Highly Asymmetric Interactions between Globin Chain during Hemoglobin Assembly Revealed by ESI Mass

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Highly Asymmetric Interactions between Globin
Chain during Hemoglobin Assembly Revealed by ESI
Mass Spectrometry

• Ming-Yih Lai
• 2006.4.20

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Introduction 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.

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Hemoglobin Assembly in Vivo
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Why 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.

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What 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

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ESI Mass spectrum of Hemoglobin (10µM) at pH8
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
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What 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).

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ESI mass spectrum at different pH
Biochemistry, Wendell P., Griffith and Igor A.
Kaltashov, 2003, 42, 10024-10033
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What 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.

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What 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.

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CD 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
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Graph 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
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Graph 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
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Graph 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
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What 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

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Where 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.

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Why 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.

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My 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 .

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Reference

• 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

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Discussion