Lecture 3:Hemoglobin (structure, function) Protein purification and characterization techniques Flashcards
(93 cards)
1
Q
What is the term for the combination β-α-β?
A
supersecondary structure
2
Q
Myoglobin and hemoglobin bind
A
heme
molecules
3
Q
Myoglobin and hemoglobin bind heme
molecules, which bind
A
O2
4
Q
Myoglobin:
A
binds and store O2
in muscle
(picks it up from the bloodstream and stores
it in muscle for use when needed)
5
Q
Hemoglobin:
A
found in the blood; transports
O2
from the lungs to tissues
6
Q
Structure of heme: The iron ion must be
A
ferrous Fe2+ to bind
O2
7
Q
The iron ion (must be ferrous Fe2+ to bind
O2 ) can form two additional bonds
A
5 th coordination site: imidazole ring of a
histidine on the protein (proximal histidine)
* 6 th coordination site: binds oxygen
8
Q
Oxygen binding changes the position of:
A
The heme iron ion
9
Q
Role of heme: binding oxygen: before oxygen binding, fe ion lies slightly:
A
outside
the plane of the porphyrin
10
Q
Role of heme: binding oxygen: after oxygen binding:
A
Fe ion moves into the plane of the
porphyrin upon oxygen binding
11
Q
Oxygen must only be released as:
A
O2
(not in the superoxide)
12
Q
superoxide is:
A
very reactive (reactive oxygen species, ROS), can
damage cells
13
Q
If oxygen is rekleased as a superoxide:
A
Iron would be left in the ferric (Fe3+) state,which can’t bind O2
14
Q
metmyoglobin
A
myoglobin bound to iron in the Fe3+ state
15
Q
Role of distal histidine
A
Distal histidine stabilizes the
bound O2
(imidazole group
hydrogen bonds with the bound
O2)
16
Q
Oxygen-binding curve:
A
plot fractional saturation
vs. the concentration of
oxygen (partial pressure
pO2
)
17
Q
P50 =
A
half-saturation of the
binding site
18
Q
for human myoglobin, p 50 =
A
~2 torr (mm Hg)
19
Q
Structure of hemoglobin:
A
tetramer
20
Q
Hemoglobin tetramer
(homodimer of
heterodimers):
A
pair of
identical αβ dimers (α1β1
and α2β2
)
21
Q
Structure of hemoglobin: tetramer; each subunit consists of:
A
a
set of α helices in the same
arrangement as the α
helices in myoglobin.
22
Q
hemoglobin p50
A
26 torr
23
Q
What does the oxygen binding curve of heoglobin look like?
A
sigmoidal
24
Q
What do we mean when we say that hemoglobin has COOPERATIVE binding of O2?
A
binding
or release at one site within the
tetramer increases the likelihood of
binding or release at another site
25
Cooperativity allows
hemoglobin to
o bind oxygen in
the lungs (high pO2
) and
release oxygen at the tissues
(low pO2
) ➔ efficient oxygen
transport
26
Hemoglobin cooperativity incorporates aspects of two
limiting models:
-the concerted model
– the sequential model
27
the quaternary structure of deoxyhemoglobin
* T (tense) state
28
= the quaternary structure of oxyhemoglobin
* R (relaxed) state
29
R (relaxed) state = the quaternary structure of oxyhemoglobin
--> O2 binding sites are
free of strain and can bind oxygen with higher affinity
30
Cooperative binding model: concerted model
31
A combination of the two models is needed to
explain the actual
cooperative behaviour of O2 binding to Hb
32
Cooperative binding model: sequential
model:
As each oxygen molecule binds to one site in the hemoglobin
tetramer, the affinities of the remaining sites increase without
inducing a full conversion from the T into the R state.
33
2,3-bisphosphoglycerate (2,3-BPG)binds to
a pocket in the Hb tetramer
(exists only in the T state)
34
2,3-BPG binds in
the central cavity of
deoxyhemoglobin (stabilizes the T-state)
35
2,3 BPG stabilizes:
T state of
hemoglobin and facilitates
the release of oxygen
36
2,3-BPG is an
allosteric effector:
a molecule that affects the binding
of another molecule from a distance
37
Fetal Hb is
a2y2 not a2B2 and has low affinity for 2,3-BPG
38
Effectors on oxygen binding and cooperativity :
1. 2,3-bisphosphoglycerate (2,3-BPG)
2. Carbon monoxide (CO)
3. Bohr effect: H+ and CO2
39
Effectors on oxygen binding and cooperativity: carbon monoxide
Carbon monoxide (CO) = a competitor for the
oxygen-binding site [binds more tightly]
* Displaces O2 even at low pO2
40
What effect will CO binding have on the oxygen-binding
curve of hemoglobin?
* leftward shift in the oxygen-binding curve
* Hb will have a higher affinity for O2
,
preventing efficient oxygen release to tissues
41
Treating carbon monoxide poisoning
* Ngb-H64Q: mutant neuroglobin
protein that binds carbon monoxide
500 times more tightly than
hemoglobin
* Exchange takes less than 60 seconds
42
H+ promotes
s the release of O2
from Hb
and stabilizes the T state
43
At lower pH, ionic bonds form tha
stabilize the T
state, leading to a greater tendency for oxygen to
be released (note His 146)
44
Carbon dioxide stimulates oxygen release by two mechanisms:
carbon dioxide reacts with water to form a bicarbonate ion and a hydrogen
ion, resulting in a drop in pH that stabilizes the T state.
* a direct chemical interaction between carbon dioxide and
hemoglobin stimulates oxygen release
45
CO2 reacts with
terminal amino groups
in hemoglobin to form carbamate
46
How is CO2
transported to the lungs?
1. Carbamate formation
2. Bicarbonate ions
47
Hemoglobin diseases: sickle cell anemia
Val on the surface of the
T-state molecule
* Can interact with other Tstate HbS molecules
(hydrophobic patches),
form aggregate
48
hemoglobin diseases: a thalissima:
α-thalassimia
Not enough α-chain made
β-chains form tetramers with no cooperativity
49
B thalissima:
Not enough β-chain formed
α-chains accumulate as insoluble aggregates
50
myoglobin: bound oxygen is stabilized through
distal histidine
51
What does lowering the pH do to oxygen affinity?
lowering the pH decreases the oxygen affiniity of hemoglobin and facilitates the release of oxygen into metabolically active tissue
52
Differential centrifugation for cell fractionation
-Step 1: disrupt the cell membranes of
intact cells, form a homogenate
* Step 2: centrifuge the homogenate at low
speed, yield a pellet consisting of heavy
material and lighter supernatant
* Step 3: centrifuge the supernatant at a
higher centrifugal force, yield another
pellet and supernatant
* This process of differential centrifugation
is repeated many times to yield several
fractions of decreasing density. One
fraction will be enriched for the desired
activity.
53
Sedimentation coefficient values of soluble
proteins correlate with
molecular weight
54
The smaller the S value, the more slowly
a
molecule moves in a centrifugal field
55
calculating s:
The smaller the S value, the more slowly a
molecule moves in a centrifugal field
S = (1- νρ)/f
ν = partial specific volume
ρ = density of medium
f = frictional coefficient
56
Dialysis:
separation of proteins from salts and
small molecules
57
Salting out:Most proteins are less soluble at
high salt
concentrations
58
The salt concentration at which a protein
precipitates
differs from one protein to another
59
Gel filtration chromatography: separation by
size
60
Gel filtration chromatography: separation by size: large proteins:
elute first
61
key property of the gel that is chosen in gel filtration chromatography
Void volume (Vo
) is a key
property of the gel that is
chosen
62
Ion-exchange chromatography: separation by
charge
63
pH of buffer is key in what type of chromatography?
Ion-exchange chromatography:the charge on the ion exchange resin and the charge on the protein must be
complementary;
64
on-exchange chromatography: separation by
charge
Proteins with the same charge as that on the column will exit
the column quickly.Proteins with the opposite charge will bind to the beads and
are released by increasing the salt concentration of the buffer
65
Affinity chromatography: separation by
affinity for
a specific ligand
66
how does affinity chromatography work?
* Proteins with affinity for
the attached group are
retained.
* Bound proteins are
released by passing a
solution enriched in the
ligand to which the protein
is bound through the
column.
67
High-pressure liquid chromatography (HPLC)
Gel filtration
High-performance liquid chromatography
(HPLC) uses very fine beads in columns and
pressure to move the liquid through the
column, which leads to sharper separations
between proteins and a more rapid
separation
68
Protein purification is achieved by c
combining
different separation step
69
specific activity =
total activity / total protein
70
percent yield=
(current total activity/original total activity ) x 100
71
percent yield =
current total activity/ original total activity
72
purification level =
current specific activity/ original specific activity
73
specific activity should rise with:
each purification step
74
SDS polyacrylamide gel electrophoresis
(SDS-PAGE)
v = Ez/f
f = 6πηr
v = velocity of migration, E = electric field strength, z = net charge of protein, f = frictional coefficient,
η = viscosity of medium, r= radius of sphere
75
SDS-PAGE gel: Coomassie blue stain
Proteins separated by SDS-PAGE are visualized by
staining the gel with dyes such as Coomassie blue
76
SDS-PAGE: estimate protein molecular weigh
Electrophoretic mobility of many proteins
in SDS–polyacrylamide gels is linearly
proportional to the logarithm of their mass.
77
Isoelectric focusing (IEF): separation according to
pI
78
isoelectric focusing =
separates proteins in a gel on the basis of their pI
79
2D gels: IEF and SDS-PAGE combined
Separates proteins in two directions:
* isoelectric focusing in a horizonal direction
* SDS-PAGE in a perpendicular (vertical) direction
80
Protein mass spectrometry
convert the analyte into gas-phase
ions and apply electrostatic
potentials to measure the massto-charge ratio (m/z).
81
Antibodies:
proteins synthesized in
response to the presence of a foreign
substance (antigen)
82
Epitope (antigenic determinant):
specific cluster of amino acids on
the target molecule that an
antibody recognizes
83
Polyclonal antibodies
heterogenous mixtures of
antibodies (derived from multiple antibodyproducing cell populations)
each antibody is specific for one of the various
epitopes on an antigen
84
Monoclonal antibodies:
: identical antibodies
produced by clones of a single antibody-producing
cell
85
ELISA: quantification of proteins using
enzymelinked antibodies
86
ELISA:
enzyme-linked immunosorbent assay (ELISA)
87
Western blot: detect
specific protein in a mixture
88
Western blotting:
proteins are separated in an SDS-PAGE gel, transferred to a polymer,
stained with a primary antibody, stained with a secondary antibody, and quantified
89
Structure determination techniques
* X-ray crystallography
* Nuclear magnetic resonance (NMR) spectroscopy
* Cryo-electron microscopy
90
Structure determination technique: X-ray
crystallography
* Electrons of the atoms scatter x-rays
* Scattered waves recombine (diffraction pattern)
* The way in which the scattered x-rays recombine
depends only on the atomic arrangement
(interpretation results in an electron density map)
91
Structure determination techniques: NMR
Spectroscopy
* Certain atomic nuclei are intrinsically magnetic and
can exist in two spin states when an external
magnetic field is applied
* Nuclei absorb electromagnetic radiation at different
frequencies (chemical shifts), which depend on the
environment of the nuclei (in proteins: the protein
structure)
92
The Nuclear Overhauser Effect (NOE) identifies
pairs of protons that
are in close proximity (interaction is proportional to distance)
93
Structure determination techniques: Cryoelectron microscopy
Cryo-EM is used to determine the structures of large protein and
macromolecular complexes
* A thin layer of the protein solution is prepared in a fine grid and quickly frozen to trap
molecules in an ensemble of orientations
* A transmission electron microscope is used to obtain two-dimensional projections, and
a computer uses these projections to build a 3Dvrepresentation of the protein/complex
