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ANANBIOANAL - 2010
Pharmaceutical R & D Summit
doi:10.4172/2155-9872.1000082
Quaternary Structure Analysis of Human Hemoglobin by a Near-
UV CD Spectroscopy
Masako Nagai
Research Center for Micro-Nano Technology, Hosei University, Tokyo, Japan
H
uman adult hemoglobin (HbA) has four subunits, namely, two-a subunits and
two b-subunits. X-ray crystallographic analysis have indicated that there are
two distinct quaternary structures, namely, the deoxy state, represented by the tense
(T), low-affinity structure, and oxy state, represented by the relaxed (R), high-affinity
structure. A characteristic spectral change of HbA in the near-UV CD occurs: from a
small positive band in the oxy-R form to a negative CD band with a distinct peak at
287nm in the deoxy-T form. This negative CD band of deoxyHbA known as T-state
marker has been supposed to derive from the changes of Tyr and Trp residues at
the α1β2 subunit interface. To identify the aromatic residue responsible for the CD
band, we have synthesized five recombinant Hbs in E. coli in which non aromatic
residue is substituted for Tyr or Trp residue; rHb (α14Trp→Leu), rHb (β15Trp→Leu),
rHb (β37Trp→His), rHb (α42Tyr→Ser), and rHb (β145Tyr→Thr). We examined the
near-UV CD spectra of these rHbs and a natural mutant, Hb Rouen (α140Tyr→His).
The CD spectra of individual aromatic residue were extracted from the difference
between Hb A and each mutant. We concluded that changes in CD bands arising
fromβ37Trp, α140Tyr, β145Tyr and α42Tyr residues contributed to the appearance
of the negative CD bands at 287nm. To examine different signals of CD band among
aromatic residues, the effects of environments on CD spectra were examined using
model compounds of Tyr and Trp dissolved in various solvants.
ANALBIOANAL-2010
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