Title: Highly Asymmetric Interactions between Globin Chain during Hemoglobin Assembly Revealed by ESI Mass
1Highly Asymmetric Interactions between Globin
Chain during Hemoglobin Assembly Revealed by ESI
Mass Spectrometry
2Introduction of Hemoglobin (a2ß2)
- consisting of two a and two ß chains which are
non-covalently bound each other. - Mw is about 64.5 kDa.
- a has 141 residues and there are 7 a- helix in
the a chain. - ß has 146 residues and there are 8 a- helix in
the ß chain. - There is one Heme () group sitting on each
chain.
3Hemoglobin Assembly in Vivo
4Why study hemoglobin assembly/dissociation?
- The dissociation of the hemoglobin molecule has a
direct effect on its oxygen-binding properties. - Hemoglobin scavengers such as haptoglobin do not
bind to the intact protein, so Hb dissociation is
an important determinant of its rapid clearing
from circulation during hemolysis or following
the transfusion of Hb-based oxygen carriers. - Numerous devastating disorders like sickle-cell
anemia is linked to abnormalities in the
hemoglobin dissociation process.
5What are the mechanisms proposed out there?
- There are three main proposed mechanisms
- a ß? aß? a2ß2
- If this is true, we should see ß.
- a ß ? aß?? aß? a2ß2
- If this is true, we should see aß
- aß? aß? aß ?a2ß2
- If this is true, we should see aß
6ESI Mass spectrum of Hemoglobin (10µM) at pH8
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
7What could we know from previous spectrum?
- The charge state distributions of protein ion in
ESI could be used to monitor the tertiary
structure. The unfold protein has higher charge
state. - Dobo, A.(2001) Anal.
Chem. 73,4763-4773. - The spectrum supports that ß is more flexible
(partially- unstructured) because of broad and
high charge state(1023). - On the contrary a is more rigid because of
narrow and low charge state (79) - The spectrum also shows there is no ß exist but
a and aß. This suggests that the a form first
then bind to ß to form aß intermediate (because
there is ß and a in the spectrum).
8ESI mass spectrum at different pH
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
9What could we know from charge state distribution?
- The average charge state of dimer (aß) is 12
but the average charge state of a and ß is 8. - The average charge state of tetramer (aß)2 is
18 but the average charge state of dimer (aß)
is 12 - This is consistent with the reduction of the
solvent accessible protein surface area due to
the dimerization. - It is also interesting that the charge state
distribution of aß and aß are basically the
same. This suggest that the flexibility of ß is
reduced by the binding to a.
10What could we know from the observed species ?
- The a exist at pH 4. This shows the strong
binding between a and heme group. - (aß)2 and aß dominate at a pH above 4.
- This is consistent with the fact that Hb is
stable down to pH 4.1 for at least 24hrs. - In the spectrum, only aß is observed (no ß and
aß). This suggests that the mechanism should be
aß? aß? aß ?a2ß2 - In the spectrum, (aß)2 is also not observed.
This suggests that aß can not form dimer.
11CD spectrum at different pH
- Protein is stable at pH 5 through pH 9
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
12Graph of Fraction of native protein a, ß, a,
,aß, aß, (aß)2 vs pH
- The data from ESI mass agree with CD spectrum data
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
13Graph Fraction of heme-liganded species a,aß,
aß, (aß)2 vs pH
- The data from ESI mass agree with CD spectrum
data but pH8 and above. The reason is that at
high pH the noncoordinating aromatic residue
around heme binding pocket is deprotonated which
make the intensity drop. It is not reliable at pH
8 and above.
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
14Graph of a/(aa) vs pH overlay with
oligomeric species vs pH
- This shows that the strong correlation between
the formation of a and oligmerization. - The author also suggest that a serves as
template for dimerization.
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
15What is the mechanism proposed by this experiment?
- a serve as template for dimerization
- ß bind to a and form aß intermediate
- With the formation of aß, the ß adapt
native-like conformation and bind to heme group. - After forming the aß complex, the dimer would
from tetramer. - aß? aß? aß ?a2ß2
16Where is asymmetry ?
- Although a and ß are similar in tertiary
structure, they react very differently. - a could form a even at low pH but ß is
unstructured until aß complex forms.
17Why is asymmetry important ?
- It is shown that the relationship between
flexibility in the structure and binding to
protein is intimate. So the ß has flexibility
which help to ß to bind a . - Those disorder in the structure will be relieve
upon the binding of the protein. The binding to
a lock the ß s structure to native-like
conformation. - This characteristic of asymmetry prevent the
monomers aggregate (a2,a4,) in a crowd cytosolic
environment of red blood cell (5mM.) This
aggregation may result in the loss of
cooperativity in charring O2.
18My opinion on this paper
- Positive If you could adjust the experiment
condition to the optimal ( no or low
fragmentation in gas phase), the MS is very good
tool to detect intermediate because the
sensitivity of mass spectrometry is very
low(10-12 to 10-15 M). - Negative If you have isomers in the
intermediate, you would have problem to determine
which one is the actual intermediate. Like aß or
aß .
19Reference
- 1. Highly Asymmetric Interactions between Globin
Chain during Hemoglobin Assembly Revealed by ESI
Mass Spectrometry Biochemistry, Wendell P.,
Griffith and Igor A. Kaltashov, 2003, 42,
10024-10033
20Discussion