin turn), requires 4 amino acids to go 180 degrees. H-bond between 1 and 4. Pro forces turn, gly at 2.
ser, asn, pro, gly are most likely to
break alpha helix
appear in beta turn
simple, no teriatry or quaternary, pure alpha helix or beta sheet, not folded or turned.
insoluble in water (lots of nonpolar aas, don't fold)
structural proteins, rope or rod-like, fibers, resist denaturation, STRONG
alpha keratin, collagen
2 alpha helixes wrap into LEFT handed coil, two chains interact via hydrophobic interactions
chains held by S-S bond (like a perm)
hair stretches when wet because water breaks H-bond
hard keratin (nails, horns, claws) have more S-S bonds than soft (hair, wool)
Two alpha helixes will wrap into what kind of coil?
left handed (2 rights make a left, two lefts make a right)
major structural protein in mammals (1/3 body protein, 35% gly)
mostly gly, pro, ala, hyp (hydroxyproline)
typical sequences of gly-X-pro or gly-X-hyp
left handed helix (hyp and pro are good in left, break right), 2x as extended as alpha so little stretch, high tensile strength.
gly at inside of triple right helix so as small as possible = tensile strength
exterior nonpolar, water insoluble
peptide bonds face inside so resist proteolysis
vitamin C and proline hydroxylase in collagen
ascorbic acid protects protein hydroxylase and prevents F2+ from becoming F3+
hydroxyl group stabilizes triple helix in collagen
resists proteolysis because peptide bond is inside and water insoluble
covalent cross-links so that even if chain is nicked, structure doesn't fall apart.
covalent crosslinks via hyl and lys stabilize structure
happens post-translation, after secretion into extracellular space
form between chains of triple helix and between helices after fibrils assemble
genetic mutations of collagen
substitute bulkier amino acid for gly, causes connective tissue defects
recurring theme or pattern of secondary structure
alpha-helix-turn-alpha-helix (180 degrees)
beta-strand-turn-beta-strand (180 degrees)
alpha-helix-turn-alpha-helix (alpha alpha)
beta-strand-turn-beta-strand (beta beta)
parallel beta sheet formed
region of polypeptide that maintains conformation and function independantly of rest of polypeptide.
made of motifs
include antibody molecules, substrate binding sites of some enzymes
loss of biological activity.
decreases water solubility to cause precipitation
caused by affecting noncovalent bonds,
detergent, urea, organic solvents that stabilize nonpolar groups at the exterior, cause unfolding
change in pH, increased temp, high salt concentration
some renature, especially small with few S-S bonds
as S-S bonds increase, chance of renaturation ______
decreases, harder to make right combo. Smaller helps.
large proteins and renaturation
large proteins can't renature, they are kinetically blocked, too many wrong ways, can't find right way.
in vivo they are folded during translation (stepwise, each step makes next step) or have chaperones
polypeptide bonding proteins, usually nonpolar bond
block incorrect interactions (prevent nonpolar from being buried before partner is translated)
guide correct folding of new proteins
quaternary structure is held together by
4 weak noncovalent interactions, occasional S-S bond
aided by chaperones
more complex structure than fibrous proteins
all have tertiary, some quaternary
more easily denatured than fibrous (fewer covalent crosslinks, less twisted)
globular protein, binds and stores O2 in muscle fro use in cellular respiration
relatively small, one polypeptide chain, no cys
alpha helix and loops (no beta or s-s)
1 domain "globin fold" around heme, polar facing out, nonpolar face in, hydrophobic pocket
prosthetic group: heme (porphyrin ring with Fe2+)
why must heme in myoglobin stay in the hydrophobic pocket?
oxidation of Fe2+ = Fe3+, met Mb (brown).
Fe3+ = ferriMb or metMb can't bind O2.
When protein is denatured this happens
How Mb binds O2
Fe2+ has 6 bonding orbitals
4 bind porphyrin N (planar)
1 binds proximal his N (perpendicular)
1 binds O2 reversibly (perpendicular to heme plane). Distal his shields O2
Mb and CO
toxic. Competes for O2 site in Mb, Hb, cytochrome a3
CO binds heme with 20,000x affinity of O2, so distal his is bent to reduce affinity. Binds with Mb at 25x and Hb with 200x
At high O2 concentration, myoglobin equilibrium shifts to _____ to form _________ and a fresh supply of O2 is delivered to muscle from lungs via arteries.
right to form MbO2
myoglobin respiration equation
Mb + O2 <---> MbO2
At low O2 concentration, myoglobin equilibrium shifts to ________ to form _________ and muscle uses up O2 via cellular respiration
left to form deoxymyoglobin
what is myoglobin's #1 function?
storage depot for O2
pO2 at which 50% of ligand-binding sites on Mb are occupied.
dissociation constant (Kd is inverse of Ka, lower means tighter binding)
p50 of Mb is very low, so high affinity for O2
shape of Mb's saturation curve
function of hemoglobin
binds and TRANSPORTS O2 throughout body.
structure of hemoglobin
4 non-identical subunits, alpha and beta (like Mb), tetrahedral, a-b by exposed hydrophobic, hemes buried and apart. Changes shape on binding O2. Each subunit can bind O2 (4 each)
"other shape". Hb changes shape upon binding O2.
deoxy form: O2sits out of porphyrin plane, T-state, low affinity for O2, salt bridges
oxy form: Fe sits in porphyrin plane, R-state, high O2 affinity, broken salt bridges
deoxy form of Hb
Fe2+ sits out of porphyrin plane, T-state "taut", salt bridges, low O2 affinity
T-state of hemoglobin
Fe2+ sits out of porphyrin plane, "taut", deoxy, salt bridges, low O2 affinity
oxy form of Hb
R-state, "relaxed", Fe2+ sits in porphyrin plane, broken salt bridges, high affinity for O2
sickle cell anemia. 1 amino acid substitution on beta chain (glu to val). neg charged to nonpolar, now hydrophobic interactions, form aggregates/tetramers, shape changes, decreases solubility, short lifespan, block capillaries.
Tell by gel electrophoresis or PCR (moves less far toward anode than HbA)
why does HbS not move as far toward the anode as HbA?
valine sub for glutamate, less negatively charged.
Hb O2 binding equation
pKa higher pH than tissues on deoxy side, pH of lungs higher than pKa on oxy side.
When O2 binds, conformational change, breaks salt bridges, lowers pKa and releases protons
cooperativity (Hb) and result
O2 binding to 1 subunit of Hb, conformational change from T to R, conformational change in other three subunits, increase O2 affinity
As a result the saturation curve is sigmoidal, not hyperbolic like Mb
Hb saturation curve is
sigmoidal (due to cooperativity/affinity)
Allows release of O gradually, even distribution throughout body, no "dump"
Low point of saturation curve for Mb and Hb
Mb goes down to 80 for land mammals, Hb goes down to 10 because it's purpose is delivery, not storage.
On saturation graph, curve more to the right has ______________ than curve on the left
affinity for O2
subcategory of ligand
a molecule that binds to a protein at a site separate from the protein's functional binding site and modulates activity of protein.
Protons are effector in Hb
Effect of H+ in Hb pathway
acts as effector
enhances uptake of O2 at lungs and delivery to tissues via Bohr effect.
Higher H+ = lower pH.
at lungs shifts equation to right so CO2 can be blown out. Low CO2 increases affinity for O2
high CO2 at tissues, binds and stabilizeses deoxy Hb via salt bridge, shifts eq to left, O2 released. Higher CO2 at tissues decreases affinity of Hb for O2
Low CO2 at lungs and Hb
low CO2 at lungs increases affinity of Hb for O2
High CO2 at tissues and Hb
high CO2 at tissues decreases affinity of Hb for O2
at low pH, Hb __________O2 so ____________
at low pH, Hb has less affinity for O2 so curve shifts to right, less O2 bound at same pO2
mechanism of CO2 transport, binds ~13% in blood, formed when Hb binds CO2 at N-terminal (releases H+), usually at tissues
three ways that CO2 is transported through the blood
dissolved CO2 (least)
2,3 BPG (bisphosphoglycerate)
VERY negative, made in RBC, crosslinks 2 beta chains of deoxyHb via salt bridge (to lys/his).
Shifts O2 binding equilib to left, decreases affinity of Hb for O2 (shifts right). Stabilizes T form
CAUSES SIGMOID CURVE, enables unloading of O2
physiological responses to low environmental O2
increase 2,3-BPG (or increased RBC).
LOWERS affinity, lowers saturation, but makes proportion/delivery close to sea level.
evolutionary response to low O2 in utero, HbF has HIGHER AFFINITY for O2 than HbA, takes O2 from mom. Myoglobin even higher.
O2 in utero: HbA --> HbF--> MbF
family of Y-shaped proteins produced and secreted by B-lymphocytes with binding sites for antigens, effector that determines response.
tetramer H2L2 joined by disulfide bonds.
Domains made of antiparallel beta pleated sheets called IMMUNOGLOBULIN FOLDS
Variable domains (N-term of VH and VL, with Ag sites), constant domains of H chains (CH, same per class, contain effector), constant domains of L chains (CL, determines two types (kappa and lamda), both in all classes of Ig)
cleavage of Igs
can be cleaved at hing region into 2 fragments: Fab (antigen binding domains) and Fc (fragment crystallizable)
site on Ag recognized by Ab (idiotype)
site on Ab that recognizes Ag (epitope)
epitope to idiotope
specific but based on shape so cross-reactivity. TIGHT, high affinity for Ag.
large polymers/foreign cells (or haptens linked to polymer), contain epitope to be recognized and bound
antibodies can cross-link 2 antigens
can also be multivalent and make larger aggregates that precipitate out of solution
strongest known affinity
antibody to antigen
small molecules made antigenic by linking to a large polymer.
produced first (primary immune response), activates complement
low affinity individ but high avidity due to structure
5 units in circle, linked by J chains/disulfide bonds
total binding strength--more weak bonds in IgM
MOST, produced after IgM, main serum ab.
Crosses PLACENTA and goes into MILK, passive immunity
cascade of proteases activated by Ab-Ag complexes (inflammation, lyses pathogen)
major secretory Ig, monomer (blood) or dimer (secretions, J chain)
transcytosis (enters epithelial cells, secreted via apical membranes)
defense against infection
binds mast cells in tissues and basophils to release histamine, eosinophils for parasites.
cell surface form (naive B cell with IgM) and secretory form (binds basophils and induces antimicrobial and B-cell-activating factors. NOT HISTAMINE release
enzyme-linked immunosorbant assay
assay for Ag or Ab
bind to polystyrene, wash, block unoccupied, wash, add Ag or Ab, wash, substrate forms color.
ELISA for HIV
pure HIV Ag bound in production. add blood as Ab. Positive suggests infection, INDIRECT, not definitive. Testing for Ab not Ag.
False positives due to cross-reactivity, so there is a more specific
tests for Ab in blood, not Ag.
capture assay. Ab on plate, Ag binds, another Ag on top, sandwich.
specific HIV test, 3 different matches make false positive/negative unlikely. (PCR too)
SDS gel electorphoresis
nitrocellulose membrane, innoculated with Ab. Specific for antigenicity and MW.